the first two micrornas ( mirnas ) discovered , lin-4 and let-7 from caenorhabditis elegans , were described in 1993 and 2000 , respectively .
these two mirnas , which are both 21 nucleotides long , were found to be endogenous regulators of genes involved in developmental timing [ 1 , 2 ] . since then , hundreds of mirnas have been identified in plants , animals , human beings and viruses by molecular approaches and bioinformatics predictions ; meanwhile , mirnas have emerged as crucial players in regulating gene expression in a variety of organisms [ 35 ] .
mirnas are a broad class of small non - coding rnas usually 2125 nucleotides in length that regulate gene expression at the post - transcriptional level .
most mirnas are transcribed by rna polymerase ii from individual mirna genes , from the introns of protein coding genes , or from poly - cistronic transcripts that often encode multiple related mirnas .
these long primary transcripts , usually thousands of nucleotides , generate a stem - loop containing primary mirna ( pri - mirna ) . in animals , pri - mirnas are transcribed from the chromosome and then are cleaved into mirna precursors ( pre - mirnas ) by a multiprotein complex made up of drosha ribonuclease iii and dgcr8 , a double - stranded rna - binding protein [ 810 ] .
pre - mirnas of about 6070 nucleotides fold into an imperfect stem - loop structure and are then transported to the cytoplasm via an exportin-5 and ran - gtp - dependent mechanism .
pre - mirnas are next processed by another endonuclease rnase iii enzyme dicer into an imperfect dsrna duplex made of two complementary strands approximately 22 nucleotides in length : mirna and mirna*. these represent the mature mirna strand and its complementary strand [ 8 , 12 ] .
the stem loop in pre - mirnas contributes to the strand selection ; however , the mirna * can also be processed to its mature form and used in gene regulation .
it was originally proposed that the double stranded small mature rnas could be separated by an atp - dependent helicase and the single - stranded form of the mirna then incorporated into the risc ( rna - induced silencing complex ) [ 14 , 15 ] .
however , the current concept is that argonaute family proteins at the core of the risc receive double stranded small rnas , and the selection of the rna strand to be incorporated is controlled by the thermodynamic profile of the small rna duplex termini [ 1618 ] .
once incorporated , the mirna - programmed forms of risc ( mirisc ) participate in post - transcriptional regulation [ 19 , 20 ] . as part of the risc ,
the mature mirna guides the complex to its mrna targets , with which it interacts using complementary base - pairing . in animals , mirnas typically target sequences in the 3 untranslated regions ( 3utrs ) of mrna that are partially complementary to the mirna , leading to repression of protein synthesis . if the complementary base - pairing interaction between the mirna and target mrna 39utrs happens to be perfect or near - perfect , the target mrna can be cleaved and degraded .
more than 700 human mirnas have been discovered , and it is estimated that these mirnas regulate approximately 30% of all protein - coding genes .
mirnas have been found to participate in almost every cellular process investigated , including such diverse biological functions and processes as development , differentiation , metabolism , growth , proliferation and apoptosis ; they are currently the centre of attention in molecular and cell biology research [ 1 , 6 ] .
dysregulation of mirnas is thought to contribute to different human pathologies including cancer , heart and neurodegenerative diseases [ 7 , 2225 ] .
recently , mirna profiling studies have indicated that mirna-196 ( mir-196 ) is overexpressed in several tumour tissue samples .
in addition , increasing numbers of reports indicate that mir-196 plays important roles in development and immunity through targeting of specific genes . in this review , we discuss the known functions and molecular mechanisms of mir-196 in normal development and the pathogenesis of cancer . further investigation of these newly discovered functional roles of mir-196 may lead to potential clinical applications of mir-196 in the management of several human diseases .
the gene families for mir-10 , mir-196 and mir-615 are located in the regions of homeobox ( hox ) clusters within the genome of vertebrates .
hox genes encode homeodomain - containing transcription factors that are essential for embryonic development . in many species ,
activation and silencing of hox genes requires preservation of their native order within each cluster , and the evolutionary patterns of both mir-10 and mir-196 closely resemble that of the hox genes in vertebrates .
the mir-196a-1 gene is located on chromosome 17 ( 17q21.32 ) at a site between hoxb9 and hoxb10 genes , and the mir-196a-2 gene is located at a region between hoxc10 and hoxc9 on chromosome 12 ( 12q13.13 ) ( fig .
the gene for mir-196b is located in a highly evolutionarily conserved region between hoxa9 and hoxa10 genes , on chromosome 7 ( 7p15.2 ) in human beings and chromosome 6 ( 6qb3 ) in mice ( table 1 and fig .
1 ) . mir-196a-1 and mir-196a-2 genes transcribe the same functional mature mirna sequence ( 3-ggguuguuguacuuugauggau-5 ) , whereas mir-196b gene produces a small rna ( 3-ggguuguuguccuuugauggau-5 ) , which differs from the sequence of mir-196a by one nucleotide .
many mirnas are fairly well - conserved among vertebrates ; in fact , they are as conserved as the most conservative phylogenetic footprints .
although mirnas are very small in size , they carry a strong phylogenetic signal [ 28 , 31 ] .
information on human mir-196 genes was obtained from the ensembl genome browser ( hppt://www.ensembl.org ) .
the mir-196a-1 gene is located in the region between hoxb9 and hoxb13 on chromosome 17 , mir-196a-2 in the region between hoxc10 and hoxc9 on chromosome 12 , and mir-196b in the region between hoxa9 and hoxa10 coding on chromosome 7 .
mir-196 genes and mature sequences * the mature sequences of the hsa - mir-196a-1 , hsa - mir-196a-2 and hsa - mir-196b were acquired from the mirbase sequence database ( http://microrna.sanger.ac.uk ) .
