cystic fibrosis ( cf ) is a multifaceted autosomal recessive disease caused by mutations in the cf transmembrane conductance regulator ( cftr ) gene .
although its pulmonary manifestations are responsible for the major morbidity and mortality associated with the disease , cf is also characterised by a multitude of clinical extrapulmonary manifestations .
in addition , the great heterogeneity in disease severity among people with cf means that the design of therapeutic interventions is particularly challenging .
micrornas ( mirna ) are a class of regulatory biomolecules with important functions in numerous biological processes and are aberrantly expressed in many human diseases .
therefore , it is important to elucidate the roles of these molecules in cf pathophysiology .
although the term  microrna  was first coined in 2001 , the first mirna , lin-4 , was discovered eight years earlier by lee and colleagues , in the nematode caenorhabditis elegans .
having been initially discovered to play important roles in developmental biology , interest in these small rnas has dramatically increased since this time as they have been found to have significant roles in a range of other biological processes such as proliferation and apoptosis . the latest version of mirbase ( http://www.mirbase.org/ , v21 ) , the most comprehensive microrna bioinformatics repository , contains entries from 223 species corresponding to over 35,000 micrornas .
expression levels of mirnas vary greatly between cells and tissues , and aberrant levels of mirna are associated with many diseases in humans . as a rule ,
mammalian mirnas are initially transcribed in the nucleus into longer primary mirna (  pri - mir  ) of up to 1000 nucleotides in length .
these stem - loop structured pri - mirs are generally transcribed by rna polymerase ii and subsequently undergo cleavage in two sequential steps .
the initial processing occurs in the nucleus by the rna endonuclease ( rnase ) type iii enzyme drosha with the involvement of other proteins , as part of the  microprocessor complex . 
drosha cleaves the pri - mir liberating shorter hairpin pre - mirna structures (  pre - mir  ) , which are approximately 70100 nucleotides in length , and these are actively transported into the cytoplasm via a process involving the protein exportin 5 .
once in the cytoplasm , the pre - mir is further processed by the rnase iii enzyme dicer , resulting in a mature mirna duplex with 5 phosphate and two - nucleotide 3 overhangs .
generally , micrornas regulate gene expression posttranscriptionally by binding in a sequence - specific manner to mirna responsive elements ( mres ) , particularly in the 3 untranslated region ( utr ) , of a target mrna .
they are recruited by argonaute ( ago ) proteins , particularly ago2   to form the multiprotein rna induced silencing complex ( risc ) [ 8 , 9 ] .
mirnas can guide the risc to a target mrna which then induces cleavage degradation or translational repression of that mrna [ 10 , 11 ] .
although most mirna studies have largely focused on mirna - mrna interactions in the 3utr of target mrna , these interactions can also occur in the 5utr and coding sequence ( cds ) [ 12 , 13 ] . as a single mirna
can regulate many target mrnas and each mrna may harbour several mres , validation of targets can be difficult and time consuming
however , many computational tools are currently available for predictions and these are continuously improving .
these are the quality of seed match , evolutionary conservation of a particular microrna , thermodynamics ( specifically free energy ) of mirna::mrna target binding , and site accessibility or mrna secondary structure .
various online tools aid in these predictions and some well - known examples include targetscan , pictar , diana - microt , microrna.org , rna22 , and rnahybrid , which all utilise different algorithms and different sources of mrna sequences .
yet , bioinformatic target prediction databases have high false positive and false negative rates , and experimental validation is ultimately required to truly determine mirna::target mrna binding and biological function .
it has been proposed that the expression and function of micrornas themselves are regulated at three levels : transcription , processing , and subcellular localization . at the level of transcription
, mirna expression can be controlled by many factors such as chromatin modifications , dna methylation , and activity of transcription factors to name a few .
mirna processing can be affected by intrinsic or acquired alterations in the mirna microprocessor machinery , thereby controlling mirna function .
these are termed  mirna sponges ,  given their ability to soak up mirnas and reduce their interactions with target mrnas . additionally , single nucleotide polymorphisms ( mirsnps ) that affect mirna binding and function are being increasingly reported .
analysis of multiple organs and tissues suggests that mirnas have dual roles as both regulators of development and in maintenance of homeostasis [ 9 , 16 , 17 ] .
widespread changes in mirna expression have been observed during lung development , and dicer knockout mice , who have disrupted mirna processing , display a lethal phenotype as a result of impaired lung growth .
