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## Meet the editor

Dr. Lütfi Tutar is currently an assistant professor in the Department of Molecular Biology and Genetics, Faculty of Art and Sciences, Kırşehir Ahi Evran University, Turkey. His interdisciplinary research focuses on bioinformatics analysis of high-throughput data, microRNAs, small RNAs, and heat shock proteins (HSPs) in human diseases and other multicellular organisms.

### Contents

## Preface

Noncoding RNAs (ncRNAs), which play important roles in regulating gene expression in animals, plants, and various human diseases, have had a huge impact on RNA biology. This book provides an overview of current ncRNA research dealing with miRNA- and ncRNA-related human diseases and molecular diagnosis, plant ncRNAs, and the role of lncRNAs in cancer.

The introductory chapter provides an overview of ncRNAs, and the subsequent chapters constitute an essential resource for researchers in the field of ncRNAs, miRNAs and lncRNAs. Chapter 2 discusses the origin of lncRNAs, their biogenesis, their mechanism of action and their role in many biological and pathological processes such as epigenetics, genome imprinting, several cancers and autoimmune diseases. Chapter 3 examines the advantage of noncoding RNA in molecular diagnosis and looks in detail at the roles of miRNAs in various cancers. The processes leading to the identification of their target molecules are described, and the latest diagnostic strategies using miRNAs are discussed with specific examples. Chapter 4 focuses on the role of ncRNAs in lung cancer pathogenesis as well as their potential use as biomarkers or therapies against lung cancer. Chapters 5 and 6 describe the roles of miRNAs in pancreatic ß cell functional modulation and breast cancer. Chapter 5 discusses the multiple effects of different types of microRNAs on ß cell physiology in the context of maintenance and function in type 2 diabetes. Chapter 6 describes the SNP prediction in mature miRNAs dysregulated in breast cancer. Chapter 7 describes the roles and molecular mechanisms of lncRNAs in neural stem cell self-renewal, neurogenesis, gliogenesis and synaptogenesis over the course of neural development. Chapter 8 describes lncRNA and miRNA interactions in digestive system tumors and the vital role played by ncRNAs (both miRNA and lncRNA) in some digestive system tumors, either as oncogene promoting cancer viability, invasiveness, proliferation, and metastasis or as tumor suppressor inhibiting tumorigenicity or inducing apoptosis. Chapter 9 looks at the miRNA-mediated regulatory mechanism related to male infertility. The final chapter summarizes current knowledge on miRNA involvement in oil palm plant response to abiotic stress and suggests possible miRNA-based strategies for the genetic improvement of oil palm salinity and drought stress tolerance. Overall, this book provides a unique perspective for scientists in the field of ncRNA biology.

> **Lütfi Tutar** Kırşehir Ahi Evran University, Kırşehir, Turkey

**1**

**Chapter 1**

*Lütfi Tutar*

**1. Introduction**

era for noncoding RNAs (ncRNAs).

(https://rfam.xfam.org/) [9].

**2. miRNAs (microRNAs)**

long noncoding RNAs (lncRNAs) are longer than 200 nts.

Introductory Chapter: Noncoding

The discovery of ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) in 1960s led to first steps of noncoding RNA (ncRNA) research [1–3]. In 1980, 90–220 nucleotides long small nuclear RNAs (snRNAs) were involved in splicing reported by Lerner *et al.* [4]. When microRNAs (miRNAs) were discovered in the late 1990s, the noncoding RNA research gained a momentum [5, 6]. Furthermore, development of the next-generation sequencing technologies and advanced bioinformatics opens a new

Ubiquitously, ncRNAs are present in all three domains of life and less than 2% of the human genome code for proteins. Generally, ncRNAs do not code for proteins, but there is some exceptions coding for small bioactive peptides [7, 8]. The two major groups of ncRNAs may be separated from each other by having less or more 200 nucleotides (nts): small noncoding RNAs (sncRNAs) are shorter than 200 nts, and

Noncoding RNAs may be grouped into some categories; tRNA (transfer RNA), rRNA (ribosomal RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), Telomerase RNA, tRF (transfer RNA-derived RNA fragments), and tiRNA (tRNA-derived stress-induced RNAs) are all "housekeeping" RNAs. "Regulatory" RNAs are miRNA (microRNA), siRNA (small interfering RNA), piRNA (P-elementinduced wimpy testis (PIWI) interacting RNA), eRNA (enhancer RNA), lncRNA (long noncoding RNA), circRNA (circular RNA), YRNA, crasiRNA (centromere repeat associated short interacting RNA), and TelsRNA (telomere-specific small RNA). Based on structure, LincRNA (long intergenic noncoding RNA), TUCRNA (transcribed ultraconserved RNA), eRNA, and NAT (natural antisense transcript) are "linear" RNAs while ciRNA, ecircRNA (exonic circular RNA), and elciRNA (exon–intron circRNA) are "circular" RNAs. Based on the location, RNAs may be grouped as sense, antisense, intronic, intergenic, and bidirectional RNAs. Some of the ncRNA families are briefly reviewed below. Rfam is a public online database, which provides information for all RNA families, each represented by multiple sequence alignments, consensus secondary structures, and covariance models

miRNAs are ~20 nts long molecules that generally regulate gene expression by inducing mRNA degradation or translational repression. miRNAs originated from

RNAs–A Brief Introduction

#### **Chapter 1**

## Introductory Chapter: Noncoding RNAs–A Brief Introduction

*Lütfi Tutar*

#### **1. Introduction**

The discovery of ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) in 1960s led to first steps of noncoding RNA (ncRNA) research [1–3]. In 1980, 90–220 nucleotides long small nuclear RNAs (snRNAs) were involved in splicing reported by Lerner *et al.* [4]. When microRNAs (miRNAs) were discovered in the late 1990s, the noncoding RNA research gained a momentum [5, 6]. Furthermore, development of the next-generation sequencing technologies and advanced bioinformatics opens a new era for noncoding RNAs (ncRNAs).

Ubiquitously, ncRNAs are present in all three domains of life and less than 2% of the human genome code for proteins. Generally, ncRNAs do not code for proteins, but there is some exceptions coding for small bioactive peptides [7, 8]. The two major groups of ncRNAs may be separated from each other by having less or more 200 nucleotides (nts): small noncoding RNAs (sncRNAs) are shorter than 200 nts, and long noncoding RNAs (lncRNAs) are longer than 200 nts.

Noncoding RNAs may be grouped into some categories; tRNA (transfer RNA), rRNA (ribosomal RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), Telomerase RNA, tRF (transfer RNA-derived RNA fragments), and tiRNA (tRNA-derived stress-induced RNAs) are all "housekeeping" RNAs. "Regulatory" RNAs are miRNA (microRNA), siRNA (small interfering RNA), piRNA (P-elementinduced wimpy testis (PIWI) interacting RNA), eRNA (enhancer RNA), lncRNA (long noncoding RNA), circRNA (circular RNA), YRNA, crasiRNA (centromere repeat associated short interacting RNA), and TelsRNA (telomere-specific small RNA). Based on structure, LincRNA (long intergenic noncoding RNA), TUCRNA (transcribed ultraconserved RNA), eRNA, and NAT (natural antisense transcript) are "linear" RNAs while ciRNA, ecircRNA (exonic circular RNA), and elciRNA (exon–intron circRNA) are "circular" RNAs. Based on the location, RNAs may be grouped as sense, antisense, intronic, intergenic, and bidirectional RNAs. Some of the ncRNA families are briefly reviewed below. Rfam is a public online database, which provides information for all RNA families, each represented by multiple sequence alignments, consensus secondary structures, and covariance models (https://rfam.xfam.org/) [9].

#### **2. miRNAs (microRNAs)**

miRNAs are ~20 nts long molecules that generally regulate gene expression by inducing mRNA degradation or translational repression. miRNAs originated from pri-miRNA and mature miRNAs regulate protein coding genes. Understanding the regulation of gene expression is changed after the discovery of first miRNA called lin-4 on *Caenorhabditis elegans* in 1993 [5]. Furthermore, miRNAs are potential biomarkers for several human diseases since they are responsible for many biological processes in the cell such as cell differentiation, cell proliferation, cell death, and development by inducing mRNA degradation or translational repression. Especially, oncomirs such as mir-21 are generally upregulated in cancer patients, and miRNAs attract researchers because of their potential as diagnostic and prognostic biomarkers. miRNA gene sequences are publicly deposited on miRBase catalogs (https://www.mirbase.org/) [10].

#### **3. siRNAs (small interfering RNAs)**

siRNAs are about 20–22 nts in size and evolved from double-stranded RNAs. They perform silent transcription of genes via inducing mRNA degradation. siRNAs are very attractive for therapeutic applications in cancer and other diseases [11]. Especially, siRNAs target genes within the tumor cells to inhibit expansions of tumors by RNAi (RNA interference) technology [12]. Moreover, RNAi is a powerful tool in agriculture for crop improvement, including development against biotic or abiotic stress, seedless fruit development, improvement of nutritional quality, and induction of male sterility [13]. Furthermore, a database of siRNA sequences called siRNAdb located on http://sirna.cgb.ki.se/ provides information for siRNAs [14].

#### **4. piRNAs (P-element-induced wimpy testis (PIWI) interacting RNAs)**

piRNAs are small noncoding RNAs, which originated from long single chain precursor transcripts. These RNAs are about 21–35 nts long and interact with PIWI proteins to form the piRNA silencing complex (piRISC). piRNAs play key roles in transposon repression, DNA methylation, silencing transposable elements, regulating gene expression, and fighting with viral infections. Since their abnormal expression is reported for many cancer types, they have potential to be diagnostic tools, prognostic markers, and therapeutic targets for cancer [15]. There are two important databases for piRNAs on the web, piRNAdb and piRNABank. piRNAdb is a piwi-interacting RNA sequences storage and search system, providing some other relevant information such as alignments, clusters, datasets, and targets of these piRNAs on its URL (https://www.pirnadb.org/index). piRNABank provides comprehensive information on piRNAs in the three widely studied mammals, namely Human, Mouse, Rat and one fruit fly, Drosophila on the http://pirnabank.ibab.ac.in/ web address [16].

#### **5. lncRNAs (long noncoding RNAs)**

lncRNAs with longer than 200 nts are not translated into functional proteins and evolved from multiple ways and mainly transcribed by RNA polymerase II (and also by other RNA polymerases). lncRNAs regulate gene expressions in multiple ways including chromatin structure modulation, regulation of function and the transcription of neighboring and distant genes, and alteration of RNA splicing, stability and translation by interacting with DNA, RNA, and proteins. Moreover, lncRNAs can play roles in the formation and regulation of organelles [17]. Furthermore, they take roles in chromatin remodeling, cancer cell invasion and metastasis, and cell differentiation

*Introductory Chapter: Noncoding RNAs–A Brief Introduction DOI: http://dx.doi.org/10.5772/intechopen.108165*

by acting as cis- or trans-regulators in biological processes [18]. lncRNAs are linked to some human diseases including hepatocellular carcinoma, Alzheimer's disease, and diabetes [19]. Owing to their key roles in diseases, lncRNAs have a potential to be therapeutic targets. Recently, antisense oligonucleotides (ASOs) technology is used to perform therapeutic lncRNA targeting [20, 21]. However, since lncRNAs lack of functional open reading frames, targeting them with CRISPR–Cas system is more difficult compared with targeting protein-coding genes [22]. There are too many lncRNA databases on the internet, but most comprehensive lncRNA database is RNAcentral located at https://rnacentral.org/ on the World Wide Web [23]. RNAcentral offers integrated access to a comprehensive and up-to-date set of ncRNA sequences.

#### **6. Future perspectives**

Mounting data, evidence, and recent discoveries of novel short and long regulatory noncoding RNAs have changed the understanding of genome and transcriptome in the cell. Advanced bioinformatics, public databases, and sequencing technologies led to fast understanding of biogenesis and function of ncRNA types and families. ncRNAs are potential biomarkers to perform therapeutic applications for diseases and to improve crops for agriculture. Further illumination of function, biogenesis, and interactions of ncRNAs will empower our understanding of natural dogma with the aid of developing bioinformatics, sequencing, and biochemical techniques.

### **Author details**

Lütfi Tutar Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, Kırşehir Ahi Evran University, Kırşehir, Turkey

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Crick FHC. The origin of the genetic code. Journal of Molecular Biology. 1968;**38**(3):367-379

[2] Crick FHC, Barnett L, Brenner S, Watts-Tobin RJ. General nature of the genetic code for proteins. Nature. 1961;**192**(4809):1227-1232

[3] Nirenberg M, Leder P. RNA codewords and protein synthesis. Science (80-.). 1964;**145**(3639):1399-1407

[4] Lerner MR, Boyle JA, Mount SM, Wolin SL, Steitz JA. Are snRNPs involved in splicing? Nature. 1980;**283**(5743):220-224

[5] Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993;**75**(5):843-854

[6] Tutar L, Tutar E, Tutar Y. MicroRNAs and cancer; an overview. Current Pharmaceutical Biotechnology. 2014;**15**(5):430-437

[7] Anderson DM et al. A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell. 2015;**160**(4):595-606

[8] Kondo T et al. Small peptides switch the transcriptional activity of Shavenbaby during Drosophila embryogenesis. Science. 2010;**329**(5989):336-339

[9] Kalvari I et al. Non-coding RNA analysis using the Rfam database. Current Protocols in Bioinformatics. 2018;**62**(1):e51

[10] Kozomara A, Birgaoanu M, Griffiths-Jones S. miRBase:

From microRNA sequences to function. Nucleic Acids Research. 2019;**47**(D1):D155-D162

[11] Nam N-H, Parang K. Current targets for anticancer drug discovery. Current Drug Targets. 2003;**4**(2):159-179

[12] Senapati D et al. Promising approaches of small interfering RNAs (siRNAs) mediated cancer gene therapy. Gene. 2019;**719**:144071

[13] Rajam MV. RNA silencing technology: A boon for crop improvement. Journal of Biosciences. 2020;**45**(1):118

[14] Chalk AM. siRNAdb: A database of siRNA sequences. Nucleic Acids Research. 2004;**33**(Database issue):D131-D134

[15] Guo B, Li D, Du L, Zhu X. piRNAs: Biogenesis and their potential roles in cancer. Cancer Metastasis Reviews. 2020;**39**(2):567-575

[16] Sai Lakshmi S, Agrawal S. piRNABank: A web resource on classified and clustered Piwi-interacting RNAs. Nucleic Acids Research. 2008;**36**(suppl\_1):D173-D177