high - mobility group a ( hmga ) proteins are able to directly bind to the mir-101b and mir-196a-2 upstream region and regulate the expression of these mirnas .
several mirnas such as mir-196a-2 , mir-101b , mir-331 and mir-29a are significantly decreased in homozygous hmga1-knockout murine embryonic fibroblasts in comparison with wild - type cells .
also , a series of reporter transgenes (  sensors ) in mouse embryos have been created for visualizing the tissue - specific expression of several mirnas during embryogenesis , including mir-10a and mir-196a , which are both encoded by genes embedded in hox clusters .
it was found that mir-196a was influenced by regulatory controls imposed on the hox clusters .
however , the hoxb9 gene , located immediately upstream of mir-196a-1 , actually has more restricted expression in the head and anterior truck of embryos than mir-196 ; this suggests that mir-196 family members are not regulated simply by control elements shared with the nearest hox genes .
furthermore , mir-196a negatively regulates target gene hoxb8 , indicating that its restricted expression pattern probably reflects a role in the hox complex expression and function .
identification of putative mrna targets is important in order to understand the specific functions of mirnas .
however , this can be very challenging because mirnas are usually imperfectly complementary to the 3utr region of their targets mrnas . in animals ,
the most consistent requirement for mirna and target mrna interaction is a contiguous and perfect base pairing of the 5mirna nucleotides 2 to 8 , called the  seed region . in many cases
, the seed region seems to determine which targets the mirna will recognize . in other cases ,
for example , a specific mrna 3utr sequence can have a reasonable complementarity to the 3 half of a mirna which allows the interaction to be stabilized , while mismatches are present in the central region of the mirna - mrna .
however , it is possible that a single mrna may be regulated by multiple mirnas , while a single mirna may target multiple transcripts within a cell type ; this amplifies the scope of putative mirna regulation of gene expression . thus ,
the particular cellular environment of a given mirna will determine its functions in a specific cell type .
a given mirna such as mir-196 may regulate different genes in different types of cells or under different conditions .
mammals have four hox clusters ( hox a to d ) containing a total of 39 genes organized into 13 paralogous subgroups . with the exception of a single g : u mismatch , pairing between mir-196a and the human hoxb8 39utr is perfect ( fig .
this conserved , near - perfect pairing suggests that hoxb8 mrna is targeted by mir-196a for cleavage .
indeed , mir-196 is known to direct the cleavage of hoxb8 mrna in mouse embryos and also regulates the expression of hoxc8 , hoxd8 and hoxa7 by transcriptional inhibition ; however , the mechanisms are not fully understood [ 28 , 36 ] .
the individual target sites of the hoxb8 mrna 3utr are conserved across several vertebrate species ( fig .
the short genomic distances between mir-196 and mir-10 and their targets are remarkable ; the target genes are located in close proximity to these mirnas [ 26 , 37 ] .
mir-10c is encoded 25 kb from its target sites in hoxb3a and 48 kb from those in hoxb1a , and a mir-196 paralogue is located only 18 kb from hoxb8 and hoxc8 and 14 kb from hoxa7 . in this case , the mirna may act in an auto - regulatory fashion on parts of its original precursor .
kawasaki and colleagues showed that mir-196 inhibits hoxb8 expression within myeloid differentiation of hl60 cells .
transfection with the vector - expressed mir-196 repressed the expression of hoxb8 by cleaving the mrnas .
thus , the mirnas appear to fine - tune the expression of the hox genes by regulating the hox genes themselves and also by regulating the downstream targets of hox genes . predicted mir-196 target recognition sites in the hoxb8 3utr in human beings and other species .
the predicted mir-196 target recognition sites in human beings and other species were obtained from the targetscanhuman 5.1 program ( http://www.targetscan.org ) .
hmga2 gene product was identified as a putative target of mir-196a-2 , suggesting that hmga1 can down - regulate the expression of hmga family members through mir-196a because hmga1 could up - regulate mir-196a-2 expression .
two potential mir-196a target recognition sites are present at the hmga2 mrna 3utr , and the nucleotide sequence at these sites are conserved in several species ( fig .
3 ) . predicted mir-196 target recognition sites in the hmga2 3utr in human beings and other species .
annexin a1 , also known as lipocortin or p35 , is a well - characterized member of the calcium- and phospholipid - binding protein family of annexins .
a potential mir-196a target recognition site is present at the annexin a1 mrna 3utr , and the conserved sequence is observed in three species ( fig .
4 ) . however , the homology of mir-196 recognition sites on annexin a1 among species is much lower than that of hoxb8 and hmga2 , described above .
it is not clear why this mir-196 target recognition sequence is not conserved in more species .
also , further investigation will be needed to confirm the specific interactions between mir-196 and annexin a1 in different types of cells and under different conditions . predicted mir-196 target recognition sites in the annexin a1 3utr in human beings and other species .
several other genes have been predicted to be target molecules of mir-196 , including s100 calcium - binding protein a9 , small proline - rich protein 2c , keratin 5 , clca family member 2 ( a chloride channel regulator ) , cytochrome p450 ( family 4 , subfamily b , polypeptide 1 ) , keratin 4 , ldoc1 , leukotriene a4 hydrolase , pleiotrophin , t - cell differentiation protein , tumour protein d52-like 1 , visinin - like 1 and v - ets erythroblastosis virus e26 oncogene homologue ( erg ) .
however , most of these target molecules of mir-196 have not been confirmed with experimental approaches .
experimentally verified target genes regulated by mir-196 mir-196 appears to play an important role in development .
its relationship to the hox gene family , crucial for embryonic development , is well - described . in drosophila
, there is a conserved or possibly convergent interaction between the mir-196 homologue iab-4 and the homeotic ubx hox gene .
analysed the iab-4 locus , which produces the mir - iab-45p and mir - iab-43p and found that mir - iab-45p could directly inhibit ubx activity in vivo .