various studies demonstrate that mirna expression remains relatively constant over time in the adult lung , supporting the notion that mirnas play a central role in maintenance of lung homeostasis in the developed lung
. however , expression of mirna is altered in pathological states , such as lung inflammation and disease .
mirnas have been shown to play important roles in the regulation of innate immunity and inflammation . at the most basic level , mirnas are important in haematopoiesis and differentiation of immune cells [ 20 , 21 ] .
numerous mirnas are induced in innate immune cells , with mirs-155 , -146 , and -21 being expressed at particularly high levels [ 22 , 23 ] . with known roles in regulation of inflammation , mirnas are increasingly being examined within the context of inflammatory lung diseases such as cf .
lining the airway epithelium in the cf lung is a depleted airway surface liquid layer ( asl ) and more mucus than normal .
impaired mucociliary clearance promotes bacterial colonisation and generates a highly proinflammatory environment wherein innate immune responses are frequently activated .
another characteristic of the cf airway lumen is the high numbers of infiltrating neutrophils which are inherently dysfunctional and contribute to the preexisting protease - antiprotease imbalance .
accumulation of misfolded cftr may contribute to endoplasmic reticulum ( er ) stress responses in the airway epithelium , and collectively these features are central to the pathology and physiology of cf lung disease .
numerous micrornas had altered expression between cf and non - cf bronchial epithelium ; the altered mirnas were predicted to regulate expression of proteins involved innate immunity , inflammation , ion conductance , and er stress , amongst others .
these cells contribute to the barrier function via three essential components : intercellular tight and adherens junctions ( regulating epithelial permeability ) , secreted antimicrobial factors , and the mucociliary escalator .
furthermore , it acts as a key mediator of both innate and adaptive immune responses toward invading pathogens .
toll - like receptors ( tlrs ) are a key group of pattern recognition receptors which mediate the recognition of and response to microbial infections and are highly expressed on myeloid cells .
the expression of tlrs is , however , not confined to immune cells , and these receptors are also expressed at high levels on other cell types , including airway epithelial cells ( aecs ) such as tracheal , bronchial , and alveolar type ii cells . in the cf lung ,
tlrs expressed by aecs contribute to the airway immune response by regulating the expression and secretion of cytokines , chemokines , and antimicrobial peptides and through enhancing the expression of cell surface adhesion molecules .
target of myb1 ( tom1 ) is a tollip - binding protein recently shown to act as a negative regulator of tlr2 , tlr4 , and il-1 induced signalling pathways in cf bronchial epithelial cells .
tom1 was predicted to be regulated by mir-126 , a mirna that is significantly downregulated in cf bronchial brushings compared to controls . to validate this observation the coexpression of mir-126 and tom1
was evaluated in cf and non - cf bronchial epithelial samples and cell lines , and a reciprocal expression pattern was evident ; the effect of overexpression of mir-126 on tom1 gene and protein levels was examined in a cf bronchial epithelial cell line , and a mir-126::tom1 mrna interaction was functionally validated using a reporter system .
this was the first report of altered mirna expression affecting innate immune responses in the cf lung and suggests that decreased mir-126 may engender a tlr hyporesponsive state which could be important at times of infective exacerbations where a rapid and robust response is required .
in addition to the epithelium , bone marrow derived cells such as monocytes , macrophages , neutrophils , and dendritic cells are important in the cf lung . these are constantly recruited to the infected cf lung to clear pulmonary pathogens , but numerous studies have suggested an impairment of these cells in the context of the cf .
it has been well established that the cf lung is dominated by a neutrophilic inflammation .
although neutrophils are required for antimicrobial defense , their accumulation over periods of time and poorly controlled release of their toxic granular content can lead to parenchymal lung tissue damage [ 27 , 28 ] .
neutrophils from people with cf have been found to release more elastase   and have defective phagocytic capacity and oxidative burst compared to controls .
impaired bacterial killing by cf neutrophils has been shown to be a result of excessive protease cleavage of important molecules such as the il-8 chemokine receptor cxcr1 on neutrophils   and also impaired cftr - dependent phagosomal chlorination .
recent work has shown that neutrophils from people with cf have altered cytosolic ion concentrations resulting in impaired degranulation .
monocytes originate from precursors in the bone marrow and circulate in the bloodstream , until they are attracted to infection or inflammatory signals in particular tissues , such as the lung , where they differentiate into macrophage or dendritic cell populations .