[17] Statello L, Guo C-J, Chen L-L, Huarte M. Gene regulation by long noncoding RNAs and its biological functions. Nature Reviews. Molecular Cell Biology. 2021;**22**(2):96-118

[18] Dhanoa JK, Sethi RS, Verma R, Arora JS, Mukhopadhyay CS. Long non-coding RNA: Its evolutionary relics and biological implications in mammals: A review. Journal of Animal Science and Technology. 2018;**60**:25

*Introductory Chapter: Noncoding RNAs–A Brief Introduction DOI: http://dx.doi.org/10.5772/intechopen.108165*

[19] DiStefano JK. "The emerging role of long noncoding RNAs in human disease". 2018:91-110

[20] Lee J-S, Mendell JT. Antisensemediated transcript knockdown triggers premature transcription termination. Molecular Cell. 2020;**77**(5):1044-1054.e3

[21] Lai F, Damle SS, Ling KK, Rigo F. Directed RNase H cleavage of nascent transcripts causes transcription termination. Molecular Cell. 2020;**77**(5):1032-1043.e4

[22] Esposito R, Bosch N, Lanzós A, Polidori T, Pulido-Quetglas C, Johnson R. Hacking the cancer genome: Profiling therapeutically actionable long non-coding RNAs using CRISPR-Cas9 screening. Cancer Cell. 2019;**35**(4):545-557

[23] Petrov AI et al. RNAcentral: A comprehensive database of non-coding RNA sequences. Nucleic Acids Research. 2017;**45**(D1):D128-D134

#### **Chapter 2**

## Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological and Pathological Processes

*Ishteyaq Majeed Shah, Mashooq Ahmad Dar, Kaiser Ahmad Bhat, Tashook Ahmad Dar, Fayaz Ahmad and Syed Mudasir Ahmad*

#### **Abstract**

RNA or ribonucleic acid constitutes of nucleotides, which are ribose sugars coupled to nitrogenous bases and phosphate groups. Nitrogenous bases include adenine, guanine, cytosine and uracil. Messenger RNA, ribosomal RNA and Transfer RNA are three main types of RNA that are involved in protein synthesis. Apart from its primary role in synthesis of protein, RNA comes in variety of forms like snRNA, miRNA, siRNA, antisense RNA, LncRNA etc., that are involved in DNA replication, post-transcriptional modification, and gene regulation etc. LncRNAs regulate gene expression by various ways including at, transcriptional, post-transcriptional, translational, post-translational and epigenetic levels by interacting principally with mRNA, DNA, protein, and miRNA. Among other biological functions, they are involved in chromatin remodelling, transcriptional interference, transcriptional activation, mRNA translation and RNA processing. In this chapter we shall be discussing the origin of lncRNAs, their biogenesis, their mechanism of action and their role in many biological and pathological processes like epigenetics, genome imprinting, several cancers and autoimmune diseases.

**Keywords:** long non-coding RNAs, epigenetics, chromatin remodelling, gene expression regulation, cancer, autoimmune diseases

#### **1. Introduction**

The genome of higher organisms contains less than 3% of protein coding genes while rest of genome is known as junk/or non-coding. With recent advances in science and technology we are learning more about the complexity of organisms, which has led to the discovery of the remarkable complexity of RNA. Large-scale projects such as ENCODE and FANTOM, for the systematic annotation and functional depiction of genes have pointed that 80% of genomic DNA of mammals

are effectively transcribed and intricately controlled with majority belonging to noncoding RNA genes [1]. The figure of ncRNA genes varies by species. Frequency of ncRNA genes but not protein-coding genes, is strongly linked with the complexity of an organism emphasising the growing relevance of ncRNAs [2]. On the basis of molecular size ncRNAs can be classified into two groups. One group includes short RNAs that are less than 200 nucleotides in length such as microRNAs (20–25 nucleotides Length), piwi-interacting RNAs etc. The other group includes Long-non coding RNAs of around 200 nucleotides or more in length. Among the ncRNAs, long non coding RNAs represent a greater portion. Regulatory non coding RNAs with length ≥ 200 nucleotides belong to long non coding RNAs (lncRNAs). Due to their low expression levels, lncRNAs in the beginning were considered to be transcription noise. According to HUMAN GENCODE statistics, there are around 16,000 lncRNA genes, although some estimates put the number at over 100,000 [3, 4]. RNA polymerase II is primarily responsible for lncRNA transcription, but other RNA polymerases are also involved. The resultant lncRNAs are frequently capped at their 5′ ends with 7-methyl guanosine (m7G), polyadenylated at their 3′ ends, and spliced similarly to mRNAs. lncRNAs, in contrast, have a median turnover of 3.5 h, while it is 5.1 h for mRNAs. After transcription, the widely held lncRNAs stay in the nucleus [5]. LncRNAs regulate gene expression at different domains including the ones at epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels by interacting principally with DNA, mRNA, protein, and miRNA [5]. LncRNAs regulate gene transcription by promoting or inhibiting the formation of transcription loops and recruiting or blocking regulators. LncRNAs also operate as precursors to other ncRNAs, such as microR-NAs (miRNAs), and influence mRNA splicing [6]. They are involved in RNA processing, transcriptional interference, chromatin remodelling, transcriptional activation, and mRNA translation, among other biological processes. Besides these roles they also act as oncogenes or tumour suppressors by regulating different signalling cascades [7]. Based on their functions lncRNAs are classified into three types: non-functional lncRNAs, these are products of transcriptional noise; lncRNAs for which the act of transcription is adequate for their activity but the transcript itself is not required and functional lncRNAs that act in both cis and trans ways [8]. In recent years, lncRNAs which account for the immense majority of non-coding RNAs (ncRNAs), have become a hot topic in disease diagnostics and target therapeutics in recent years.

#### **2. Biogenesis of lncRNAs**

Understanding the biosynthesis of these lncRNAs is not only crucial but also ineluctable in order to decipher their functional value, relevance, and differentiation from other forms of RNAs. LncRNA biogenesis is both cell type and stage specific, regulated by stage and cell specific stimuli [9]. Enhancers, promoters, and intergenic regions are among the DNA components in eukaryotic genomes that transcribe diverse types of lncRNAs [10]. LncRNA biogenesis involve ribonuclease P for cleavage to create mature ends, the production of protein (snoRNP) complex caps at their ends, small nucleolar RNA, and the development of circular structures [11, 12]. During specific lncRNAs biogenesis, "paraspeckles" (sub-nuclear structures) have been discovered [13]. The identity of 4 paraspeckle proteins (PSPs) required for paraspeckle formation was made possible by RNAi analyses of 40

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

paraspeckle proteins [14]. Overall, the biology dealing with regulation, synthesis of various lncRNAs is still unknown.

#### **3. Origin of LncRNAs**

Compared to protein-coding genes, very less information regarding origins and evolution of lncRNAs is available. They reveal that among mammals, sequence conservation is poor and evolution is fast [15]. For the origin of lncRNAs, various evolutionary assumptions have been considered. The first idea is that the protein-coding gene undergoes transformation as a result of a gene-duplication process [16, 17]. Throughout evolution, one copy of a protein-coding gene acquires mutations and loses its capacity to code for proteins. Then, among other coding fractions, a new functional lncRNA gene is generated with polyadenylation sequences, splicing signals, regulatory elements and exon sequences [16, 18].

X inactive-specific transcript (XIST), meant for dosage compensation in mammals is assumed to have arisen from the chicken protein-coding Lnx3 gene [16, 17, 19], was classified as a pseudo gene [18]. The 5-UTR of Lnx3 exons 1 and 2 were used to create the XIST promoter region. Lnx3 exons 4 and 11 were also used to create exons 4 and 5 of the XIST gene of humans [20]. Other lncRNA genes, such as short and long noncoding RNAs, can duplicate segments or whole genes to generate lncRNAs. According to genomic research large homologous protein-coding gene families, protein coding gene duplication is ubiquitous; yet apart from protein coding genes, there is minimal proof for the entire duplication of lncRNAs. This might be owing to the fast sequence divergence of lncRNAs. The duplicated lncRNA mouse nuclear-enriched abundant transcript 2 is paralogous to non exonic sequences in the mouse genome [18]. The generation of lncRNAs appears to be aided by segmental gene duplication within antisense non-coding RNAs (ancRNAs).

De novo creation is another option for lncRNA origins. Examples include genome changes viz. chromosomal rearrangement, creation of (proto-) splice sites and (proto-) promoters transformed non-functional genomic stretches into functional lncRNAs [16]. Genesis of lncRNAs via insertion of transposable elements is the most recent and final hypothesis [17, 18, 21]. The bulk of human lncRNAs have TE segments as related to other genes such as pseudo genes, tiny lncRNAs, and proteincoding genes. Internal exons, transcription start sites, polyadenylation (polyA) sites, or a mix of these components can all include TEs. According to this research at least 75% of human lncRNAs contain at least one exon containing partial TE origin.

#### **4. Mechanism of action of LncRNAs**

#### **4.1 LncRNAs as chromatin regulators**

The figure of lncRNAs with known roles is gradually increasing, and the majority of the studies focus on their controlling potential. Histone modifications, DNA methylation, and chromatin remodelling are all functional steps where lncRNAs regulate chromatin structure.

LncRNAs are frequently used as important regulators (modulators) of protein coding gene expression by acting in cis and trans ways [22]. Binding of lncRNAs to histone modification complexes like PRC1 and PRC2 [in-specific] causes methylation of lysine 27 on histone 3, a histone signature connected to suppressed transcriptional state. Xist, a lncRNA abundantly generated from inactive X chromosomes in females (Xi), enhances PRC2 recruitment to the Xi to mute gene expression, according to studies on mammalian X chromosome inactivation [23, 24]. Other example includes HOTAIR lncRNA, synthesised from the HoxC gene cluster but targets histone H3K4me1/2 demethylase LSD1 and PRC2 complex to induce transcriptional gene suppression at the HoxD locus in trans [25]. Some well-studied lncRNAs, including ANRIL and KCNQ1OT1, bring epigenetic modifiers to particular loci, allowing for chromatin remodelling. For example, KCNQ1OT1, binds to PRC2 as well as the methyl-transferase G9A, whereas ANRIL binds to both polycomb repressive complexes [26]. Many different lncRNAs act as scaffolds and work by leading restrictive histone modifying complexes to particular loci [26]. By forming complexes with the trithorax group (TrxG) and the polycomb repressive complex 2, lncRNA steroid receptor RNA activator also plays a role in transcriptional control [27].

Examples of lncRNA that interact with PRC1 complex include FAL1 RNA (focally amplified lncRNA on chromosome 1). BMI1, a PRC1 subunit, interacts with FAL1. FAL1 controls not only the stability of BMI1, but also its connection with target promoter regions, altering target gene expression [28].

#### **4.2 Transcriptional regulation**

At gene promoters transcription is thought to be regulated by the interaction of transcription factors and chromatin modifying factors. LncRNAs regulate gene expression by interacting with other molecules such as Proteins, RNA and DNA near their target genes promoters or enhancers. LncRNAs have a number of ways for controlling transcription.

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

various proteins. HOTAIR aids in the demethylation of H3K4 by interacting with LSD1 (lysine-specific histone demethylase 1A), REST (restriction element 1-silencing transcription) factor & REST corepressor1 at the 3′ end. HOTAIR (At the 5′ end) is a transcriptional gene silencing factor derived from the HOXC locus that leads to transcriptional gene silencing in trans across 40 kb of the HOXD locus by inducing a repressive chromatin state, recruiting PRC2, & reinforcing H3K27 methylation [33].

Long non-coding RNAs also function as cofactors, influencing the activity of transcription factors. For example, the ncRNA Evf2 is produced from a conserved distal enhancer and attracts DlX2 (TF) to the same enhancer, causing neighbouring protein-coding genes to be expressed.

#### **4.3 Regulation after transcription**

Because ncRNAs can recognise complementary sequences, they can affect how mRNAs are processed after transcription, including capping, splicing, editing, transport, translation, destruction, and stability. LncRNAs compete with microRNAs to impact mRNA levels by changing mRNA stability, degradation, and translation [34].

damage-induced noncoding RNA (lncRNA) that is p53-dependent, affects p53 stability [40]. DINO participates in p53-mediated phenotypes such as Apoptosis and cell cycle arrest with respect to DNA damage. Eminently, DINO binds to and stabilises p53, allowing induced p53 to favourably regulate downstream targets like DINO. Because of its capacity to stabilise the p53 protein, DINO is an important component of the DNA damage-p53 regulation network. lncRNA GUARDIN is another paragon, which is likewise a p53-responsive lncRNA [41].

#### **4.4 LncRNA-mediated regulation of protein activity**

Several studies have discovered that lncRNAs can influence protein stability through ubiquitination or phosphorylation. DINO (lncRNA) has a function in phenotypes regulated by p53, such lncRNAs also regulate protein activity in mechanisms other than transcription. The *Caenorhabditis elegans* lncRNA rncs-1 found by Hellwig and Bass (2008) and functions in the processing of short RNAs by binding to and blocking the Dicer enzyme [42]. Marchese FP et al. established the involvement of lncRNA CONCR in sister chromatid cohesion control [43]. CONCR is a cell cycle-regulated lncRNA that is essential for DNA replication and cell cycle advancement and is activated by MYC. CONCR interacts with DDX11 (DNA-dependent transcription factor) and regulates its activity. ATPase and helicase have an impact on DNA replication and sister chromatid cohesion. Liu et al. showed how lncRNA regulates kinase signalling in the setting of metabolic stress response. The LKB1-AMPK pathway promotes the expression of lncRNA neighbour of BRCA1 gene 2 (NBR2), which has also been found to interact with AMPK in the context of energy stress [44]. The association

#### **Figure 1.**

*Schematic representation of few mechanisms employed by lncRNAs in regulation gene expression. (A) Regulation by chromatin remodelling (B) Regulation through signalling (C) lncRNA sponging mRNA (D) By acting as scaffolds.*

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

of lncRNA NBR2 with AMPK enhances its kinase activity under energy stress. AMPK activation is decreased in NBR2 loss, resulting in aberrant cell cycle, altered apoptosis/autophagy responses, and increase in tumour formation. The hypoxiaregulated lncRNA linc-p21 has been found to bind both HIF-1 and VHL, altering the VHL-HIF-1 interaction and increasing HIF-1 accumulation [45]. The importance of lincRNA-p21 in the control of the Warburg effect in tumour cells is highlighted by this positive feedback loop between HIF-1 and lincRNA-p21, which accelerates glycolysis under hypoxia. Certain lncRNAs control transcription factor activity in addition to impacting gene expression via mRNA interactions. As an alternatively spliced form of Evf-1 RNA, the lncRNA Evf2 is generated from the Dlx-5/6 ultra-conserved region. The lncRNA Evf2 forms a stable complex with the transcription factor Dlx2, boosting Dlx-5/6 enhancer transcriptional activation mediated by Dlx2 [46].