iab-45p attenuated endogenous ubx protein accumulation and induced a classical homeotic mutant phenotype : halteres transformed into wings . in chicken , the interaction between mir-196 and hoxb8 has been implicated in the mechanism that abolishes the competence of mesoderm to undergo limb induction by retinoic acid .
mir-196 may act at the upstream of hoxb8 and sonic hedgehog ( shh ) in vivo in the context of limb development , specifically in the hindlimb .
endogenous mir-196a is expressed at the posterior trunk during xenopus development and its expression level increases in later stages .
overexpression of mir-196a by microinjection of synthetic mammalian mir-196a precursor into xenopus embryo during early development led to dose - dependent eye anomalies and changes in the expression of several critical genes .
hmga are nuclear architectural factors that play critical roles in a wide range of biological processes .
two variants of hmga proteins , hmga1 and hmga2 , have been identified , and both proteins are encoded by two distinct genes and have three dna binding motifs called at - hooks .
both hmga genes are highly expressed during embryogenesis , and there is evidence that the absence of hmga 2 protein causes growth retardation in mice [ 46 , 47 ] .
it is interesting that hmga1 is able to regulate mir-196 expression , whereas mir-19a can control hmga2 mrna translation , which may affect development .
defined the expression pattern of mirna precursors in pancreatic cancer and compared it with those of normal pancreas and chronic pancreatitis using microarray analysis ; by looking also at the pattern of mirna expression with respect to long - term ( 24 months ) survival , it was found that mirna-196a levels are inversely correlated with survival in pancreatic adenocarcinoma patients .
it has been reported that an analysis of the expression of mir-196a along with mir-217 can be used to classify benign and malignant pancreatic tissues in pancreatic fine - needle aspirates .
also , the plasma levels of mir-196a along with other mirnas including mir-21 , mir-210 and mir-155 have been found to be significantly higher in patients with pancreatic adenocarcinoma than healthy controls .
thus , mir-196a may represent an important factor in the pathogenesis of pancreatic cancer and may prove useful in the diagnosis , prognosis and/or treatment of this devastating disease .
likewise , we recently reported that the expression of mir-196a was significantly up - regulated by real time pcr analysis in the majority of pancreatic cancer tissues ( 70% ) and cell lines ( 100% ) compared normal pancreatic tissues and cells .
performed mirna expression analysis by using taqman low density arrays with quantitative real - time pcr confirmation in 40 archival formalin - fixed paraffin - embedded breast lumpectomy specimens , and found that a panel of mirnas was consistently dysregulated in breast cancer compared with normal breast samples ; this included up - regulation of previously reported breast cancer - related mirnas such as mir-21 , mir-155 , mir-191 and mir-196a as well as down - regulation of mir-125b and mir-221 .
the quality of these data validate conducting global mirnas profiling using ffpe samples , thereby offering enormous opportunities to evaluate such materials linked to clinical databases in order to rapidly acquire insight into the pathogenic roles of mirnas . in leukaemia
, levels of mir-10a , mir-10b and mir-196a-1 showed a clear correlation with hox gene expression in 30 cases of primary adult acute myeloid leukaemia ( aml ) with normal karyotype , according to quantitative real - time pcr assay .
in addition to the hox genes , nearly 30% of the genes with a high correlation with mir-10a , mir-10b and mir-196a-1 have been shown to have oncogenic potential [ 5355 ] . by quantifying expression of 19 selected mirna genes , schotte et al . found that the expression of mir-196b was 500-fold higher in myeloid / lymphoid or mixed lineage leukaemia ( mll)-rearranged cases and 800-fold higher in 5/15 t - lineage acute lymphoblastic leukaemia ( t - all ) cases compared with expression levels in precursor b cell acute lymphoblastic leukaemia ( b - all ) cases .
these data indicate that the expression profiles of these mirna are all subtype - specific rather than linked to the differentiation status associated with these subtypes .
showed that the mll gene normally regulates expression of mir-196b in a pattern similar to that of the surrounding 5 hox genes , hoxa9 and hoxa10 , during stem cell differentiation .
again , overexpression of mir-196b was found specifically in patients with mll - associated leukaemia based on analysis of 55 primary leukaemia samples ; these demonstrated increased proliferative capacity and survival , as well as a partial block in differentiation in bone marrow progenitor cells .
meanwhile , leukemogenic mll fusion proteins cause over expression of mir-196b , which may be necessary for mll fusion - mediated immortalization .
a recent report demonstrated that mir-196b expression is significantly more up - regulated in aml than all and that , again , mir-196b up - regulation is negatively associated with overall survival of aml patients .
in addition to the mir-196b deregulation observed in leukaemia , mir-196b has been reported to induce changes in myeloid development , and the co - expression of mir-196b and mir-21 reportedly block granulocyte colony - stimulating factor - induced granulopoiesis completely .
highly elevated levels of mir-196a have also been detected in oesophageal adenocarcinoma , based on analysis of microdissected paraffin - embedded tissues from 11 patients .
this study found that mir-196a may have growth - promoting and anti - apoptotic functions ; further , it may be considered a marker of progression from barrett s oesophagus to low - grade dysplasia , high - grade dysplasia and oesophageal adenocarcinoma .
mir-196a levels were inversely correlated with the predicted target keratin 5 , small proline - rich protein 2c and s100 calcium - binding protein a9 mrna levels in oesophageal adenocarcinoma .
the expression of hmga proteins is usually low or absent in most of normal adult tissues ; however , overexpression of hmga proteins has been observed in several human malignancies and has been found to correlate with occurrence of metastasis and poor prognosis .
moreover , transgenic mice overexpressing either hmga1 or hmga2 develop lymphoma and pituitary adenomas , which strongly suggest oncogenic property of these proteins [ 5962 ] .