the monocyte / macrophage lineage of myeloid cells has three primary roles in the immune response : phagocytosis , antigen presentation , and immunomodulation . in the lungs , monocytes primarily differentiate into alveolar macrophages .
their numbers are increased in balf of young noninfected cf patients   and similarly in cf mouse models [ 37 , 38 ] .
emerging evidences suggests that these cells are hyperresponsive in people with cf , when exposed to bacterial agonists [ 3941 ] .
cf macrophages also appear to be defective in intracellular bacterial killing [ 4244 ] and efferocytosis ( i.e. , scavenging of apoptotic neutrophils ) [ 4547 ] .
hector and colleagues have examined mirna expression in cf myeloid cells ( neutrophils and mononuclear cells ) and found changes in specific mirnas including decreased mir-9 in cf neutrophils and increased mir-126 in cf mononuclear cells versus the same cells from healthy control cells ( andreas hector , university of tuebingen , personal communication ) .
functional studies will define if these changes in mirna expression impact on dysfunctional processes such as those described above .
the cf lung is a high protease milieu and bacterial - derived proteases can contribute to this protease burden .
for example , pseudomonas aeruginosa secretes the metalloproteases pseudomonas elastase ( pse ) and alkaline protease ( apr ) , capable of cleaving a wide range of host proteins and of altering the physiology of the cf airways [ 4850 ] .
high numbers of neutrophils contribute significantly to the abnormally high concentrations of neutrophil - derived proteases , for example , neutrophil elastase [ 5153 ] , proteinase 3 , and cathepsin g ; however a range of other endogenously expressed cysteine , metallo- , and aspartyl proteases generated by other cell types are also important .
these include the cysteinyl protease cathepsin s   which can be expressed by bronchial epithelial cells and antigen presenting cells such as macrophages and dendritic cells .
weldon and colleagues   have recently found that the expression and activity of cathepsin s is increased in the balf of children with cf , including a cohort of ps .
aeruginosa - negative preschool children , compared to non - cf children with recurrent infection , indicating that upregulation of cathepsin s may be cf - specific .
interestingly , they illustrated that this is due , in part , to decreased mir-31 which they have shown regulates the transcription factor interferon regulatory factor 1 ( irf-1 ) , which controls cathepsin s expression .
levels of mir-31 were lower in cf versus non - cf cell lines , primary bronchial epithelial cells , and bronchial brushings .
other studies have looked at alternative roles of mirna in other aspects of inflammation in cf .
aeruginosa induces the production of proinflammatory cytokines such as il-8 in the cf airway epithelium .
fabbri et al .   found that mir-93 is decreased in cf bronchial epithelial ib3 - 1 cells during infection with this cf pathogen .
they also demonstrated that the decrease in mir-93 expression is correlated with an increase in il-8 levels and that mir-93 directly targeted il-8 mrna .
the cftr gene encodes a membrane bound ion transport protein that belongs to the atp - binding cassette ( abc ) superfamily of transporter proteins .
the gene , containing 27 exons , was mapped by positional cloning in 1985 to the long arm of chromosome 7 ( 7q31 ) .
its protein product , which is 1480 amino acids in length , primarily functions as an ion channel that , in concert with the ca - activated cl channel ( cacc ) , works to secrete cl and fluid required to hydrate the airway mucus but has the additional ability to transport bicarbonate   and glutathione .
although the mechanism is still not fully understood , evidence is emerging for the role of cftr in regulating the epithelial na channel ( enac ) and the failure of mutated forms of cftr in restricting salt absorption through enac .
therefore , in the cf airway , epithelial cftr dysfunction leads to airway surface liquid volume depletion due to an imbalance between cftr - mediated cl secretion and enac - mediated na absorption . indeed transgenic mice overexpressing the -subunit of enac develop a cf - like lung pathology   and have been used as a model of cf lung disease . 