Despite the fact that lncRNAs are distinct from mRNAs exported to cytoplasm for protein synthesis [47, 48], few lncRNAs are confined in sub-nuclear compartments, suggesting possible activities in these compartments. The schematic figure that represents few mechanisms employed by lncRNAs in gene expression regulation (**Figure 1**).

#### **5. Roles and functions of lncRNAs in different biological processes**

An emerging view of lncRNAs is that they are key players in many biological processes. These lncRNAs have been discovered to be master regulators in different biological and pathological processes and their dysregulations leads to many life-threatening diseases including different cancers. Some of the pivotal roles and functions carried out by different lncRNAs are well documented.

#### **5.1 Long non-coding RNAs in genomic imprinting: regulation of allelic expression**

In layman's terms genomic imprinting represents a condition in which one of the alleles in an inherited paternal pair is active while the other one remains inactive. It is the parent of origin which will determines the differential expression of inherited parental alleles; in some cases, a genes allele is paternally imprinted, while in others, it is maternally imprinted.

Dna Methylation and Genomic imprinting: The process of adding a methyl (CH3) group to a cytosine known as DNA methylation is commonly located at CpG dinucleotides in mammals and is believed to regulate gene expression. CpG sites are very rare in the genome with the exceptions of CpG islands (which have high CpG amount/ density). Generally found near or around promoter region these islands are usually unmethylated. Outside of CpG islands, CpG sites are often methylated, resulting in a bimodal methylation pattern across the genome. A family of DNA methyltransferases catalyses the acquisition of DNA methylation. DNMT1 is a DNA methyltransferase homologue that has an affinity for hemi-methylated DNA and is responsible for sustaining methylation following DNA replication. DNMT3A and DNMT3B catalyse de novo DNA methylation, while DNMT3L is a cofactor with no methyltransferase activity [49]. DNA methylation leads to epigenetic mechanisms and epigenetics leads to allow the transcriptional machinery of the cell to distinguish the two parental chromosomes at imprinted loci.

Imprinting is typically accomplished by altering the histone and/or DNA of a given locus, however lncRNAs have recently been identified to have a role in these phenomena [50]. Imprinted genes must be regulated by cis-regulatory mechanisms since expressed and repressed alleles share the same nucleus. The breakthrough in genomic imprinting was established with the discovery of gametic differentially methylated regions (these are actually control elements) that contain the "imprinting mark" acquired in oocytes and sperm. These markers are then transmitted down through the generations, guiding parental-specific allelic expression in children and embryos.

Control of imprinted lncRNAs is achieved by shared regulatory mechanisms such as parent-of-origin-dependent differentially methylated regions (DMRs) & lncRNAs within a single imprinted cluster [51]. Allele-specific DNA methylation occurs in a separate ICR in the germline in well-studied imprinted clusters, termed as principal DMRs or germline-derived DMRs and persists beyond fertilisation. Epigenetic changes (parent-of-origin-specific) such as DNA methylation, influence parentally inherited allele expression patterns in ICRs in imprinted clusters [52]. There are approximately 35 imprinted gDMRs in the human & 24 in Mouse genome (Monk et al., 2018). For embryonic development to govern imprinted gene expression, gDMRs must be established on paternal or maternal alleles [53]. In early primordial germ cells epigenetic markers including DNA methylation and histone modifications are substantially erased genome-wide. Depending on the parent-of-origin, DNA methylation of ICRs is reinstated in germline cells in gametes. After fertilisation, gDMRs are impervious to subsequent global epigenetic reprogramming. The data on DNA methylation at the ICRs of imprinted areas is retained. Imprinted loci gDMRs establish themselves strongly throughout germline development and are hence resistant to genomic reprogramming after fertilisation. Imprinting markings, on the other hand, are passed down from one generation to the next [54]. The allele-specific methylation statuses of gDMRs are generally identified by transcription regulators in maintaining parent-of-origin specific regulation of imprinted genes for example ZFP57 protein. The development of monoallelic gene expression requires differential methylation statuses of gDMRs on parental alleles. In early embryonic and adult lineages, imprinting control regions regulate DNA methylation and chromatin organisation, resulting in the survival of imprinting patterns through generations and their perpetuation in adult tissues [55].

To explain the control of gene regulation within an imprinted cluster, two fundamental methods have been proposed [56]. The lncRNA model is the earliest and arguably most frequent model. According to this concept, imprinted lncRNAs control imprinted gene expression. In this model, imprinted lncRNAs are closely linked to ICRs. Imprinted lncRNAs are defined by their ability to suppress imprinted genes in the same cluster [57]. As shown by the imprinted cluster Kcnq1/Kcnq1ot1. On paternal allele, the constantly expressed imprinted lncRNA Kcnq1ot1 can repress multiple imprinted genes bidirectionally throughout their gene area. The insulator paradigm, wherein parental allele-specific epigenetic changes at ICRs contribute to topological variations of imprinted gene areas, resulting in gene silence or activation of certain alleles, has been observed in additional imprinted regions. The insulin-like growth factor 2/H19 locus controls imprinted genes mechanistically, according to this concept. The following are some well-known lncRNAs and their roles in genomic imprinting:

I.**Airn lncRNA:** 108-kb nuclear-localised transcript that is synthesised in the opposite direction from 3.7-kb ICE of Igf type-2 receptor gene [58]. This lncRNA have been shown to influence the regulation of three parent of origin-specific genes viz. Igf2r, Slc22a2 & Slc22a3. Mice with an ICE deletion show biallelic expression of 3 genes (including Igf2r) and are smaller at birth [59].

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

#### **5.2 X-chromosome inactivation**

Sex in most animals including humans is generally determined by X and Y chromosome system with men carrying XY and females XX chromosomes. In order to have the balance of products of sex-linked and autosomal genes, dosage compensation is required. X-chromosome inactivation (XCI) is a special kind of dosage compensation present in mammalian females involving random selection and transcriptional repression in one among the two X -chromosomes during the early stages of embryonic development [67, 68]. However, humans lack imprinted XCI and instead have XCD (X chromosome dampening) [69–71]. Most of the research with respect to this has been carried on mouse model and regulated by interactions of many lncRNAs [72]. At the heart of XCI/XCR (X-chromosome reactivation) regulation is a region of X chromosome namely Xic X-inactivation centre (containing many noncoding RNA genes whose expression is regulated by pluripotency factors) [73]. Developmental phase of mice includes two forms of XCI; one being imprinted and another random. Imprinted XCI, in which the paternally inherited X (Xp) is always inactivated, occurs during preimplantation development in the early embryo, when Xist becomes expressed on the Xp from the 2-cell stage onwards and is maintained in the placenta's extraembryonic tissues [74].

Xist lncRNA regulates all three stages (initiation, establishment, and maintenance) of XCI [75]. The X-linked minimum genetic region (XIC) contains many elements and genes like Tsix & Xist which are meant for XCI initiation [76]. Among the lncRNA loci reported in a 100–500 kb region of the mouse X chromosome and a 2.3 Mb syntenic

region of the human X chromosome are RepA Jpx, Xist, Ftx, Tsix, & Xite [75, 76]. XIC contains the lncRNA Xist, and is located 15 kb downstream of Tsix antisense [77].

In first phase, complex factors Xite, OCT4, Tsix & CTCF among others bind Xi and Xa independently to promote X chromosome pairing and counting in the embryo following fertilisation [78]. After counting and pairing, Tsix, Xist, and other genes are elevated, which is regulated by a network of genetic interactions including OCT4, Jpx, Rnf12, and RepA Tsix, Sox2 & PRDM14 [79]. They use alternative transcription fates when full initiation of XCI occurs, with one becoming the Xa and the other Xi chromosome [72]. RNF12, Tsix, and RepA, as well as pluripotency factors (NANOG, OCT4, SOX2) impact lncRNA Xist activation and expression in Xi. Xist binds to Polycomb repressive complex 2 via Repeat A, producing the Xist-PRC2 complex, while YY1 tethers the PRC2-Xist complex to the Xi nucleation centre via Repeat C, where RNA polymerase II gets the lncRNA Xist-PRC2 complex [80]. Upon completion of initiation phase lncRNA Xist recruits different protein complex factors (including heterogeneous nuclear protein U, meant for lncRNA Xist localization, heterogeneous nuclear ribonucleoprotein K for Xist-mediated chromatin modifications) and gene-silencing factor Spen which binds to C, B, F, and A repeats at the 5′ end of the lncRNA Xist and causes the lncRNA Xist topologically associated domains meant for (epigenetic modification and chromatin compaction) to spread along the Xi chromosome at the established phase [76, 81]. ATRX directs PRC1 and PRC2 that cause epigenetic silencing by acetylation of histone H3 and H4 and methylation of CpG islands [56]. The protein complexes SHARP, HDAC3, LBR, Airn and Kcnq1ot1, U1 snRNP, Rsx, and CdK8 are all implicated in the lncRNA Xist spreading process. LncRNA Xist attracts restrictive complexes (like H3K27me3, H2AK119Ub, & the CpG island), which cause immediate histone modifications and DNA methylation, and coats the Xi to generate Xi [82]. Continuous synthesis of lncRNA Xist RNA in an inactive state has been used to establish and maintain the Xi.

#### **6. Role of LncRNAs in cancer**

The deadliest disease on the planet is linked to abnormal gene expression. Non-coding areas of the genome have been related to the majority of malignancies. Cancer genomic mutations are found in areas that do not code for proteins [83]. These areas, however, are frequently translated into long non-coding RNAs (lncRNAs). Anomalies in these lncRNAs is thought to have the tumour suppressor or carcinogenic effects and thus play a vital role in tumour establishment. Since lncRNAs have expression that are unique to a tissue, expressed in regulated manner and in association with other gene sets impact cell cycle regulations, immunological responses, survival etc. all of which affect cancer cell transformation [84]. The role of lncRNA is associated with their specific subcellular location. LncRNAs regulate gene expression by different interactions both in the nucleus as well as in the cytoplasm. In nucleus regulate expression at both epigenetic and transcriptional levels including histone modifications [24, 85], DNA methylation regulation [86], chromatin remodelling [87, 88], chromatin modification complexes [89, 90], transcription regulators [91] and proteins present in nucleus [92] while in the cytoplasm these lncRNAs regulate gene expression at both transcriptional and translational levels including interplay with proteins present in the cytoplasm [93], Control of mRNA metabolism [94], as endogenous competitive RNA (ceRNA) interacts with microRNA [95]. As a result, lncRNA play a significant role in cancer cell proliferation, transition, invasion, and treatment resistance [96, 97].

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

LncRNAs like LUCAT1, KCNQ1OT, HOTAIR, ANRIL, MALAT1(metastasisassociated lung adenocarcinoma transcript 1), Taurine-upregulated gene 1, LINC00152, RP11-385 J1.2 and TUBA4B bring different epigenetic modifiers at their respective loci and modify the chromatin shape and their dysregulation is strongly associated with establishment of different tumours including LUCAT1: digestive system tumours, ANRIL: prostate cancer, MALAT1: breast, liver, and colon cancer [98], HOTAIR: breast cancer KCNQ1OT1: colorectal cancer etc.

Some of known lncRNAs with links to cancer is represented below in a table:

One of the first lncRNAs to be discovered as having a functional role in cancer formation was the HOTAIR. HOTAIR lncRNAs enhance cancer spread by triggering epigenetic alterations in the chromatin status of tumour cells [104]. Interestingly, various lncRNAs have been reported to originate from the HOX locus, implying that it plays a global regulatory role [21]. In tumours, lncRNAs are also implicated in the control of the epithelial-mesenchymal transition (EMT) [99]. TGF-activated lncRNA (lncRNA-ATB) stimulated the EMT cascade by upregulating the levels of zinc finger

E-box-binding homeobox (ZEB1 and ZEB2) [105]. Recent research has discovered that the transcription factor PNUTS has a corresponding lncRNA-PNUTS, which plays a role in breast cancer metastasis by influencing the EMT process [106]. lncRNA human ortholog RNA of Dreh (hDREH), inhibitor of EMT is seen downregulated in many types of cancers. Some tumour suppressor lncRNAs, on the other hand, are expressed at low levels in tumours [107]. It is now possible to increase the expression of these lncRNAs in order to treat cancer. lncRNAs have been implicated in stemness-related signalling pathways in a number of studies. The tumour suppressor long noncoding RNA (TSLNC8), for example, inhibit the STAT3 signalling pathway and thus acted as a tumour suppressor [108]. lncRNA MST1/2-antagonising for YAP activation (MAYA) participate in the Hippo-YAP signalling pathway. These lncRNAs might be directly regulating cell stemness [109]. Thus, these lncRNAs act as potential targets for cancer treatment.

#### **7. Role of LncRNAs in immunology**

The study of lncRNA biology is still in its early stages, but it has already revealed key roles in the formation and functions of immune cells. However, because the functions of most lncRNAs are unknown, the field must fill a significant gap in order to comprehend the breadth of their roles in immunity. The function of non-long coding RNAs in the immunology of humans even though in its infancy stage has become hot topic in recent research. In immune system, lncRNAs have a role in immune cell formation, survival, cell fate determination, differentiation, amplification, and activation. Although the functions of most lncRNAs are unknown, novel protein-protein interactions or partnering with RNA or DNA have been discovered to influence innate and adaptive immune responses transcriptionally or post-transcriptionally. According to the recent research lncRNAs have been found in many immune cells including T cells and B cells. Regulation of these lncRNAs including expression levels is considered to be linked to immune cell development, differentiation, and activation [110].

To carry out their functions these IncRNAs can associate with transcription regulators and signalling molecules (NF-B, STAT3) [111, 112], RNA binding proteins including (hnRNP, HuR) [113, 114], and chromatin remodelling components (PRC2, WDR5).

#### **7.1 Long non-coding RNA in T cells**

LncRNAs, such as TMEVPG1 (also known as LincR-Ifng-3′AS), have been identified in both mouse and human CD8+ T cells and demonstrated to be positioned inside a cluster of cytokines genes, and have been shown to control Theiler's virus load in CNS infection [115]. T-bet/Stat, a transcription factor exclusive to Th1 cells that collaborates with TMEVPG1, promotes interferon gamma expression [116]. Interaction of lncRNA called NeST with WDR5, significant component of MLL H3K4 methyltransferases, leads to increase in methylation state of histones at Ifng gene in CD8+/Th1 cells [117]. Hundreds of lncRNAs were found in both CD8+ T cells of animal and human spleens after a genome-wide expression examination utilising a proprietary array, indicating that lncRNAs are important for lymphocyte differentiation and stimulation [118]. To get clearer picture of the role of lncRNAs in T cell growth and differentiation, Hu et al. used RNA-Seq to recognise 1524 genomic regions that produce lincRNAs in 42 subsets of mature peripheral T cells and thymocytes;

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

specific transcription regulators including T-bet, STAT4 for CD8+ descendants, & GATA-3 and STAT6 for CD4+ lineages seemed to be substantially responsible for lineage-specific expression of T cell lincRNAs (LincR-Ccr2-5′AS) [119].