it could promote tumour progression if hmga1 dominantly up - regulates mir-196 expression ; however , if mir-196 significantly inhibits hmga2 protein synthesis , it could inhibit tumour progression . thus , ultimate impact of mir-196 on the tumour pathogenesis will be dependent on the balance of regulation of mir-196 expression and mir-196 targeting molecules ( oncogenes or tumour suppression genes ) in specific cells
. these regulation mechanisms of mir-196 expression and functions are largely unknown and warrant further investigation . increased levels of mir-196a have also been found in 12 cancer cell lines of oesophageal , breast and endometrial origin and 10 oesophageal adenocarcinomas ; furthermore , levels of mir-196a have been demonstrated to have a significant inverse correlation with annexin a1 mrna levels in these cancers .
annexin a1 has been postulated to act as tumour suppressor in different cancers such as prostate , breast , oesophageal and b - cell non - hodgkin s lymphoma [ 6367 ] . as annexin a1
could be a target molecule of mir-196a , increased mir-196a may mediate down - regulation of annexin a1 , contributing to tumorigenesis .
however , overexpression of annexin a1 has been reported in pancreatic , hepatic and stomach cancers , suggesting that the functional role of annexin a1 may be tissue- and cell type - specific [ 6870 ] . of note
, the majority of pancreatic cancer tissues and cell lines demonstrate a high expression level of mir-196a . the relationship and functional interactions of mir-196a and annexin a1 warrant further investigation . mir-196a
the mir-196a expression profile in colorectal cancer , mucosal samples and diverse cancer cell lines has been quantified by rt - pcr , and mir-196b has been found to be up - regulated in colonic cancer .
high levels of mir-196a were observed to activate the akt signalling pathway ; promote cancer cell detachment , migration , invasion and chemosensitivity ; and increase the development of lung metastases in mice after tail vein injection [ 71 , 72 ] .
although most studies on mir-196 suggest its oncogenic function in cancer , it is important to point out that mir-196 may play a tumour suppressive role as well .
for example , strongly reduced expression of mir-196a was observed in melanoma cells when compared to healthy control melanocytes , and the low mir-196a expression in melanoma cells led to high hoxb7 gene expression , which subsequently raised ets-1 and bmp4 activity and thereby enhanced migratory potential of these melanoma cells .
moreover , a recent report by li et al . showed that enforced expression of mir-196a or mir-196b reduced in vitro invasion and in vivo spontaneous metastasis of breast cancer cells , indicating that the members of mir-196 family are potent metastasis suppressors .
if mir-196 has a dominant effect on the inhibition of oncogenic molecules , mir-196 will play a tumour suppressor function ; although if mir-196 mainly targets tumour suppressors , mir-196 will demonstrate oncogenic effects .
mirnas are likely important players in the pathogenesis of cancer , and dysregulation of mirna expression in cancer is an important field of investigation .
both genetic and epigenetic alterations may affect mirna biogenesis and activity [ 75 , 76 ] .
for example , one single - nucleotide polymorphism ( snp ) of mir-196a-2 ( rs11614913 ) has been associated with survival in chinese individuals with lung cancer ; those patients with the cc genotype had reduced survival .
this particular snp affects the binding efficiency of mir-196a-2 to its target mrna [ 77 , 78 ] .
the presence of the cc genotype also correlated with increased susceptibility to lung cancer in the same patient population . consistently
, this variant is also associated with increased gastric cancer risk in chinese patients . in two separate studies , the tt genotype of this snp was found to be associated with decreased risk of breast cancer in a chinese population , whereas the cc genotype again was associated with an increased risk of breast cancer in chinese patients [ 80 , 81 ] . meanwhile ,
the opposite relationship has been described in glioma , where the cc genotype was associated with a decreased risk .
this discrepancy highlights the need for further investigation of this snp in different tissues , tumours and patient populations . of note ,
the major allele in the caucasian population is the c allele , whereas in the chinese population , the major allele is t. so , interestingly , the 
variant homozygote cc genotype as described in chinese population studies is actually the dominant genotype in caucasians ; this exemplifies the limitations of attributing the terms  wild - type and  variant to the alleles when describing snps .
endometriosis is a gynaecological disorder in females in which endometrial - like cells grow in areas outside the uterine cavity .
a mirna expression study identified mir-196b as one of 8 down - regulated mirna in ectopic endometrial tissues as compared to paired eutopic tissues .
mir-196 may also play a role in the pathogenesis of severe congenital neutropenia ( scn ) , a rare haematological disorder characterized by an abnormally low number of neutrophils .
mutations in several genes , including growth factor independent protein 1 ( gfi1 ) , have been reported to be responsible for scn .
gfi1 is a transcriptional repressor that is required for normal myelopoiesis , and gfi1 loss - of - function mutations are found in some patients with scn .
it has been shown that mir-196b is negatively regulated by gfi1 ; its expression is up - regulated in gfi1 mice and gfi1n382s scn patients .
thus , up - regulation of mir-196 may mediate some of the effects of this disease .
indeed , cd56 t cells may be able to up - regulate the expression of mir-196a , which shows anti - hepatitis c virus ( hcv ) properties [ 84 , 85 ] .
furthermore , antiviral cytokine interferon  can induce numerous cellular mirnas , including mir-196 , which have sequence - predicted targets within hcv genomic rna .
overexpression of these mirnas in infected liver cells leads to a marked attenuation of viral replication , demonstrating the importance of mirnas in anti - viral immunity .
since viruses also play a role in the pathogenesis of several tumour types , mirnas may mediate viral  host interactions that have important implications not only for infectious diseases but also for cancer pathogenesis .