transcription can begin at different start sites depending on the tissue or developmental stage in question .
for example , cftr is positively regulated by a selection of transcription factors including c / ebp proteins and foxa factors , amongst others ; cftr is also posttranscriptionally regulated by mirnas .
a number of studies have examined the role of micrornas in the control of cftr expression and various micrornas were demonstrated to regulate cftr [ 6775 ] .
although different experimental situations were examined , such as different cell lines and response to cigarette smoke , mir-101 , mir-145 , mir-494 , and mir-509 - 3p have been repeatedly implicated in many of these studies , strongly highlighting their roles in regulating cftr expression . for example , oglesby and colleagues   demonstrated that mir-145 , mir-223 and mir-494 were upregulated in cf bronchial brushings and cell lines , inversely correlated with cftr levels , and were shown to directly target cftr mrna .
ramachandran et al .   showed that mir-494 and mir-509 - 3p are increased in cf primary airway epithelial cells , regulate cftr , and are regulated by nf-b .
in the most recent study , viart et al . identified mirnas that participate in cftr downregulation in the lung after birth . having compared the mirna expression profiles of adult and foetal lungs , three mirnas in particular ( mir-145 , mir-150 , and mir-451 ) were found to have a temporal effect , being significantly upregulated in the adult lung and therefore contributing to downregulation of cftr .
they also demonstrated how inhibitors based on these mirnas can affect cftr gene expression and function in air - liquid interface culture and suggest that these may be developed as tools for cftr correction in people with cf .
the er is the site of protein translation , folding , and processing for transport to secretory vesicles .
misfolded variants of cftr , for example , the class ii p.phe508del-cftr protein , accumulate in the er and fail to reach the apical surface of epithelial cells to function as anion channels .
er perturbation can lead to er stress and the initiation of signalling networks aimed at restoring er equilibrium .
recent evidence has implicated mirnas in regulation of the upr , in contexts other than cf [ 7780 ] .
however one recent study has examined whether altered mirna expression regulates expression of upr genes in cf airway epithelium .
activating transcription factor 6 ( atf6 ) is an er resident transcription factor and a key component of the upr .
its activation leads to transcriptional induction of atf6-regulated genes which function primarily to restore correct protein folding in the er . the role of mirna in basal regulation of atf6 was investigated in cf and non - cf bronchial epithelial cells in vitro and in vivo .
three of these , mir-145 , mir-221 , and mir-494 , were upregulated in a p.phe508del-cftr versus non - cf bronchial epithelial cell line and also in p.phe508del-cftr versus non - cf bronchial brushings .
after experimentally validating atf6 as a molecular target of these mirnas through the use of a luciferase reporter vector containing the full length 3utr of atf6 , the human studies were complemented by analysing the expression of key mirnas in a mouse model of cf lung disease .
expression of mir-221 , which is also predicted to regulate murine atf6 , was significantly increased in native airway tissues of enac - overexpressing transgenic mice with cf - like lung disease versus wild type littermates , demonstrating structural and functional conservation between humans and mice .
these findings implicate enac - overexpressing transgenic mice as a useful animal model for studies manipulating mir-221 levels in vivo using mirna overexpression strategies to limit er stress - mediated inflammation .
in this review , we have discussed current data regarding mirna studies in cystic fibrosis .
what is clear is that mirna dysregulation exists in cf , with many studies highlighting an altered mirna expression profile in the cf lung , be it in cell lines , primary cell cultures , or bronchial brush samples .
some of these aberrantly expressed mirnas have been demonstrated to be involved in the regulation of key components of inflammatory signalling and , more recently , the upr .
although this is an emerging field , some work is beginning to be carried out with respect to the development of strategies to ultimately modulate mirna levels in vivo in the cf lung , through the use of mirna mimics and inhibitors .
finally , the potential of mirna as biomarkers of cf disease progression remains underexplored in comparison to other diseases such as cancers .
the expression of these may become particularly useful for predicting and determining cf lung disease in infants and children , where currently used surrogate markers and biomarkers are of little use .
 the cystic fibrosis lung is a complex milieu comprising multiple factors that coordinate its physiology . 
 micrornas are regulatory factors involved in most biological processes and it is becoming increasingly clear that they play a key role in the development and manifestations of cf lung disease . these small noncoding rnas act 
 posttranscriptionally to inhibit protein production . 
 their involvement in the pathogenesis of cf lung disease stems from the fact that their expression is altered in vivo in the cf lung due to intrinsic and extrinsic factors ; to date defective chloride ion conductance , endoplasmic reticulum stress , inflammation , and infection have been implicated in altering endogenous mirna expression in this setting . here 
 , the current state - of - the - art and biological consequences of altered microrna expression in cystic fibrosis are reviewed . 
1. Introduction
2. MicroRNA
3. Concluding Remarks and Perspective