#### **7.2 Long non-coding RNA in B cells**

B lymphocytes after being activated by antigen interaction, the major roles are to make antigen-specific antibodies, operate as APCs, and develop into memory cells. In compared to T cells, less is known regarding lncRNA roles in B cells. Bolland et al. [120] have documented how lncRNAs play their part in the chromatin remodelling that occurs during V/D/J gene recombination, which is required for the production of epitopes (Ig or TCR). Further study discovered that the synthesis of such antisense and sense lncRNAs is related to VH portions looping next to DJH areas during pro-B cell recombination, and as a result, it has been dubbed the Igh locus whole transcriptome of sense and antisense transcripts [121]. SAS-ZFAT gene expression which is restricted to CD19+ B cells of peripheral blood lymphocytes may be important for B cell function and AITD development [122].

#### **7.3 Long non-coding RNA in macrophages**

Macrophages are white blood cells that surround and destroy pathogens, destroy dead cells and stimulate other immune system cells. Macrophages as APCs also contribute to the optimal operation of both intrinsic and adaptive immune responses. LncRNA acknowledgement and purposes in monocytes/macrophages have received little attention. Macrophages have capital baseline level of ptprj/CD148 (a tyrosine phosphatase with tumour inhibitor-like role), as expected; its level is modulated by LPS, TLR, and CSF-1 treatments in various animals. Dave et al. investigated ptprj-as1, a 1006-nt lncRNA species that is produced antisense to ptprj. Transcripted ptprj-as1 is highly regulated in tissues augmented with macrophages that has been temporarily activated by TLR ligands, similar to ptprj [123]. Thus, the ptprj coding transcript may guide to inflammatory alteration that is promptly connected to macrophages. When myeloid and CD11c + dendritic cells are stimulated by lipopolysaccharide, a TLR4 signalling activator via NFkB, they express lncRNAs-COX2 (Ptgs2) [84]. Li et al. investigated how lincRNA expression changed when innate immune signalling was activated in THP1 macrophages and discovered that an unannotated LincRNA called THRIL was a critical factor in TNF- control and that its activity was clearly lower during the acute period of Kawasaki disease [114]. LncRNA has been observed to regulate healthy and pathological inflammatory immune responses through an RNA-protein complex with hnRNPL [124]. Many lncRNAs discovered as unique binding ally for lincRNA-cox2 in the nucleus and cytoplasm of macrophages, are key switch of immune genes [113].

#### **7.4 Long non-coding RNA in natural-killer cells**

Wright et al. uncovered an intron 2 promoter in many KIR genes that generate spliced antisense transcripts. KIR antisense lncRNA is found in progenitor cell lines, and its abundance in NK cells results in the under expression of KIR protein coding genes. KIR's antisense lncRNA corresponds to exons 1 and 2 of the KIR gene, and also the proximal promoter upstream of KIR. Myeloid zinc finger one (MZF-1) appears to impact KIR antisense lncRNA transcription, resulting in KIR silence via an unknown mechanism [125].

#### **7.5 Functions of immune related lncRNAs**

The following table represents the functions of some immune related lncRNAs:

#### **8. LncRNAs in auto-immune diseases**

Auto-immune diseases are conditions in which our immune system wrongly assaults our bodies, assumed to be the result of a complex interplay of genetic, immunological and environmental variables.

#### **8.1 Systemic lupus erythematosus (SLE)**

TLR4 signalling dysregulation has been connected to the progression of SLE. A lncRNA has been associated to SLE and is controlled by TLR4 signalling. Nuclear enriched abundant transcript 1 (NEAT1), a lncRNA, has been connected to the

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

pathogenesis of SLE [136]. In a study NEAT1 levels were found to be elevated in PBMCs of SLE patients in comparison with healthy individual. In LPS-stimulated human monocytic cell lines, silencing NEAT1 with siRNA results in lower production of Interleukin-6, CCL2, and CXCL10, all of which have been related to SLE pathogenesis. NEAT1 regulates the stimulation of late mitogen-activated protein kinase signalling pathways, which play a role in the TLR4-driven inflammatory response. SLE patients have lower Gas5 expression in plasma, and in both CD4+ T cells and B cells than in healthy people [137].

#### **8.2 Rheumatoid arthritis (RA)**

Is an autoimmune illness characterised by inflammation and proliferation of synovial joint resulting in significant joint damage. Several recent investigations have found that dysregulated lncRNAs play a significant part in aetiology of RA. In 2015, Song et al. discovered that PBMCs and serum exosomes of RA patients have higher expression of HOTAIR than in the healthy controls. Furthermore, increasing HOTAIR increases active macrophage migration, whereas decreased HOTAIR suppresses the synthesis of matrix metalloproteinase (MMP)-2 and MMP-13 in developed osteoclasts and rheumatoid synoviocytes. These results suggest that abnormal HOTAIR expression could perform a part in the development of RA [138]. Low levels of lincRNA-p21 have been linked to increased NF-B activity in persons with Arthritis. Spurlock et al. observed that amount of phosphorylated p65, a hallmark of NF-B activation, was greater in the whole blood of patients suffering from RA however the expression of lincRNA-p21 was found to be downregulated. In comparison with MTX-treated RA patients, untreated counter parts had reduced levels of lincRNA-p21 activity and upregulated levels of p65 [139]. Lu et al. observed that T cells of patients suffering from RA have upregulated lncRNAs LOC100652951 and LOC100506036 in comparison with healthy individuals [140].

#### **8.3 Sjogren's Syndrome (SS)**

Chronic systemic auto-immune illness characterised by symptoms of driest mouth and eyes caused by gland inflammation (exocrine). The lncRNA Tmevpg1, which modulates Th1 responses, may have a role in the aetiology of SS, according to one research. The researchers discovered a connection between SS and the lncRNA Tmevpg1 [141].

#### **8.4 Polymyositis and dermatomyositis (PM/DM)**

Muscle inflammatory illness that is persistent. PM/DM has been connected to the expression of the RNA component of signal recognition particles, lncRNA 7SL [142]. Using microarray research, Peng et al. discovered 1198 differently transcribed lncRNAs in diabetes patients muscles compared to normal ones. uc011ihb.2, ENST00000583156.1, ENST00000551761.1, ENST00000541196.1 & linc-DGCR6-1, were among the five lncRNAs confirmed. As per their computational predictions, the linc-DGCR6-1 gene controls USP18, a kind of type 1 interferon-inducible gene found predominantly in the perifascicular portions of muscle fibres of diabetes patients [143].

#### **9. Conclusion**

Although there has been immense research going on to understand the role of lncRNAs, still there are many questions that need to be addressed. Efforts are being carried out to elucidate the role of lncRNAs as potential regulators in different biological and pathological processes. The advancement in technologies will further help to pave way in clarifying the mechanisms underlying of lncRNA influence in different diseases. Studying lncRNA role in regulation of host-pathogen interactions can be helpful in identifying different lncRNAs that can serve as potential drug targets and can also serve as novel biomarkers.

### **Author details**

Ishteyaq Majeed Shah1 , Mashooq Ahmad Dar2,3, Kaiser Ahmad Bhat4 , Tashook Ahmad Dar4 , Fayaz Ahmad1 and Syed Mudasir Ahmad3 \*

1 Department of Zoology, University of Kashmir, Kashmir, India

2 Department of Biochemistry/Clinical Biochemistry, University of Kashmir, Kashmir, India

3 Division of Animal Biotechnology, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology, Kashmir, India

4 School of Biosciences and Biotechnology, Baba Ghulam Shah Badshah University, Rajouri, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

#### **References**

[1] Hon CC, Ramilowski JA, Harshbarger J, Bertin N, Rackham OJ, Gough J, et al. An atlas of human long non-coding RNAs with accurate 5′ ends. Nature. 2017;**543**(7644):199-204

[2] Kapusta A, Feschotte C. Volatile evolution of long noncoding RNA repertoires: Mechanisms and biological implications. Trends in Genetics. 2014;**30**(10):439-452

[3] Uszczynska-Ratajczak B, Lagarde J, Frankish A, Guigó R, Johnson R. Towards a complete map of the human long non-coding RNA transcriptome. Nature Reviews Genetics. 2018;**19**(9):535-548

[4] Fang S, Zhang L, Guo J, Niu Y, Wu Y, Li H, et al. NONCODEV5: A comprehensive annotation database for long non-coding RNAs. Nucleic Acids Research. 2018;**46**(D1):D308-D314

[5] Quinn JJ, Chang HY. Unique features of long non-coding RNA biogenesis and function. Nature Reviews Genetics. 2016;**17**(1):47-62

[6] Jarroux J, Morillon A, Pinskaya M. History, discovery, and classification of lncRNAs. Long Non Coding RNA Biology. 2017;**1008**:1-46

[7] Huarte M. The emerging role of lncRNAs in cancer. Nature Medicine. 2015;**21**(11):1253-1261

[8] Kornienko AE, Guenzl PM, Barlow DP, Pauler FM. Gene regulation by the act of long non-coding RNA transcription. BMC Biology. 2013;**11**(1):1-14

[9] Akerman I, Tu Z, Beucher A, Rolando DM, Sauty-Colace C, Benazra M, et al. Human pancreatic β cell lncRNAs control cell-specific regulatory networks. Cell Metabolism. 2017;**25**(2):400-411

[10] Wu H, Yang L, Chen LL. The diversity of long noncoding RNAs and their generation. Trends in Genetics. 2017;**33**(8):540-552

[11] Vicens Q, Westhof E. Biogenesis of circular RNAs. Cell. 2014;**159**(1):13-14

[12] Chen LL. The biogenesis and emerging roles of circular RNAs. Nature Reviews Molecular Cell Biology. 2016;**17**(4):205-211

[13] Naganuma T, Hirose T. Paraspeckle formation during the biogenesis of long non-coding RNAs.  Nature Structural & Molecular Biology. 2017;**24**(10):783-784

[15] Kutter C, Watt S, Stefflova K, Wilson MD, Goncalves A, Ponting CP, et al. Rapid turnover of long noncoding RNAs and the evolution of gene expression. PLoS Genetics. 2012;**8**(7):e1002841

[16] Kaessmann H. Origins, evolution, and phenotypic impact of new genes. Genome Research. 2010;**20**(10):1313-1326

[17] Kapusta A, Kronenberg Z, Lynch VJ, Zhuo X, Ramsay L, Bourque G, et al. Transposable elements are major contributors to the origin, diversification, and regulation of vertebrate long noncoding RNAs. PLoS Genetics. 2013;**9**(4):e1003470

[18] Ponting CP, Oliver PL, Reik W. Evolution and functions of long

noncoding RNAs. Cell. 2009;**136**(4): 629-641

[19] Gribnau J, Grootegoed JA. Origin and evolution of X chromosome inactivation. Current Opinion in Cell Biology. 2012;**24**(3):397-404

[20] Elisaphenko EA, Kolesnikov NN, Shevchenko AI, Rogozin IB, Nesterova TB, Brockdorff N, et al. A dual origin of the Xist gene from a proteincoding gene and a set of transposable elements. PLoS One. 2008;**3**(6):e2521

[21] Rinn JL, Chang HY. Genome regulation by long noncoding RNAs. Annual Review of Biochemistry. 2012;**81**:145-166

[22] Mao YS, Sunwoo H, Zhang B, Spector DL. Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs. Nature Cell Biology. 2011;**13**(1):95-101

[23] Plath K, Fang J, Mlynarczyk-Evans SK, Cao R, Worringer KA, Wang H, et al. Role of histone H3 lysine 27 methylation in X inactivation. Science. 2003;**300**(5616):131-135

[24] da Rocha ST, Boeva V, Escamilla-Del-Arenal M, Ancelin K, Granier C, Matias NR, et al. Jarid2 is implicated in the initial Xist-induced targeting of PRC2 to the inactive X chromosome. Molecular Cell. 2014;**53**(2):301-316

[25] Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell. 2007;**129**(7):1311-1323

[26] Lee JT. Gracefully ageing at 50, X-chromosome inactivation becomes a paradigm for RNA and chromatin control. Nature Reviews Molecular Cell Biology. 2011;**12**(12):815-826

[27] Wongtrakoongate P, Riddick G, Fucharoen S, Felsenfeld G. Association of the long non-coding RNA steroid receptor RNA activator (SRA) with TrxG and PRC2 complexes. PLoS Genetics. 2015;**11**(10):e1005615

[28] Hu X, Feng Y, Zhang D, Zhao SD, Hu Z, Greshock J, et al. A functional genomic approach identifies FAL1 as an oncogenic long noncoding RNA that associates with BMI1 and represses p21 expression in cancer. Cancer Cell. 2014;**26**(3):344-357

[29] Li W, Notani D, Rosenfeld MG. Enhancers as non-coding RNA transcription units: Recent insights and future perspectives. Nature Reviews Genetics. 2016;**17**(4):207-223

[30] Kim TK, Hemberg M, Gray JM, Costa AM, Bear DM, Wu J, et al. Widespread transcription at neuronal activity-regulated enhancers. Nature. 2010;**465**(7295):182-187

[31] Andersson R, Gebhard C, Miguel-Escalada I, Hoof I, Bornholdt J, Boyd M, et al. An atlas of active enhancers across human cell types and tissues. Nature. 2014;**507**(7493):455-461

[32] Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science. 2010;**329**(5992):689-693

[33] Luco RF, Pan Q, Tominaga K, Blencowe BJ, Pereira-Smith OM, Misteli T. Regulation of alternative splicing by histone modifications. Science. 2010;**327**(5968):996- 1000

[34] Dykes IM, Emanueli C. Transcriptional and post-transcriptional gene regulation by long non-coding RNA. Genomics, Proteomics & Bioinformatics. 2017;**15**(3):177-186

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

[35] Tripathi V, Ellis JD, Shen Z, Song DY, Pan Q, Watt AT, et al. The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Molecular Cell. 2010;**39**(6):925-938

[36] Pandey RR, Mondal T, Mohammad F, Enroth S, Redrup L, Komorowski J, et al. Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatinlevel regulation. Molecular Cell. 2008;**32**(2):232-246