a recent study revealed that mir-196a plays an important role in mesenchymal stem cell differentiation . specifically , overexpression of mir-196a inhibited the proliferation of human adipose tissue - derived mesenchymal stem cells while enhancing osteogenic differentiation .
mir-196a , along with three other mirnas , displayed an increased expression in red blood cells up to day 20 after blood collection , after which expression subsequently decreased .
thus , mirnas may be potentially useful markers to predict storage - related lesions during blood storage .
although the mature nucleotide sequence is identical for mir-196a-1 and mir-196a-2 , mature mir-196b differs from mir-196a by one nucleotide .
the expression levels of mir-196 appear to be strictly controlled under physiologic conditions , whereas dysregulation of mir-196 can be observed in many disease conditions .
mir-196 may play critical roles in normal development and cancer pathogenesis by targeting several regulation molecules including hoxb8 , hmga2 and annexin a1 .
meanwhile , it may also play roles in viral immunity and the cell differentiation processes that , when dysregulated , lead to endometriosis and scn
. however , the detailed functions and molecular mechanisms whereby mir-196 contributes to the pathophysiology of these diseases remain largely unknown . for example , it is not completely understood how mir-196a is regulated ; how mir-196a controls cell proliferation or differentiation ; and how mir-196a differentially recognizes its target molecules in a cell- or tissue - specific fashion . addressing these critical questions will help to establish potential clinical applications of mir-196-directed technology in the diagnosis , treatment , and/or prognosis of development defects , viral infections and different types of human cancers .
the gene families for mir-10 , mir-196 and mir-615 are located in the regions of homeobox ( hox ) clusters within the genome of vertebrates .
hox genes encode homeodomain - containing transcription factors that are essential for embryonic development . in many species ,
they are organized in tightly linked clusters along the chromosome . activation and silencing of hox genes
requires preservation of their native order within each cluster , and the evolutionary patterns of both mir-10 and mir-196 closely resemble that of the hox genes in vertebrates .
the mir-196a-1 gene is located on chromosome 17 ( 17q21.32 ) at a site between hoxb9 and hoxb10 genes , and the mir-196a-2 gene is located at a region between hoxc10 and hoxc9 on chromosome 12 ( 12q13.13 ) ( fig .
the gene for mir-196b is located in a highly evolutionarily conserved region between hoxa9 and hoxa10 genes , on chromosome 7 ( 7p15.2 ) in human beings and chromosome 6 ( 6qb3 ) in mice ( table 1 and fig .
1 ) . mir-196a-1 and mir-196a-2 genes transcribe the same functional mature mirna sequence ( 3-ggguuguuguacuuugauggau-5 ) , whereas mir-196b gene produces a small rna ( 3-ggguuguuguccuuugauggau-5 ) , which differs from the sequence of mir-196a by one nucleotide .
many mirnas are fairly well - conserved among vertebrates ; in fact , they are as conserved as the most conservative phylogenetic footprints .
although mirnas are very small in size , they carry a strong phylogenetic signal [ 28 , 31 ] .
information on human mir-196 genes was obtained from the ensembl genome browser ( hppt://www.ensembl.org ) .
the mir-196a-1 gene is located in the region between hoxb9 and hoxb13 on chromosome 17 , mir-196a-2 in the region between hoxc10 and hoxc9 on chromosome 12 , and mir-196b in the region between hoxa9 and hoxa10 coding on chromosome 7 .
mir-196 genes and mature sequences * the mature sequences of the hsa - mir-196a-1 , hsa - mir-196a-2 and hsa - mir-196b were acquired from the mirbase sequence database ( http://microrna.sanger.ac.uk ) .
high - mobility group a ( hmga ) proteins are able to directly bind to the mir-101b and mir-196a-2 upstream region and regulate the expression of these mirnas .
several mirnas such as mir-196a-2 , mir-101b , mir-331 and mir-29a are significantly decreased in homozygous hmga1-knockout murine embryonic fibroblasts in comparison with wild - type cells .
also , a series of reporter transgenes (  sensors ) in mouse embryos have been created for visualizing the tissue - specific expression of several mirnas during embryogenesis , including mir-10a and mir-196a , which are both encoded by genes embedded in hox clusters .
it was found that mir-196a was influenced by regulatory controls imposed on the hox clusters .
however , the hoxb9 gene , located immediately upstream of mir-196a-1 , actually has more restricted expression in the head and anterior truck of embryos than mir-196 ; this suggests that mir-196 family members are not regulated simply by control elements shared with the nearest hox genes .
furthermore , mir-196a negatively regulates target gene hoxb8 , indicating that its restricted expression pattern probably reflects a role in the hox complex expression and function .
identification of putative mrna targets is important in order to understand the specific functions of mirnas .
however , this can be very challenging because mirnas are usually imperfectly complementary to the 3utr region of their targets mrnas . in animals ,
the most consistent requirement for mirna and target mrna interaction is a contiguous and perfect base pairing of the 5mirna nucleotides 2 to 8 , called the  seed region . in many cases
, the seed region seems to determine which targets the mirna will recognize . in other cases ,
for example , a specific mrna 3utr sequence can have a reasonable complementarity to the 3 half of a mirna which allows the interaction to be stabilized , while mismatches are present in the central region of the mirna - mrna .
however , it is possible that a single mrna may be regulated by multiple mirnas , while a single mirna may target multiple transcripts within a cell type ; this amplifies the scope of putative mirna regulation of gene expression .
thus , the particular cellular environment of a given mirna will determine its functions in a specific cell type .
a given mirna such as mir-196 may regulate different genes in different types of cells or under different conditions .
mammals have four hox clusters ( hox a to d ) containing a total of 39 genes organized into 13 paralogous subgroups . with the exception of a single g : u mismatch , pairing between mir-196a and the human hoxb8 39utr
this conserved , near - perfect pairing suggests that hoxb8 mrna is targeted by mir-196a for cleavage .