[37] Gonzalez I, Munita R, Agirre E, Dittmer TA, Gysling K, Misteli T, et al. A lncRNA regulates alternative splicing via establishment of a splicing-specific chromatin signature. Nature Structural & Molecular Biology. 2015;**22**(5):370-376

[38] Lan Y, Xiao X, He Z, Luo Y, Wu C, Li L, et al. Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer. Nucleic Acids Research. 2018;**46**(11):5809-5821

[39] Huang J, Zhang A, Ho TT, Zhang Z, Zhou N, Ding X, et al. Linc-RoR promotes c-Myc expression through hnRNP I and AUF1. Nucleic Acids Research. 2016;**44**(7):3059-3069

[40] Schmitt AM, Garcia JT, Hung T, Flynn RA, Shen Y, Qu K, et al. An inducible long noncoding RNA amplifies DNA damage signaling. Nature Genetics. 2016;**48**(11):1370-1376

[41] Hu WL, Jin L, Xu AN, Wang YF, Thorne RF, Zhang XD, et al. GUARDIN is a p53-responsive long non-coding RNA that is essential for genomic stability. Nature Cell Biology. 2018;**20**(4):492-502

[42] Hellwig S, Bass BL. A starvationinduced noncoding RNA modulates

expression of dicer-regulated genes. Proceedings of the National Academy of Sciences. 2008;**105**(35):12897-12902

[43] Marchese FP, Grossi E, Marín-Béjar O, Bharti SK, Raimondi I, González J, et al. A long noncoding RNA regulates sister chromatid cohesion. Molecular Cell. 2016;**63**(3):397-407

[44] Liu X, Xiao ZD, Han L, Zhang J, Lee SW, Wang W, et al. LncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress. Nature Cell Biology. 2016;**18**(4):431-442

[45] Yang F, Zhang H, Mei Y, Wu M. Reciprocal regulation of HIF-1α and lincRNA-p21 modulates the Warburg effect. Molecular Cell. 2014;**53**(1):88-100

[46] Feng J, Bi C, Clark BS, Mady R, Shah P, Kohtz JD. The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator. Genes & Development. 2006;**20**(11):1470-1484

[47] Kanduri C. Kcnq1ot1: a chromatin regulatory RNA. Seminars in Cell & Developmental Biology. 2011;**22**(4): 343-350

[48] Clark MB, Johnston RL, Inostroza-Ponta M, Fox AH, Fortini E, Moscato P, et al. Genome-wide analysis of long noncoding RNA stability. Genome Research. 2012;**22**(5):885-898

[49] Adalsteinsson BT, Ferguson-Smith AC. Epigenetic control of the genome—Lessons from genomic imprinting. Genes. 2014;**5**(3):635-655

[50] Kanduri C. Long noncoding RNAs: Lessons from genomic imprinting. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms. 2016;**1859**(1):102-111

[51] Peters J. The role of genomic imprinting in biology and disease: An expanding view. Nature Reviews Genetics. 2014;**15**(8):517-530

[52] da Rocha ST, Gendrel AV. The influence of DNA methylation on monoallelic expression. Essays in Biochemistry. 2019;**63**(6):663-676

[53] Elbracht M, Mackay D, Begemann M, Kagan KO, Eggermann T. Disturbed genomic imprinting and its relevance for human reproduction: Causes and clinical consequences. Human Reproduction Update. 2020;**26**(2):197-213

[54] Henckel A, Chebli K, Kota SK, Arnaud P, Feil R. Transcription and histone methylation changes correlate with imprint acquisition in male germ cells. The EMBO Journal. 2012;**31**(3):606-615

[55] Monk D, Mackay DJ, Eggermann T, Maher ER, Riccio A. Genomic imprinting disorders: Lessons on how genome, epigenome and environment interact. Nature Reviews Genetics. 2019;**20**(4):235-248

[56] Chen Z, Zhang Y. Maternal H3K27me3- dependent autosomal and X chromosome imprinting. Nature Reviews Genetics. 2020;**21**(9):555-571

[57] Tucci V, Isles AR, Kelsey G, Ferguson-Smith AC, Bartolomei MS, Benvenisty N, et al. Genomic imprinting and physiological processes in mammals. Cell. 2019;**176**(5):952-965

[58] Wutz A, Barlow DP. Imprinting of the mouse Igf2r gene depends on an intronic CpG island. Molecular and Cellular Endocrinology. 1998;**140**(1-2):9-14

[59] Sleutels F, Zwart R, Barlow DP. The non-coding air RNA is required for silencing autosomal imprinted genes. Nature. 2002;**415**(6873):810-813

[60] Mohammad F, Mondal T, Guseva N, Pandey GK, Kanduri C. Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1. Development. 2010;**137**(15):2493-2499

[61] Ripoche MA, Kress C, Poirier F, Dandolo L. Deletion of the H19 transcription unit reveals the existence of a putative imprinting control element. Genes & Development. 1997;**11**(12):1596-1604

[62] Monnier P, Martinet C, Pontis J, Stancheva I, Ait-Si-Ali S, Dandolo L. H19 lncRNA controls gene expression of the imprinted gene network by recruiting MBD1. Proceedings of the National Academy of Sciences. 2013;**110**(51):20693-20698

[63] Brannan CI, Dees EC, Ingram RS, Tilghman SM. The product of the H19 gene may function as an RNA. Molecular and Cellular Biology. 1990;**10**(1):28-36

[64] Edwards CA, Mungall AJ, Matthews L, Ryder E, Gray DJ, Pask AJ, et al. The evolution of the DLK1-DIO3 imprinted domain in mammals. PLoS Biology. 2008;**6**(6):e135

[65] McMurray EN, Schmidt JV. Identification of imprinting regulators at the Meg3 differentially methylated region. Genomics. 2012;**100**(3):184-194

[66] Royo H, Cavaillé J. Non-coding RNAs in imprinted gene clusters. Biology of the Cell. 2008;**100**(3):149-166

[67] Bar S, Seaton LR, Weissbein U, Eldar-Geva T, Benvenisty N. Global characterization of X chromosome inactivation in human pluripotent stem cells. Cell Reports. 2019;**27**(1):20-29

[68] Strehle M, Guttman M. Xist drives spatial compartmentalization of DNA

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

and protein to orchestrate initiation and maintenance of X inactivation. Current Opinion in Cell Biology. 2020;**64**:139-147

[69] Wang F, Shin J, Shea JM, Yu J, Bošković A, Byron M, et al. Regulation of X-linked gene expression during early mouse development by Rlim. eLife. 2016;**5**:e19127

[70] Wu H, Luo J, Yu H, Rattner A, Mo A, Wang Y, et al. Cellular resolution maps of X chromosome inactivation: Implications for neural development, function, and disease. Neuron. 2014;**81**(1):103-119

[71] Gendrel AV, Heard E. Noncoding RNAs and epigenetic mechanisms during X-chromosome inactivation. Annual Review of Cell and Developmental Biology. 2014;**30**:561-580

[72] Vallot C, Rougeulle C. Long non-coding RNAs and human X-chromosome regulation: A coat for the active X chromosome. RNA Biology. 2013;**10**(8):1262-1265

[73] Payer B, Lee JT. Coupling of X-chromosome reactivation with the pluripotent stem cell state. RNA Biology. 2014;**11**(7):798-807

[74] Huynh KD, Lee JT. Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos. Nature. 2003;**426**(6968):857-862

[75] Sidorenko J, Kassam I, Kemper KE, Zeng J, Lloyd-Jones LR, Montgomery GW, et al. The effect of X-linked dosage compensation on complex trait variation. Nature Communications. 2019;**10**(1):1-11

[76] Loda A, Heard E. Xist RNA in action: Past, present, and future. PLoS Genetics. 2019;**15**(9):e1008333

[77] Monfort A, Wutz A. The B-side of Xist. 2020;**F1000Research**:9

[78] Kung JT, Kesner B, An JY, Ahn JY, Cifuentes-Rojas C, Colognori D, et al. Locus-specific targeting to the X chromosome revealed by the RNA interactome of CTCF. Molecular Cell. 2015;**57**(2):361-375

[79] Khamlichi AA, Feil R. Parallels between mammalian mechanisms of monoallelic gene expression. Trends in Genetics. 2018;**34**(12):954-971

[80] Chigi Y, Sasaki H, Sado T. The 5′ region of Xist RNA has the potential to associate with chromatin through the A-repeat. RNA. 2017;**23**(12):1894-1901

[81] Dossin F, Pinheiro I, Żylicz JJ, Roensch J, Collombet S, Le Saux A, et al. SPEN integrates transcriptional and epigenetic control of X-inactivation. Nature. 2020;**578**(7795):455-460

[82] Mira-Bontenbal H, Gribnau J. New Xist-interacting proteins in X-chromosome inactivation. Current Biology. 2016;**26**(8):R338-R342

[83] Maurano MT, Humbert R, Rynes E, Thurman RE, Haugen E, Wang H, et al. Systematic localization of common disease-associated variation in regulatory DNA. Science. 2012;**337**(6099):1190-1195

[84] Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, et al. Chromatin signature reveals over a thousand highly conserved large noncoding RNAs in mammals. Nature. 2009;**458**(7235):223-227

[85] Li L, Liu B, Wapinski OL, Tsai MC, Qu K, Zhang J, et al. Targeted disruption of Hotair leads to homeotic transformation and gene derepression. Cell Reports. 2013;**5**(1):3-12

[86] Venkatraman A, He XC, Thorvaldsen JL, Sugimura R, Perry JM, Tao F, et al. Maternal imprinting at

the H19–Igf2 locus maintains adult haematopoietic stem cell quiescence. Nature. 2013;**500**(7462):345-349

[87] Zhang J, Zhang P, Wang L, Piao HL, Ma L. Long non-coding RNA HOTAIR in carcinogenesis and metastasis. Acta Biochimica et Biophysica Sinica. 2014;**46**(1):1-5

[88] Chen D, Zhang Z, Mao C, Zhou Y, Yu L, Yin Y, et al. ANRIL inhibits p15INK4b through the TGFβ1 signaling pathway in human esophageal squamous cell carcinoma. Cellular Immunology. 2014;**289**(1-2):91-96

[89] Hasegawa Y, Brockdorff N, Kawano S, Tsutui K, Tsutui K, Nakagawa S. The matrix protein hnRNP U is required for chromosomal localization of Xist RNA. Developmental Cell. 2010;**19**(3):469-476

[90] Hacisuleyman E, Goff LA, Trapnell C, Williams A, Henao-Mejia J, Sun L, et al. Topological organization of multichromosomal regions by the long intergenic noncoding RNA firre. Nature Structural & Molecular Biology. 2014;**21**(2):198-206

[91] Jiang W, Liu Y, Liu R, Zhang K, Zhang Y. The lncRNA DEANR1 facilitates human endoderm differentiation by activating FOXA2 expression. Cell Reports. 2015;**11**(1):137-148

[92] Li M, Gou H, Tripathi BK, Huang J, Jiang S, Dubois W, et al. An Apela RNAcontaining negative feedback loop regulates p53-mediated apoptosis in embryonic stem cells. Cell Stem Cell. 2015;**16**(6):669-683

[93] Chu C, Zhang QC, Da Rocha ST, Flynn RA, Bharadwaj M, Calabrese JM, et al. Systematic discovery of Xist RNA binding proteins. Cell. 2015;**161**(2):404-416

[94] Giovarelli M, Bucci G, Ramos A, Bordo D, Wilusz CJ, Chen CY, et al. H19 long noncoding RNA controls the mRNA decay promoting function of KSRP. Proceedings of the National Academy of Sciences. 2014;**111**(47):E5023-E5028

[95] Kallen AN, Zhou XB, Xu J, Qiao C, Ma J, Yan L, et al. The imprinted H19 lncRNA antagonizes let-7 microRNAs. Molecular Cell. 2013;**52**(1):101-112

[96] Zhang J, Liu SC, Luo XH, Tao GX, Guan M, Yuan H, et al. Exosomal long noncoding RNA s are differentially expressed in the Cervicovaginal lavage samples of cervical cancer patients. Journal of Clinical Laboratory Analysis. 2016;**30**(6):1116-1121

[97] Xia T, Chen S, Jiang Z, Shao Y, Jiang X, Li P, et al. Long noncoding RNA FER1L4 suppresses cancer cell growth by acting as a competing endogenous RNA and regulating PTEN expression. Scientific Reports. 2015;**5**(1):1-9

[98] Gutschner T, Hämmerle M, Diederichs S. MALAT1—A paradigm for long noncoding RNA function in cancer. Journal of Molecular Medicine. 2013;**91**(7):791-801

[99] Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 2010;**464**(7291): 1071-1076

[100] Wilusz JE, Freier SM, Spector DL. 3′ end processing of a long nuclearretained noncoding RNA yields a tRNA-like cytoplasmic RNA. Cell. 2008;**135**(5):919-932

[101] Poliseno L, Haimovic A, Christos PJ. Deletion of PTENP1 pseudogene in human melanoma. The Journal of Investigative Dermatology. 2011;**131**(12):2497

*Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

[102] Sánchez Y, Segura V, Marín-Béjar O, Athie A, Marchese FP, González J, et al. Genome-wide analysis of the human p53 transcriptional network unveils a lncRNA tumour suppressor signature. Nature Communications. 2014;**5**(1):1-13

[103] Zheng Z, Zhao F, Zhu D, Han J, Chen H, Cai Y, et al. Long non-coding RNA LUCAT1 promotes proliferation and invasion in clear cell renal cell carcinoma through AKT/GSK-3β signaling pathway. Cellular Physiology and Biochemistry. 2018;**48**(3):891-904

[104] Guffanti A, Iacono M, Pelucchi P, Kim N, Soldà G, Croft LJ, et al. A transcriptional sketch of a primary human breast cancer by 454 deep sequencing. BMC Genomics. 2009;**10**(1):1-17

[105] Yuan JH, Yang F, Wang F, Ma JZ, Guo YJ, Tao QF, et al. A long noncoding RNA activated by TGF-β promotes the invasion-metastasis cascade in hepatocellular carcinoma. Cancer Cell. 2014;**25**(5):666-681

[106] Grelet S, Link LA, Howley B, Obellianne C, Palanisamy V, Gangaraju VK, et al. A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumour progression. Nature Cell Biology. 2017;**19**(9):1105-1115

[107] Huang JF, Guo YJ, Zhao CX, Yuan SX, Wang Y, Tang GN, et al. Hepatitis B virus X protein (HBx) related long noncoding RNA (lncRNA) down-regulated expression by HBx (Dreh) inhibits hepatocellular carcinoma metastasis by targeting the intermediate filament protein vimentin. Hepatology. 2013;**57**(5):1882-1892

[108] Zhang J, Li Z, Liu L, Wang Q, Li S, Chen D, et al. Long noncoding RNA TSLNC8 is a tumor suppressor that inactivates the interleukin-6/

STAT3 signaling pathway. Hepatology. 2018;**67**(1):171-187

[109] Li C, Wang S, Xing Z, Lin A, Liang K, Song J, et al. A ROR1–HER3– lncRNA signalling axis modulates the hippo–YAP pathway to regulate bone metastasis. Nature Cell Biology. 2017;**19**(2):106-119

[110] Atianand MK, Fitzgerald KA. Long non-coding RNAs and control of gene expression in the immune system. Trends in Molecular Medicine. 2014;**20**(11):623-631

[111] Rapicavoli NA, Qu K, Zhang J, Mikhail M, Laberge RM, Chang HY. A mammalian pseudogene lncRNA at the interface of inflammation and anti-inflammatory therapeutics. elife. 2013;**2**:e00762

[112] Wang P, Xue Y, Han Y, Lin L, Wu C, Xu S, et al. The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation. Science. 2014;**344**(6181):310-313

[113] Carpenter S, Aiello D, Atianand MK, Ricci EP, Gandhi P, Hall LL, et al. A long noncoding RNA mediates both activation and repression of immune response genes. Science. 2013;**341**(6147):789-792

[114] Li Z, Chao TC, Chang KY, Lin N, Patil VS, Shimizu C, et al. The long noncoding RNA THRIL regulates TNFα expression through its interaction with hnRNPL. Proceedings of the National Academy of Sciences. 2014;**111**(3):1002-1007

[115] Vigneau S, Rohrlich PS, Brahic M, Bureau JF. Tmevpg1, a candidate gene for the control of Theiler's virus persistence, could be implicated in the regulation of gamma interferon. Journal of Virology. 2003;**77**(10):5632-5638

[116] Collier SP, Collins PL, Williams CL, Boothby MR, Aune TM. Cutting edge: Influence of Tmevpg1, a long intergenic noncoding RNA, on the expression of Ifng by Th1 cells. The Journal of Immunology. 2012;**189**(5):2084-2088

[117] Gomez JA, Wapinski OL, Yang YW, Bureau JF, Gopinath S, Monack DM, et al. The NeST long ncRNA controls microbial susceptibility and epigenetic activation of the interferon-γ locus. Cell. 2013;**152**(4):743-754

[118] Pang KC, Dinger ME, Mercer TR, Malquori L, Grimmond SM, Chen W, et al. Genome-wide identification of long noncoding RNAs in CD8+ T cells. The Journal of Immunology. 2009;**182**(12):7738-7748

[119] Hu G, Tang Q, Sharma S, Yu F, Escobar TM, Muljo SA, et al. Expression and regulation of intergenic long noncoding RNAs during T cell development and differentiation. Nature Immunology. 2013;**14**(11):1190-1198

[120] Bolland DJ, Wood AL, Johnston CM, Bunting SF, Morgan G, Chakalova L, et al. Antisense intergenic transcription in V (D) J recombination. Nature Immunology. 2004;**5**(6):630-637

[121] Verma-Gaur J, Torkamani A, Schaffer L, Head SR, Schork NJ, Feeney AJ. Noncoding transcription within the Igh distal VH region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells. Proceedings of the National Academy of Sciences. 2012;**109**(42):17004-17009

[122] Shirasawa S, Harada H, Furugaki K, Akamizu T, Ishikawa N, Ito K, et al. SNPs in the promoter of a B cell-specific antisense transcript, SAS-ZFAT, determine susceptibility to autoimmune thyroid disease. Human Molecular Genetics. 2004;**13**(19):2221-2231

[123] Dave RK, Dinger ME, Andrew M, Askarian-Amiri M, Hume DA, Kellie S. Regulated expression of PTPRJ/CD148 and an antisense long noncoding RNA in macrophages by proinflammatory stimuli. PLoS One. 2013;**8**(6):e68306

[124] Huarte M, Guttman M, Feldser D, Garber M, Koziol MJ, Kenzelmann-Broz D, et al. A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell. 2010;**142**(3):409-419

[125] Wright PW, Huehn A, Cichocki F, Li H, Sharma N, Dang H, et al. Identification of a KIR antisense lncRNA expressed by progenitor cells. Genes & immunity. 2013;**14**(7):427-433

[126] Imamura K, Akimitsu N. Long non-coding RNAs involved in immune responses. Frontiers in Immunology. 2014;**5**:573

[127] Bei L, Lu Y, Bellis SL, Zhou W, Horvath E, Eklund EA. Identification of a HoxA10 activation domain necessary for transcription of the gene encoding β3 integrin during myeloid differentiation. Journal of Biological Chemistry. 2007;**282**(23):16846-16859

[128] Ilott NE, Heward JA, Roux B, Tsitsiou E, Fenwick PS, Lenzi L, et al. Long non-coding RNAs and enhancer RNAs regulate the lipopolysaccharideinduced inflammatory response in human monocytes. Nature Communications. 2014;**5**(1):1-14

[129] Campbell M, Kim KY, Chang PC, Huerta S, Shevchenko B, Wang DH, et al. A lytic viral long noncoding RNA modulates the function of a latent protein. Journal of Virology. 2014;**88**(3):1843-1848

[130] Kotzin JJ, Spencer SP, McCright SJ, Kumar DBU, Collet MA, Mowel WK, et al. *Long Non-Coding RNAs: Biogenesis, Mechanism of Action and Role in Different Biological… DOI: http://dx.doi.org/10.5772/intechopen.104861*

The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan. Nature. 2016;**537**(7619):239-243

[131] Sehgal L, Mathur R, Braun FK, Wise JF, Berkova Z, Neelapu S, et al. FAS-antisense 1 lncRNA and production of soluble versus membrane FAS in B-cell lymphoma. Leukemia. 2014;**28**(12):2376-2387

[132] Atianand MK, Hu W, Satpathy AT, Shen Y, Ricci EP, Alvarez-Dominguez JR, et al. A long noncoding RNA lincRNA-EPS acts as a transcriptional brake to restrain inflammation. Cell. 2016;**165**(7):1672-1685

[133] Spurlock CF, Tossberg JT, Guo Y, Collier SP, Crooke PS, Aune TM. Expression and functions of long noncoding RNAs during human T helper cell differentiation. Nature Communications. 2015;**6**(1):1-12

[134] Ranzani V, Rossetti G, Panzeri I, Arrigoni A, Bonnal RJ, Curti S, et al. The long intergenic noncoding RNA landscape of human lymphocytes highlights the regulation of T cell differentiation by linc-MAF-4. Nature Immunology. 2015;**16**(3):318-325

[135] Jiang R, Tang J, Chen Y, Deng L, Ji J, Xie Y, et al. The long noncoding RNA lnc-EGFR stimulates T-regulatory cells differentiation thus promoting hepatocellular carcinoma immune evasion. Nature Communications. 2017;**8**(1):1-15

[136] Zhang F, Wu L, Qian J, Qu B, Xia S, La T, et al. Identification of the long noncoding RNA NEAT1 as a novel inflammatory regulator acting through MAPK pathway in human lupus. Journal of Autoimmunity. 2016;**75**:96-104

[137] Wu GC, Li J, Leng RX, Li XP, Li XM, Wang DG, et al. Identification of long

non-coding RNAs GAS5, linc0597 and lnc-DC in plasma as novel biomarkers for systemic lupus erythematosus. Oncotarget. 2017;**8**(14):23650

[138] Song J, Kim D, Han J, Kim Y, Lee M, Jin EJ. PBMC and exosome-derived Hotair is a critical regulator and potent marker for rheumatoid arthritis. Clinical and Experimental Medicine. 2015;**15**(1):121-126

[139] Spurlock CF III, Tossberg JT, Matlock BK, Olsen NJ, Aune TM. Methotrexate inhibits NF-κB activity via long intergenic (noncoding) RNA–p21 induction. Arthritis & Rheumatology. 2014;**66**(11):2947-2957

[140] Lu MC, Yu HC, Yu CL, Huang HB, Koo M, Tung CH, et al. Increased expression of long noncoding RNAs LOC100652951 and LOC100506036 in T cells from patients with rheumatoid arthritis facilitates the inflammatory responses. Immunologic Research. 2016;**64**(2):576-583

[141] Wang J, Peng H, Tian J, Ma J, Tang X, Rui K, et al. Upregulation of long noncoding RNA TMEVPG1 enhances T helper type 1 cell response in patients with Sjögren syndrome. Immunologic Research. 2016;**64**(2):489-496

[142] Satoh T, Okano T, Matsui T, Watabe H, Ogasawara T, Kubo K, et al. Novel autoantibodies against 7SL RNA in patients with polymyositis/ dermatomyositis. The Journal of Rheumatology. 2005;**32**(9):1727-1733

[143] Peng QL, Zhang YM, Yang HB, Shu XM, Lu X, Wang GC. Transcriptomic profiling of long non-coding RNAs in dermatomyositis by microarray analysis. Scientific Reports. 2016;**6**(1):1-11

#### **Chapter 3**

## Advantages of Noncoding RNAs in Molecular Diagnosis

*Tomomi Fujii, Tomoko Uchiyama and Maiko Takeda*

#### **Abstract**

Noncoding RNAs contribute to physiological processes by regulating many intracellular molecules participating in the life-supporting mechanisms of development, differentiation, and regeneration as well as by disrupting various signaling mechanisms such as disease development and progression and tumor growth. Because microRNAs (miRNAs) target and regulate the functions of key proteins, it is very useful to identify specific miRNAs that contribute to cellular functions and to clarify the roles of their target molecules as diagnostic and therapeutic strategies for cancer prognosis and treatment. In this section, the roles of miRNAs in various cancers and the processes leading to the identification of their target molecules are described, and the latest diagnostic strategies using miRNAs are discussed with specific examples.

**Keywords:** microRNA, cancer, molecular diagnosis

#### **1. Introduction**

Noncoding RNAs (ncRNAs) comprise the majority of gene transcripts in eukaryotes. ncRNAs play little biological role, but microRNAs (miRNAs), first reported in 1993, have important roles in gene expression [1, 2]. One of the most well-known miRNAs, let-7, a heterochronic gene in C. elegans, is considered important for developmental differentiation and regulates the larva-to-adult transition during larval development [3]. As a summary of the biosynthetic process of miRNAs, double-stranded RNA, the source of RNA interference, is sequentially processed into single-stranded RNA fragments of 21–23 nucleotides (nt) by the action of various enzymes [4, 5]. Let-7 encodes a small 21-nt ncRNA that binds to the 3′-untranslated region (UTR) of lin genes, resulting in RNA–RNA interactions and negative regulation of mRNA [6]. More than 30,000 miRNAs have been identified from more than 250 species, ranging from prokaryotes to eukaryotes, invertebrates to vertebrates, and even viruses [7, 8]. More than 5000 of these miRNAs have been identified in humans [miRBase: The microRNA database; cited 2018 Aug; Available from: http://www. mirbase.org].

miRNAs function as posttranscriptional regulators (negative regulators) of various gene expressions by binding to complementary sequences, primarily in the 3′-UTR of their target genes. miRNAs can target a large number of genes, and the mRNAs targeted by miRNAs mRNAs targeted by miRNAs account for more than 60% of all genes. Most miRNAs are encoded within the introns of coding mRNAs, but

some can also be found within the 3′-UTR sequences of noncoding or coding mRNAs but can also be found in the 3′-UTR sequence of noncoding or coding mRNAs [9]. When miRNAs bind to target mRNAs, these are destabilized by deadenylation factors becoming more susceptible to degradation. As a result, mRNA is degraded; however, sometimes the mRNA is not degraded and translation is regulated, a mechanism that is not yet unclear. Alterations in the genetic structure of miRNA-associated regions are known to occur in many pathologies, especially cancer. Such chromosomal aberrations include amplifications (e.g. miR-26a in gliomas), deletions (e.g. miR-15a/16–1 in chronic lymphocytic leukemia), mutations (e.g. miR-125a in breast cancer), translocations [e.g. miR-125b in acute myeloid leukemia with t(2;11)(p21;q23)], single-nucleotide polymorphisms (e.g. miR-608 in colorectal adenocarcinoma), and heterozygous deletions (such as the 14q32 cluster in acute lymphoblastic leukemia) [10–15]. These changes can occur not only in the sequence of the mature miRNA itself but also in the promoter region/primary transcript (pri-miRNA) sequence or in the miRNA binding site of the target gene, and as somatic or germline mutations [16–18]. A number of transcription factors, as well as vital protein-coding genes, influence the expression levels of miRNAs, and this regulatory action is assumed to be particularly important for tissue specificity and developmental stage control. Conversely, loss of Dicer, DGCR8, Drosha, and Argonaute2(Ago2), components of pathways involved in the miRNA biosynthesis mechanism, is known to result in the inability to sustain life and early pregnancy death due to severe developmental defects. [19–22]. Loss of reproductive function due to the deletion of components involved in miRNA biosynthesis, and developmental defects in the heart, lungs, and neuromuscular tissue have been reported [23–30]. Dicer mutations have also been reported to cause tumor development and progression [31, 32]. Dysfunction of the miRNA biogenesis pathway causes the disruption of important life-support mechanisms associated with various diseases, among which cancer development and progression are strongly influenced. In particular, abnormal expression of Dicer and Drosha is known to lead to poor prognosis in various cancers [33–39]. Although there is a vast amount of evidence that miRNAs play fundamental roles in the mechanisms of many diseases, including cancer, unfortunately, they have not yet been fully exploited in diagnostic and therapeutic settings. It is therefore necessary to determine how miRNAs can potentially be utilized in clinical practice in the future. miRNAs likely have the greatest and most direct potential as novel biomarkers of diagnosis and prognosis, as well as predictors of therapeutic efficacy. It is also clear that miRNAs regulate or disrupt the expression of numerous target mRNAs, resulting in maintenance of vital activity or disease, which cannot be resolved by a single miRNA. Therefore, gene expression analysis by miRNA expression profiling in disease must accurately discriminate between disease diagnosis and tumor stage of development [40].