indeed , mir-196 is known to direct the cleavage of hoxb8 mrna in mouse embryos and also regulates the expression of hoxc8 , hoxd8 and hoxa7 by transcriptional inhibition ; however , the mechanisms are not fully understood [ 28 , 36 ] .
the individual target sites of the hoxb8 mrna 3utr are conserved across several vertebrate species ( fig .
the short genomic distances between mir-196 and mir-10 and their targets are remarkable ; the target genes are located in close proximity to these mirnas [ 26 , 37 ] .
mir-10c is encoded 25 kb from its target sites in hoxb3a and 48 kb from those in hoxb1a , and a mir-196 paralogue is located only 18 kb from hoxb8 and hoxc8 and 14 kb from hoxa7 . in this case , the mirna may act in an auto - regulatory fashion on parts of its original precursor .
kawasaki and colleagues showed that mir-196 inhibits hoxb8 expression within myeloid differentiation of hl60 cells .
transfection with the vector - expressed mir-196 repressed the expression of hoxb8 by cleaving the mrnas
thus , the mirnas appear to fine - tune the expression of the hox genes by regulating the hox genes themselves and also by regulating the downstream targets of hox genes . predicted mir-196 target recognition sites in the hoxb8 3utr in human beings and other species .
the predicted mir-196 target recognition sites in human beings and other species were obtained from the targetscanhuman 5.1 program ( http://www.targetscan.org ) .
hmga2 gene product was identified as a putative target of mir-196a-2 , suggesting that hmga1 can down - regulate the expression of hmga family members through mir-196a because hmga1 could up - regulate mir-196a-2 expression .
two potential mir-196a target recognition sites are present at the hmga2 mrna 3utr , and the nucleotide sequence at these sites are conserved in several species ( fig .
3 ) . predicted mir-196 target recognition sites in the hmga2 3utr in human beings and other species .
annexin a1 , also known as lipocortin or p35 , is a well - characterized member of the calcium- and phospholipid - binding protein family of annexins .
a potential mir-196a target recognition site is present at the annexin a1 mrna 3utr , and the conserved sequence is observed in three species ( fig .
4 ) . however , the homology of mir-196 recognition sites on annexin a1 among species is much lower than that of hoxb8 and hmga2 , described above .
it is not clear why this mir-196 target recognition sequence is not conserved in more species .
also , further investigation will be needed to confirm the specific interactions between mir-196 and annexin a1 in different types of cells and under different conditions . predicted mir-196 target recognition sites in the annexin a1 3utr in human beings and other species .
several other genes have been predicted to be target molecules of mir-196 , including s100 calcium - binding protein a9 , small proline - rich protein 2c , keratin 5 , clca family member 2 ( a chloride channel regulator ) , cytochrome p450 ( family 4 , subfamily b , polypeptide 1 ) , keratin 4 , ldoc1 , leukotriene a4 hydrolase , pleiotrophin , t - cell differentiation protein , tumour protein d52-like 1 , visinin - like 1 and v - ets erythroblastosis virus e26 oncogene homologue ( erg ) .
however , most of these target molecules of mir-196 have not been confirmed with experimental approaches .
its relationship to the hox gene family , crucial for embryonic development , is well - described . in drosophila ,
there is a conserved or possibly convergent interaction between the mir-196 homologue iab-4 and the homeotic ubx hox gene .
analysed the iab-4 locus , which produces the mir - iab-45p and mir - iab-43p and found that mir - iab-45p could directly inhibit ubx activity in vivo .
iab-45p attenuated endogenous ubx protein accumulation and induced a classical homeotic mutant phenotype : halteres transformed into wings . in chicken , the interaction between mir-196 and hoxb8 has been implicated in the mechanism that abolishes the competence of mesoderm to undergo limb induction by retinoic acid .
mir-196 may act at the upstream of hoxb8 and sonic hedgehog ( shh ) in vivo in the context of limb development , specifically in the hindlimb .
endogenous mir-196a is expressed at the posterior trunk during xenopus development and its expression level increases in later stages .
overexpression of mir-196a by microinjection of synthetic mammalian mir-196a precursor into xenopus embryo during early development led to dose - dependent eye anomalies and changes in the expression of several critical genes .
hmga are nuclear architectural factors that play critical roles in a wide range of biological processes .
two variants of hmga proteins , hmga1 and hmga2 , have been identified , and both proteins are encoded by two distinct genes and have three dna binding motifs called at - hooks .
both hmga genes are highly expressed during embryogenesis , and there is evidence that the absence of hmga 2 protein causes growth retardation in mice [ 46 , 47 ] .
it is interesting that hmga1 is able to regulate mir-196 expression , whereas mir-19a can control hmga2 mrna translation , which may affect development .
defined the expression pattern of mirna precursors in pancreatic cancer and compared it with those of normal pancreas and chronic pancreatitis using microarray analysis ; by looking also at the pattern of mirna expression with respect to long - term ( 24 months ) survival , it was found that mirna-196a levels are inversely correlated with survival in pancreatic adenocarcinoma patients .
it has been reported that an analysis of the expression of mir-196a along with mir-217 can be used to classify benign and malignant pancreatic tissues in pancreatic fine - needle aspirates .
also , the plasma levels of mir-196a along with other mirnas including mir-21 , mir-210 and mir-155 have been found to be significantly higher in patients with pancreatic adenocarcinoma than healthy controls .
thus , mir-196a may represent an important factor in the pathogenesis of pancreatic cancer and may prove useful in the diagnosis , prognosis and/or treatment of this devastating disease .
likewise , we recently reported that the expression of mir-196a was significantly up - regulated by real time pcr analysis in the majority of pancreatic cancer tissues ( 70% ) and cell lines ( 100% ) compared normal pancreatic tissues and cells .