A more accurate diagnosis of tumors can be obtained by morphological histopathology using formalin-fixed paraffin-embedded (FFPE) tissues and profiling of expressed proteins via immunohistochemical staining. Genetic analysis using FFPE is also emerging as being important for therapeutic selection. However, the degradation of nucleic acids is a problem for FFPE, and nucleic acids extracted from aged tissues are degraded [41]. A particularly attractive property of miRNAs is their stability against chemical and enzymatic degradation. This implies that they can be purified from routinely prepared FFPE biopsy materials and measured robustly [42]. As a result, retrospective miRNA expression studies are underway, using FFPE as a conserved and valuable resource. The stability and clinical utility of miRNAs are due to their presence in extracellular biological fluids, including blood, as well as in

#### *Advantages of Noncoding RNAs in Molecular Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.105525*

tissues and cells. Tumor-associated miRNAs are found at higher levels in the serum and plasma of cancer patients than in healthy controls. Therefore, the use of miRNAs as potential noninvasive biomarkers of disease has attracted much attention, and miRNAs have been detected in many biological fluids, including plasma, serum, tears, urine, cerebrospinal fluid, breast milk, and saliva [43, 44]. The widespread use of biomarker testing, which is noninvasive and more reliable than the conventional histopathological diagnosis of cancer that invasive to the patient, could pave the way for screening programs, improve the early detection rate of disease, and enhance cancer prevention. Despite the growing functional importance of miRNAs as diagnostic and therapeutic strategies for cancer, there are still many knowledge gaps despite the potential to develop miRNA markers in the future.

#### **2. Role of miRNAs as oncogenes and tumor suppressor genes**

The role of miRNAs in regulating the genes involved in cancer growth and progression may be to suppress cancer progression primarily through suppressing target gene expression or to inhibit the function of genes that suppress cancer progression, therefore positively affecting cancer progression. The results obtained by detailed profiling of miRNA expression in cancer according to organ or cancer differentiation level revealed that the expression pattern of miRNAs varies depending on the organ, histological type, and differentiation level of cancer, indicating that the role of miRNAs is highly segmented [40]. Many miRNAs function in a suppressive manner in cancer. The expression of the aforementioned let-7 miRNA is downregulated in lung cancer, targeting and suppressing the expression of Ras, an important oncogene. This indicates that let-7 may function as a cancer suppressor gene [45]. The expression of miR-15 and miR-16 is absent or decreased in chronic lymphocytic leukemia, suggesting they act in a tumor-suppressive manner by targeting B cell lymphoma-2, an antiapoptotic factor [46]. Human miR-373 binds to the E-cadherin (CDH1) promoter and induces gene expression [47]. In addition to gene silencing through base pairing with DNA and mRNA target sequences, miRNAs are known to inhibit the function of regulatory proteins (decoy activity). miR-328 binds to poly C-binding protein 2 (PCBP2), also known as heterogeneous ribonucleoprotein E2. This binding does not involve the seed region of the miRNA, but it inhibits its interaction with the target mRNA. In chronic myeloid leukemia, downregulation of miR-328 inhibits myeloid differentiation via PCBP2, leading to tumor progression [48]. It is also important to understand the molecular mechanisms of miRNAs to define the mechanism of their suppressive properties against cancer. miRNAs primarily target the 3′-UTR of mRNAs and downregulate the cytoplasmic expression of protein-coding genes. As evidence for this, miRNAs are usually localized in the nucleus [49]. In addition to the 3′-UTR, other genetic regions at the DNA and RNA levels, that is, the 5′-UTR, promoter, and coding regions, as well as proteins can be targeted (**Figure 1**) [50–52]. By binding to various functional regions of genes, miRNAs can upregulate or downregulate gene expression, interact with other ncRNAs and RNA transcripts, and modulate biological networks [53, 54]. It has become clear that the actions of these miRNAs on their target molecules lead to the regulation of gene expression [55]. It has been shown that miRNAs not only bind to key sites in other RNAs but also to promoter regions at the DNA level, thereby affecting transcriptional activity. As a specific example, in terms of localization sites, miRNAs have been shown to function both in the cytoplasm and nucleus [56]. Investigation of the subcellular localization of miR-29b revealed

**Figure 1.**

*Predicted binding sites for miRNAs in genes. miRNAs have binding sites not only in the 3*′*UTR but also in every site of the gene.*

it was mostly localized to the nucleus. The characteristic hexanucleotide terminal motif of miR-29b functions as a metastable nuclear localization element that induces the nuclear localization of the miRNA or small-interference RNA to which it binds [49]. It has been shown that miRNAs bind to adenylate-uridylate-rich elements and upregulate translation in cells that have undergone cell cycle arrest. This suggests that miRNAs can affect not only cell differentiation and proliferation stages in a broad sense but also the cell cycle [57].

#### **3. Expression dynamics of miRNAs in cancer and expectations for clinical application**

Cancer is a malignant disease in which a population of cells with genetically abnormal protein expression proliferate autonomously, destroying host tissues as they grow, metastasize, and settle in other organs via vascular flow. As a result, cancer causes the dysfunction of normal tissues and ultimately leads to organ failure. There is evidence that carcinogenesis is a multistep process in which malignant cells accumulate epigenetic and genetic changes that gradually transform normal cells into malignant cells. Triggers for carcinogenesis include tobacco, chemical exogenous stimuli such as chemicals and dust from air pollution, and accumulation of genetic damage due to the aging of individual cells [58]. The proliferation of cancer cells progresses by causing structural changes in genes that promote cancer cell proliferation (oncogenes), genes that prevent their proliferation (tumor suppressor genes), and abnormal gene expression [59–62]. Malignant cells lose cell discipline in morphology and differentiation and acquire autonomous proliferation, antiapoptosis, and invasiveness properties. miRNAs have been analyzed in various human malignant tumors and can be involved in the pathophysiology of benign and malignant tumors in various ways. Although there are differences in miRNA expression profiles between normal and tumor tissues, many of these miRNAs are only indirectly altered by genetic changes that occur during carcinogenesis, epigenomic dynamics, and physiological changes in cell biology and do not directly trigger tumor development.  The involvement of miRNAs in the carcinogenic process may involve changes in the components of

#### *Advantages of Noncoding RNAs in Molecular Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.105525*

miRNA biosynthesis pathway, and overall suppression of miRNA maturation by mutations in DROSHA, DGCR8, or DICER has been shown to affect the transcription and translation of multiple mRNAs, ultimately promoting cell transformation and tumorigenesis [77–79]. The fact that conditional loss of DICER or DGCR8 inhibits bone and cartilage growth and differentiation in mice and in vitro has been demonstrated, suggesting that components involved in the miRNA biosynthesis mechanism are also key molecules in carcinogenesis and cancer progression [80, 81]. The expression of Dicer, Exportin-5, TRKRA, TARBP2, DGCR8, and Argonaut-2 changes dynamically with changes in miRNA expression during the oncogenic phase in pancreatic cancer, suggesting that these miRNA biosynthesis-related molecules can cause cellular changes, including carcinogenesis [82]. Decreased expression of some components that play an important role in miRNA biogenesis, such as DICER, is associated with cancer prognosis and sensitivity to molecularly targeted drugs and has been shown to correlate with short survival in non-small cell lung cancer while increasing sensitivity to gefitinib [36, 83]. The methylation of DICER and DROSHA is also a potential biomarker for lung cancer [84]. Some miRNA precursors, known as mirtrons, have the unique feature of entering the miRNA biosynthesis pathway without undergoing Drosha-mediated cleavage while mimicking the structural features of pre-miRNAs. They are susceptible to epigenetic regulation and have been shown to exert epigenetic effects through interactions between various miRNAs and host genes in urothelial cell carcinoma [85–87]. Breast cancer susceptibility gene 1 (BRCA1) promotes the processing of primary miRNA transcripts and increases the expression of both precursors and mature forms of let-7a-1, miR-16-1, miR-145, and miR-34a [88]. BRCA1 interacts with Smad3, p53, and DHX9 RNA helicases by directly binding to DROSHA and DDX5 in the DROSHA microprocessor complex that regulates miRNA maturation and recognizes RNA secondary structures through its DNA-binding domain, which interacts with pri-miRNA via the DNA-binding domain.

Thus, from their discovery to the present, miRNAs have brought great promise and success in cancer diagnosis, prognosis, and treatment both in clinical practice and experimental medicine. Powerful technologies, such as miRNA arrays, short RNA deep sequencing, specific quantitative polymerase chain reaction (PCR) of miRNAs, and antisense technologies, have revealed the functions of numerous miRNAs. It is expected that they will continue to have a major impact on clinical oncology in terms of cancer diagnosis and prognostic treatment. Because miRNAs are important factors that define cellular characteristics, direct detection of miRNAs and miRNA target molecules from algorithms based on the vast amount of data obtained by profiling analysis could be used as a valuable tool for cancer diagnosis. miRNA expression profiling has been considered more efficient and informative than conventional mRNA profiling in classifying tumors with respect to their tissue of origin and differentiation [89–93]. These findings indicate that in clinical practice, miRNAs are useful biomarkers for tracking the tissue of origin of tumors [94–97]. miRNA expression profiles can be an important source of information for cancer progression prediction and prognosis. It has been shown that high expression of miR-21 and miR-155 in lung cancer and colorectal cancer (CRC) predicts efficient recurrence, poor prognosis, and reduced survival [98–101]. Functional analysis of miRNAs through experimental processes has been found to be valuable for therapeutic strategies based on the regulation of miRNA activity. As a therapeutic strategy against miRNAs acting as oncogenes (oncomirs), anti-miRNAs are expected to exert therapeutic effects by blocking oncomirs through the action of oligonucleotides that are complementary to miRNAs. For them to function stably, they must be chemically modified to enhance their

stability and deliverability to the target organs. As is the case with many drugs used in cancer therapy, issues related to the delivery of miRNA-containing compounds, their stability in vivo, and biological defense measures against toxicity are important issues that must be resolved as miRNAs progress from experimental to clinical trials. However, because miRNAs have multiple targets, inhibition of target molecules by miRNAs may cause serious side effects. These issues are expected to be resolved and applied clinically in the future.

#### **4. Functions of miRNAs in various organs**

Given their properties, miRNAs have many target molecules and each performs different functions during cell differentiation and proliferation. Therefore, miRNAs are expected to perform characteristic functions in various organs. Here, the functions of miRNAs in typical cancers are introduced.

#### **4.1 Breast cancer**

miRNA profiling studies in breast cancer have been conducted from various perspectives.  Subsequent studies confirmed the elevated levels of miR-10b, miR-34a, and miR-155 in patients with metastatic breast cancer [103]. Furthermore, miR-10b and miR-373 were recently shown to be upregulated in lymph node-positive breast cancer [104]. In addition, miRNAs from previous miRNA profiling studies have been shown to play potential roles as tumor suppressor genes or oncogenes in breast cancer [65, 105, 106]. The decreased expression of many genes involved in cancer development and progression has become evident, and miRNAs and their target molecules that are downregulated in breast cancer tissues and cell lines have become the focus of research. Breast cancer is a cancerous tissue with high histopathological heterogeneity. Identifying miRNAs and their target molecules that are specifically expressed and function in this context and have diagnostic value is therefore difficult. miRNA profiling should facilitate the accurate classification of tumor subtypes and provide useful additional information to complement current classification methods and guide therapeutic decisions. Studies have nested to predict miRNAs in accordance with previous subtype classifications. Recent analyses of miRNAs have identified predicted miRNAs corresponding to hormone receptor and HER2 subtypes, including ER (miR-342, miR-299, miR-217, miR-190, miR-135b, and miR-218), PR (miR-520g, miR-377, miR- 527-518a, miR-520f-520c), and HER2 (miR-520d, miR-181c, miR-302c, miR-376b, and miR-30e). These expression kinetics classified cases with 100% accuracy compared to immunohistochemistry results [107]. Further classification is expected to increase miRNA profiling accuracy and the development of new diagnostic markers and therapeutic strategies based on miRNA target molecules.

#### **4.2 Prostate cancer**

Prostate cancer (PCa) is one of the most common causes of cancer-related death in men. Although most PCa progress very slowly and remain within the primary tumor,

some have an aggressive behavior that can spread to other organs via the vascular system. Prostate-specific antigen (PSA) and prostate acid phosphatase (PAP) are useful tumor markers for prostate cancer. However, PAP has low specificity and PSA, despite its superior specificity, is inadequate for predicting the tumor types in which it is not elevated and histological differentiation and progression. Many studies have indicated that miRNAs are involved in the development of PCa [108–112]. Aberrant expression of miRNAs has been detected in PCa cell lines, in experimental carcinogenesis using cancer xenografts, and in clinical samples obtained from patients [113–118]. These changes suggest that miRNAs play an important role in the pathogenesis of PCa.

Many miRNAs are expressed as oncogenes. For example, miR-375, miR-106a, and miR-194 have been reported to play important roles in tumor formation, proliferation, and progression [119–121]. When the expression of tumor suppressor miRNAs decreases, the expression of oncogenes increases, promoting cancer growth and progression. Experimental introduction of precursors of these miRNAs into cells and overexpression of these miRNAs to reproduce normal expression patterns suppressed the growth, invasion, clonogenesis, and metastatic potential of PCa cells. The most well-known miRNAs are miR-149-5p, miR-7-5p, miR-122, miR-124-3p, miR-188, and miR-129 [122–127]. Downregulation of these miRNAs is associated with increased cell proliferation, migration, and chemotherapy resistance, primarily through the regulation of cell growth and proliferation via the mitogen-activated protein kinase pathway.

Clinically practical markers in PCa include serum PSA measurement and digital rectal examination; however, their diagnostic value is limited and does not extend to prognostic prediction based on cancer progression or assessment of differentiation related to tumor growth potential. Human glandular kallikrein 2, urokinase plasminogen activator and its receptor, transforming growth factor-β1, and interleukin 6 and its receptor are biomarkers with high sensitivity and specificity for the early diagnosis of PCa. miRNAs are also clinically promising diagnostic markers for this cancer. miRNAs can be used as diagnostic and disease-monitoring markers for the presence of minimal residual disease and early detection of recurrence. Furthermore, the establishment of a diagnostic algorithm combining multiple oncomir and tumor suppressor miRNAs is expected to enable a more stepwise diagnosis of PCa. In this cancer, histopathological diagnosis by biopsy is highly invasive and often fails to detect microscopic cancers owing to the extremely small amount of visible tissue. To compensate for the shortcomings of local biopsy pathology diagnosis, the availability of profiling tests with circulating miRNAs has the potential to predict the onset of cancer and the degree of differentiation through its combination algorithm. From the time of diagnosis and initiation of treatment, the detection of circulating miRNAs could contribute to the early detection of hormone refractory PCa. The role of cancer stem cells in PCa has been elucidated in previous studies. The CD44-positive PCa stem cell population has tumorigenic and metastatic potential, which has been mentioned in conjunction with other useful markers for cancer stem cells [128]. Functional analysis of miRNAs has shown that miR-9-5p, miR-34a, miR-373, miR-708, and miR-520c directly target CD44 suppressing its expression [129–133]. The fact that the expression levels of these miRNAs are decreased in PCa suggests an increased potential for tumor invasion and metastasis. Profiling of miRNAs in PCa holds promise as a more precise diagnostic tool before performing prostate biopsy.