hui et al . performed mirna expression analysis by using taqman low density arrays with quantitative real - time pcr confirmation in 40 archival formalin - fixed paraffin - embedded breast lumpectomy specimens , and found that a panel of mirnas was consistently dysregulated in breast cancer compared with normal breast samples ; this included up - regulation of previously reported breast cancer - related mirnas such as mir-21 , mir-155 , mir-191 and mir-196a as well as down - regulation of mir-125b and mir-221 .
the quality of these data validate conducting global mirnas profiling using ffpe samples , thereby offering enormous opportunities to evaluate such materials linked to clinical databases in order to rapidly acquire insight into the pathogenic roles of mirnas . in leukaemia
, levels of mir-10a , mir-10b and mir-196a-1 showed a clear correlation with hox gene expression in 30 cases of primary adult acute myeloid leukaemia ( aml ) with normal karyotype , according to quantitative real - time pcr assay .
in addition to the hox genes , nearly 30% of the genes with a high correlation with mir-10a , mir-10b and mir-196a-1 have been shown to have oncogenic potential [ 5355 ] . by quantifying expression of 19 selected mirna genes , schotte et al . found that the expression of mir-196b was 500-fold higher in myeloid / lymphoid or mixed lineage leukaemia ( mll)-rearranged cases and 800-fold higher in 5/15 t - lineage acute lymphoblastic leukaemia ( t - all ) cases compared with expression levels in precursor b cell acute lymphoblastic leukaemia ( b - all ) cases .
these data indicate that the expression profiles of these mirna are all subtype - specific rather than linked to the differentiation status associated with these subtypes .
. showed that the mll gene normally regulates expression of mir-196b in a pattern similar to that of the surrounding 5 hox genes , hoxa9 and hoxa10 , during stem cell differentiation .
again , overexpression of mir-196b was found specifically in patients with mll - associated leukaemia based on analysis of 55 primary leukaemia samples ; these demonstrated increased proliferative capacity and survival , as well as a partial block in differentiation in bone marrow progenitor cells .
meanwhile , leukemogenic mll fusion proteins cause over expression of mir-196b , which may be necessary for mll fusion - mediated immortalization .
a recent report demonstrated that mir-196b expression is significantly more up - regulated in aml than all and that , again , mir-196b up - regulation is negatively associated with overall survival of aml patients .
in addition to the mir-196b deregulation observed in leukaemia , mir-196b has been reported to induce changes in myeloid development , and the co - expression of mir-196b and mir-21 reportedly block granulocyte colony - stimulating factor - induced granulopoiesis completely .
highly elevated levels of mir-196a have also been detected in oesophageal adenocarcinoma , based on analysis of microdissected paraffin - embedded tissues from 11 patients .
this study found that mir-196a may have growth - promoting and anti - apoptotic functions ; further , it may be considered a marker of progression from barrett s oesophagus to low - grade dysplasia , high - grade dysplasia and oesophageal adenocarcinoma .
mir-196a levels were inversely correlated with the predicted target keratin 5 , small proline - rich protein 2c and s100 calcium - binding protein a9 mrna levels in oesophageal adenocarcinoma .
the expression of hmga proteins is usually low or absent in most of normal adult tissues ; however , overexpression of hmga proteins has been observed in several human malignancies and has been found to correlate with occurrence of metastasis and poor prognosis .
moreover , transgenic mice overexpressing either hmga1 or hmga2 develop lymphoma and pituitary adenomas , which strongly suggest oncogenic property of these proteins [ 5962 ] .
it could promote tumour progression if hmga1 dominantly up - regulates mir-196 expression ; however , if mir-196 significantly inhibits hmga2 protein synthesis , it could inhibit tumour progression .
thus , ultimate impact of mir-196 on the tumour pathogenesis will be dependent on the balance of regulation of mir-196 expression and mir-196 targeting molecules ( oncogenes or tumour suppression genes ) in specific cells .
. increased levels of mir-196a have also been found in 12 cancer cell lines of oesophageal , breast and endometrial origin and 10 oesophageal adenocarcinomas ; furthermore , levels of mir-196a have been demonstrated to have a significant inverse correlation with annexin a1 mrna levels in these cancers .
annexin a1 has been postulated to act as tumour suppressor in different cancers such as prostate , breast , oesophageal and b - cell non - hodgkin s lymphoma [ 6367 ] . as annexin a1
could be a target molecule of mir-196a , increased mir-196a may mediate down - regulation of annexin a1 , contributing to tumorigenesis .
however , overexpression of annexin a1 has been reported in pancreatic , hepatic and stomach cancers , suggesting that the functional role of annexin a1 may be tissue- and cell type - specific [ 6870 ] .
of note , the majority of pancreatic cancer tissues and cell lines demonstrate a high expression level of mir-196a .
mir-196a may also have pro - oncogenic functions in colorectal cancer . the mir-196a expression profile in colorectal cancer , mucosal samples and diverse cancer cell lines has been quantified by rt - pcr , and mir-196b has been found to be up - regulated in colonic cancer .
high levels of mir-196a were observed to activate the akt signalling pathway ; promote cancer cell detachment , migration , invasion and chemosensitivity ; and increase the development of lung metastases in mice after tail vein injection [ 71 , 72 ] .
although most studies on mir-196 suggest its oncogenic function in cancer , it is important to point out that mir-196 may play a tumour suppressive role as well .
for example , strongly reduced expression of mir-196a was observed in melanoma cells when compared to healthy control melanocytes , and the low mir-196a expression in melanoma cells led to high hoxb7 gene expression , which subsequently raised ets-1 and bmp4 activity and thereby enhanced migratory potential of these melanoma cells .
moreover , a recent report by li et al . showed that enforced expression of mir-196a or mir-196b reduced in vitro invasion and in vivo spontaneous metastasis of breast cancer cells , indicating that the members of mir-196 family are potent metastasis suppressors .