#### **4.3 CRC**

CRC is a common cancer worldwide and is the second most common cause of cancer-related deaths in the Western world. Although the 5-year survival rate for early-stage cancer exceeds 90%, it decreases to approximately 70% for advanced cancer and to approximately 10% when distant metastases are present. Although it is certainly a carcinoma for which early diagnosis is very useful, in reality early detection is less than 40% due to insufficient noninvasive screening. Although direct diagnosis by endoscopy is most effective in CRC, it often relies on a fecal occult blood test because of its invasive nature. However, the fecal occult blood reaction can be positive even for inflammatory or nonneoplastic polyps and lacks specificity in terms of cancer diagnosis. If CRC can be found to have miRNA expression characteristics, analysis of miRNAs using stool and the detection of circulating miRNAs may be useful in detecting marginally present tumor cells. Therefore, we searched for miRNAs that were significantly elevated in CRC using stool and plasma and found a number of miRNAs, including miR-17, miR-18a, miR-19a, miR-21, miR-92a, miR-200c, miR-221, and miR-106a as diagnostic markers [134–138]. miR-141 is a miRNA that functions in an inhibitory manner against colon cancer cells in terms of cell proliferation, invasiveness, and migration [139–142]. As a circulating miRNA, miR-141, together with miR-200 and miR-143, could be an independent prognostic marker for advanced CRC [143, 144].

CRC progresses from adenoma to colorectal adenocarcinoma as precancerous lesions. Investigation of miRNA expression in staged histological types has revealed that the expression of several miRNAs is altered in precancerous lesions, adenomas, and CRC tissues compared to that in normal colorectal tissue. This suggests that some miRNAs are involved in the process of CRC and may be important biomarkers in the transition of tumor tissue from benign to malignant. In addition, miRNAs are now being extracted from stored FFPE tissue specimens and retrospectively profiled for miRNA expression using next-generation sequencing (NGS) to understand the origin of CRC development and metastatic disease in cases with multiple lesions. The most studied miRNAs with respect to clinical prognosis are miR-21, miR-143, and miR-145. Although miR-21 suppresses the expression of various tumor suppressor genes, it also affects cell proliferation, apoptosis, invasion, and tumor progression. Increased miR-21 expression in CRC tumor tissue correlates with increased metastatic potential of the primary tumor and is associated with shorter disease-free and overall survival. Owing to the antiapoptotic effect of miR-21, its overexpression was also found to affect therapeutic efficacy by reducing the efficacy of 5-fluorouracil (5-FU) chemotherapy [145–150]. Decreased expression of miR-143 and miR-145 in CRC tumor tissue has been observed in several studies, suggesting that they behave as tumor suppressors [132, 151–154]. Their decreased expression has been associated with increased tumor growth and angiogenesis, and it is clinically associated with shorter disease-free survival. The expression of miR-143 directly correlates with the sensitivity of the colon cancer cell line HCT116 to 5-FU treatment. miR-215 is involved in the cell proliferative capacity of CRC and is sensitive to chemotherapy and molecular-targeted therapy. miR-215 is also involved in the prognostic prediction of CRC [155–160].

Although biomarker analysis using stool as a method to directly capture tumor cells in CRC is open for consideration, the establishment of a methodology for goodquality nucleic acid extraction that enables good analysis and sample quality maintenance is required to obtain reliable data due to the heterogeneity of sample processing. In contrast, for the prediction of treatment potential and prognosis for advanced cancer, miRNA profiling can be performed by nucleic acid extraction from FFPE of excised tumor tissue, and sufficient technology is being established for accuracy and reliability.

#### **5. Analysis of circulating miRNAs for their usefulness as biomarkers**

In addition to serum and plasma, various body fluids, such as saliva, cerebrospinal fluid, and body cavity fluid, are candidates as analyzable materials for the detection of circulating miRNAs. However, in samples of these fluids from cancer patients, not only tumor cells but also blood cells, macrophages, exosomes, and microparticles are present; where the miRNAs are present, there is also the issue of how many of the targeted tumor-derived miRNAs are included or how reliable the analysis is based on which miRNAs are included. Nevertheless, regardless of the members, each of whom may possess miRNAs, if the changes caused by the coexistence of cancer can be captured for cancer patients, the usefulness of profiling will be well maintained. It is well known that analysis of exosome-derived miRNAs can detect more cancerrelevant miRNAs than analysis using untreated serum. An interesting aspect of the quantitative analysis of miRNAs in exosomes and microparticles is that they may be involved in cell–cell interactions, stimulate intracellular signaling, and modulate metabolic functions and homeostasis in a small number of stem cells. Quantitative analysis of circulating miRNAs is most useful for miRNAs quantitative real-time PCR (qRT-PCR), because it detects miRNAs from a very small amount of nucleic acids. Although NGS is superior for expression profiling, it requires a sufficient amount of nucleic acids and is likely to lack reliability due to detection errors or low sensitivity. Therefore, a single-miRNA assay using the TaqMan method should be performed on miRNAs with presumed abnormal expression at the transcriptional level, as detected from microarray analysis or NGS analysis based on FFPE, followed by qRT-PCR validation. Capturing abnormal miRNA expression in cancer is a highly useful diagnostic strategy but, because capturing circulating miRNAs involves sampling a physiological diverse environment, we may be quantifying miRNAs derived not only from cancer but also from blood or other organs. In addition, miRNAs derived not only from cancer but also from coexisting diseases may be captured; therefore, it is not always the case that miRNAs of cancer cells are captured. In other words, the possibility of observing the dynamics of miRNAs in a broad sense, including in the cancer microenvironment, must be fully understood. Nevertheless, the analysis of circulating miRNAs is highly useful as a potential candidate for blood-based biomarkers that can be tested repeatedly. Circulating miRNAs are promising diagnostic biomarkers owing to their high information content and disease-specific regulation. miRNAs inhibit the translation of various proteins involved in disease-related signaling pathways, including malignant and benign diseases, and alterations in physiological states, thereby inhibiting specific signaling pathways. Therefore, the analysis of the complex profiling of multiple miRNAs involved in complex pathways is expected to provide more specific diagnostic algorithms.

Biomarker development involves several important issues. The use of various analytical technology platforms, serum or plasma samples, extraction of miRNAs from whole serum/plasma or exosomes/microvesicles separated by specific disease-related markers, and sample collection, storage, and processing, all need to be addressed. Depending on the time point of blood sample collection (presurgery, during surgery,

and posttreatment) and patient treatment (surgery, chemotherapy, immunotherapy, and/or drug therapy), the expression profile of circulating miRNAs may change. It is also important to establish endogenous miRNA control and assess its consistency with the patient's clinicopathological data to assess prognosis.

#### **6. Conclusions**

miRNA expression profiling for cancer diagnosis can be subdivided into miRNA expression patterns based on signaling common to cancer, such as cell proliferation, invasion, migration, and antiapoptotic effects, and miRNA expression patterns according to various organ or tumor differentiation levels and tissue types. In addition, the expression patterns of miRNAs in cancers of various organs are different according to cancer differentiation levels and tissue types. In addition, the degree of differentiation and tissue classification are often heterogeneous among cancers of various organs, and the estimation of miRNAs based on the differentiation of each organ or developmental stage and the miRNAs that may be targeted may be important determinants for organ identification in so-called primary unknown cancers. A vast number of miRNA expression analyses in various cancers are expected to be reported in the near future. As more cancer-specific miRNAs become known, they will be systematically classified and miRNA expression profiling consistent with conventional immunohistochemical staining of cancer tissues is expected to have practical application.

#### **Acknowledgements**

We thank Aya Sugimoto for her excellent research skills and support of our work.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Tomomi Fujii\*, Tomoko Uchiyama and Maiko Takeda Department of Diagnostic Pathology, Nara Medical University School of Medicine, Nara, Japan

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Advantages of Noncoding RNAs in Molecular Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.105525*

#### **References**

[1] Lee RC, Feinbaum RL, Ambros V, The C. Elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993;**75**(5):843-854

[2] Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell. 1993;**75**(5):855-862

[3] Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature. 2000;**403**(6772):901-906

[4] Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;**391**(6669):806-811

[5] Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000;**101**(1):25-33

[6] Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature. 2000;**408**(6808):86-89

[7] Fan R, Xiao C, Wan X, Cha W, Miao Y, Zhou Y, et al. Small molecules with big roles in microRNA chemical biology and microRNA-targeted therapeutics. RNA Biology. 2019;**16**(6):707-718

[8] Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Research. 2006;**34**, (Database issue):D140-D144

[9] Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A. Identification of mammalian microRNA host genes and transcription units. Genome Research. 2004;**14**(10A):1902-1910

[10] Agueli C, Cammarata G, Salemi D, Dagnino L, Nicoletti R, La Rosa M, et al. 14q32/miRNA clusters loss of heterozygosity in acute lymphoblastic leukemia is associated with up-regulation of BCL11a. American Journal of Hematology. 2010;**85**(8):575-578

[11] Bousquet M, Quelen C, Rosati R, Mansat-De Mas V, La Starza R, Bastard C, et al. Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation. The Journal of Experimental Medicine. 2008;**205**(11):2499-2506

[12] Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**(24):15524-15529

[13] Huse JT, Brennan C, Hambardzumyan D, Wee B, Pena J, Rouhanifard SH, et al. The PTENregulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo. Genes & Development. 2009;**23**(11):1327-1337

[14] Li W, Duan R, Kooy F, Sherman SL, Zhou W, Jin P. Germline mutation of microRNA-125a is associated with breast cancer. Journal of Medical Genetics. 2009;**46**(5):358-360

[15] Lin M, Gu J, Eng C, Ellis LM, Hildebrandt MA, Lin J, et al. Genetic polymorphisms in MicroRNArelated genes as predictors of clinical outcomes in colorectal adenocarcinoma patients. Clinical Cancer Research. 2012;**18**(14):3982-3991

[16] Abelson JF, Kwan KY, O'Roak BJ, Baek DY, Stillman AA, Morgan TM, et al. Sequence variants in SLITRK1 are associated with Tourette's syndrome. Science. 2005;**310**(5746):317-320

[17] Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, Wojcik SE, et al. A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. The New England Journal of Medicine. 2005;**353**(17):1793-1801

[18] Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**(9):2999-3004

[19] Bernstein E, Kim SY, Carmell MA, Murchison EP, Alcorn H, Li MZ, et al. Dicer is essential for mouse development. Nature Genetics. 2003;**35**(3):215-217

[20] Kao SH, Cheng WC, Wang YT, Wu HT, Yeh HY, Chen YJ, et al. Regulation of miRNA biogenesis and histone modification by K63- Polyubiquitinated DDX17 controls Cancer stem-like features. Cancer Research. 2019;**79**(10):2549-2563

[21] Morita S, Horii T, Kimura M, Goto Y, Ochiya T, Hatada I. One Argonaute family member, Eif2c2 (Ago2), is essential for development and appears not to be involved in DNA methylation. Genomics. 2007;**89**(6):687-696

[22] Wang Y, Medvid R, Melton C, Jaenisch R, Blelloch R. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nature Genetics. 2007;**39**(3):380-385

[23] Chapnik E, Sasson V, Blelloch R, Hornstein E. Dgcr8 controls neural crest cells survival in cardiovascular development. Developmental Biology. 2012;**362**(1):50-56

[24] Chen Z, Wu J, Yang C, Fan P, Balazs L, Jiao Y, et al. DiGeorge syndrome critical region 8 (DGCR8) proteinmediated microRNA biogenesis is essential for vascular smooth muscle cell development in mice. The Journal of Biological Chemistry. 2012;**287**(23):19018-19028

[25] da Costa Martins PA, Bourajjaj M, Gladka M, Kortland M, van Oort RJ, Pinto YM, et al. Conditional dicer gene deletion in the postnatal myocardium provokes spontaneous cardiac remodeling. Circulation. 2008;**118**(15):1567-1576

[26] Gonzalez G, Behringer RR. Dicer is required for female reproductive tract development and fertility in the mouse. Molecular Reproduction and Development. 2009;**76**(7):678-688

[27] Harris KS, Zhang Z, McManus MT, Harfe BD, Sun X. Dicer function is essential for lung epithelium morphogenesis. Proceedings of the National Academy of Sciences of the United States of America. 2006;**103**(7):2208-2213

[28] O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT.

*Advantages of Noncoding RNAs in Molecular Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.105525*

c-Myc-regulated microRNAs modulate E2F1 expression. Nature. 2005; **435**(7043):839-843

[29] Shin D, Shin JY, McManus MT, Ptacek LJ, Fu YH. Dicer ablation in oligodendrocytes provokes neuronal impairment in mice. Annals of Neurology. 2009;**66**(6):843-857

[30] Tao J, Wu H, Lin Q, Wei W, Lu XH, Cantle JP, et al. Deletion of astroglial dicer causes non-cell-autonomous neuronal dysfunction and degeneration. The Journal of Neuroscience. 2011;**31**(22):8306-8319

[31] Kumar MS, Lu J, Mercer KL, Golub TR, Jacks T. Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nature Genetics. 2007;**39**(5):673-677

[32] Yang Z, Jin P, Xu S, Zhang T, Yang X, Li X, et al. Dicer reprograms stromal fibroblasts to a pro-inflammatory and tumor-promoting phenotype in ovarian cancer. Cancer Letters. 2018;**415**:20-29

[33] Chiosea S, Jelezcova E, Chandran U, Acquafondata M, McHale T, Sobol RW, et al. Up-regulation of dicer, a component of the MicroRNA machinery, in prostate adenocarcinoma. The American Journal of Pathology. 2006;**169**(5):1812-1820

[34] Faber C, Horst D, Hlubek F, Kirchner T. Overexpression of dicer predicts poor survival in colorectal cancer. European Journal of Cancer. 2011;**47**(9):1414-1419

[35] Guo X, Liao Q, Chen P, Li X, Xiong W, Ma J, et al. The microRNAprocessing enzymes: Drosha and dicer can predict prognosis of nasopharyngeal carcinoma. Journal of Cancer Research and Clinical Oncology. 2012;**138**(1):49-56

[36] Karube Y, Tanaka H, Osada H, Tomida S, Tatematsu Y, Yanagisawa K, et al. Reduced expression of dicer associated with poor prognosis in lung cancer patients. Cancer Science. 2005;**96**(2):111-115