if mir-196 has a dominant effect on the inhibition of oncogenic molecules , mir-196 will play a tumour suppressor function ; although if mir-196 mainly targets tumour suppressors , mir-196 will demonstrate oncogenic effects .
mirnas are likely important players in the pathogenesis of cancer , and dysregulation of mirna expression in cancer is an important field of investigation .
both genetic and epigenetic alterations may affect mirna biogenesis and activity [ 75 , 76 ] .
for example , one single - nucleotide polymorphism ( snp ) of mir-196a-2 ( rs11614913 ) has been associated with survival in chinese individuals with lung cancer ; those patients with the cc genotype had reduced survival .
this particular snp affects the binding efficiency of mir-196a-2 to its target mrna [ 77 , 78 ] .
the presence of the cc genotype also correlated with increased susceptibility to lung cancer in the same patient population .
consistently , this variant is also associated with increased gastric cancer risk in chinese patients . in two separate studies , the tt genotype of this snp was found to be associated with decreased risk of breast cancer in a chinese population , whereas the cc genotype again was associated with an increased risk of breast cancer in chinese patients [ 80 , 81 ] . meanwhile ,
the opposite relationship has been described in glioma , where the cc genotype was associated with a decreased risk .
this discrepancy highlights the need for further investigation of this snp in different tissues , tumours and patient populations . of note ,
the major allele in the caucasian population is the c allele , whereas in the chinese population , the major allele is t. so , interestingly , the 
variant homozygote cc genotype as described in chinese population studies is actually the dominant genotype in caucasians ; this exemplifies the limitations of attributing the terms  wild - type and  variant to the alleles when describing snps .
endometriosis is a gynaecological disorder in females in which endometrial - like cells grow in areas outside the uterine cavity .
a mirna expression study identified mir-196b as one of 8 down - regulated mirna in ectopic endometrial tissues as compared to paired eutopic tissues .
mir-196 may also play a role in the pathogenesis of severe congenital neutropenia ( scn ) , a rare haematological disorder characterized by an abnormally low number of neutrophils .
mutations in several genes , including growth factor independent protein 1 ( gfi1 ) , have been reported to be responsible for scn .
gfi1 is a transcriptional repressor that is required for normal myelopoiesis , and gfi1 loss - of - function mutations are found in some patients with scn .
it has been shown that mir-196b is negatively regulated by gfi1 ; its expression is up - regulated in gfi1 mice and gfi1n382s scn patients .
thus , up - regulation of mir-196 may mediate some of the effects of this disease .
indeed , cd56 t cells may be able to up - regulate the expression of mir-196a , which shows anti - hepatitis c virus ( hcv ) properties [ 84 , 85 ] .
furthermore , antiviral cytokine interferon  can induce numerous cellular mirnas , including mir-196 , which have sequence - predicted targets within hcv genomic rna .
overexpression of these mirnas in infected liver cells leads to a marked attenuation of viral replication , demonstrating the importance of mirnas in anti - viral immunity .
since viruses also play a role in the pathogenesis of several tumour types , mirnas may mediate viral  host interactions that have important implications not only for infectious diseases but also for cancer pathogenesis .
a recent study revealed that mir-196a plays an important role in mesenchymal stem cell differentiation . specifically , overexpression of mir-196a inhibited the proliferation of human adipose tissue - derived mesenchymal stem cells while enhancing osteogenic differentiation .
mir-196a , along with three other mirnas , displayed an increased expression in red blood cells up to day 20 after blood collection , after which expression subsequently decreased .
thus , mirnas may be potentially useful markers to predict storage - related lesions during blood storage .
although the mature nucleotide sequence is identical for mir-196a-1 and mir-196a-2 , mature mir-196b differs from mir-196a by one nucleotide .
the expression levels of mir-196 appear to be strictly controlled under physiologic conditions , whereas dysregulation of mir-196 can be observed in many disease conditions .
mir-196 may play critical roles in normal development and cancer pathogenesis by targeting several regulation molecules including hoxb8 , hmga2 and annexin a1 .
meanwhile , it may also play roles in viral immunity and the cell differentiation processes that , when dysregulated , lead to endometriosis and scn
. however , the detailed functions and molecular mechanisms whereby mir-196 contributes to the pathophysiology of these diseases remain largely unknown . for example , it is not completely understood how mir-196a is regulated ; how mir-196a controls cell proliferation or differentiation ; and how mir-196a differentially recognizes its target molecules in a cell- or tissue - specific fashion .
addressing these critical questions will help to establish potential clinical applications of mir-196-directed technology in the diagnosis , treatment , and/or prognosis of development defects , viral infections and different types of human cancers .
 abstractthe discovery of micrornas ( mirnas ) represents one of the most significant advances in biological and medical sciences in the last decade . 
 hundreds of mirnas have been identified in plants , viruses , animals and human beings , and these tiny , non - coding rna transcripts have been found to play crucial roles in important biological processes involved in human health and disease . 
 recently , many studies have demonstrated that mir-196 plays critical roles in normal development and in the pathogenesis of human disease processes such as cancer . 
 several investigations have implemented cell culture and animal models to explore the potential molecular mechanisms of mir-196 . 
 this review provides updated information about the structure of the mir-196 gene and the roles of mir-196 in development , cancer and disease formation . 
 importantly , we discuss the possible molecular mechanisms whereby mir-196 regulates cellular functions including targeting molecules and gene regulation pathways ; potential clinical applications are addressed , as well as future directions for investigation . 
 mir-196a may prove to be a novel therapeutic target for several cancers . 
Introduction
Gene structure and regulation
Target molecules
Role in development
Role in cancer
Other biological functions
Summary and perspectives
Conflict of interest