liver fibrosis is characterized by excessive scarring , caused by chronic inflammatory processes during liver diseases of different origin . in response to chronic liver injuries , various cell types get activated and transdifferentiate into myofibroblastic cells that then participate in synthesis and reorganization of connective tissue ( hautekeete and geerts , 1997 ; friedman , 1999 , 2008 ; knittel et al . , 1999 ) .
a main source of extracellular matrix ( ecm ) production are hepatic stellate cells ( hsc ) undergoing myofibroblastic transition ( hautekeete and geerts , 1997 ; friedman , 1999 , 2008 ) . myofibroblastic differentiation and matrix accumulation of hsc is heavily triggered by profibrogenic mediators such as transforming growth factor  ( tgf- ) and the -isoform of platelet - derived growth factor ( pdgf ) .
hence , in response to tgf- , hsc take center stage during fibrosis by the enhanced synthesis of ecm proteins , in particular collagen i and ii . here , we emphasize the role of dysregulated microrna ( mirna ) during liver fibrosis , their function in ecm expression and in profibrogenic signaling in hsc .
the fibrotic remodeling process and the change of gene expression during liver fibrosis are associated with an altered pattern of mirna .
mirna are small ( 1924 nucleotides long ) non - coding rna molecules inhibiting posttranscriptionally gene expression .
it is suggested that more than one - third of all human genes are regulated by mirna ( krek et al .
the primary mirna ( pri - mirna ) molecules of more than 1000 bases in length are transcribed by rna - polymerase ii .
cleavage of the pri - mirna then generates the precursor mirna ( pre - mirna ) .
this pre - mirna is exported into the cytoplasm and further processed by rnase iii and dicer resulting in the release of a mature mirna ( ruby et al . , 2007 ) .
after integration into the rna - induced silencing complex ( risc ) , interaction of mirna sequences with the untranslated region ( utr ) of transcripts causes translational repression or transcript degradation ( bartel , 2009 ) .
several algorithms predict that one mirna might bind to a multitude of mrna transcripts and , in turn , that one mrna might be targeted by a widespread panel of mirna species ( doench and sharp , 2004 ; lewis et al . , 2005 ) .
in liver , mir-122 is the most abundant mirna , accounting for more than 70% of the total mirna in hepatocytes ( jopling et al . , 2005 ) .
mir-122 is involved in cholesterol synthesis and hepatitis c virus ( hcv ) replication ( jopling et al .
is markedly decreased depending on severity of fibrosis ( cheung et al . , 2008 ;
other mirna species , that are also highly expressed in the healthy liver , e.g. , mir-125b or mir-22 , were shown to be additionally reduced in fibrotic liver biopsies of chronic hepatitis c patients .
furthermore , the members of the mir-29 family , as well as mir-194 and mir-150 are reported to be downregulated during fibrogenesis ( kwiecinski et al . , 2011 ; roderburg et al . ,
additionally , mir-19b is recently found to be diminished in fibrotic liver of rat and human ( table 1 ) ( lakner et al . , 2012 ) . whereas a wide range of mirna are reduced after fibrosis , only some mirna like the members of the mir-199 , the mir-200 , and the mir-34 family are known to be increased ( alisi et al . , 2010 ; pogribny et al . ,
2010 ; murakami et al . , 2011 ) . though mir-21 upregulation was only analyzed on fibrotic liver biopsies of a limited number of hcv patients ( marquez et al . , 2010 ) , in many other organs mir-21 is one of the most important mirna prevalently expressed after fibrosis induction ( thum et al . , 2008 ; liu et al . , 2010 ; zhu and fan , 2011 ) .
personal observation ; nafld , non - alcoholic fatty liver disease ; hfd , high fat diet . the mir-122 decrease , observed in primary hsc during culture - induced myofibroblastic activation , might be due to the dilution of contaminating hepatocyte mirna of the primary hsc isolate .
recent reports have shown that altered mirna levels are also associated with the phenotypical changes of hsc during the myofibroblastic transition process including the induction of ecm proteins ( guo et al . , 2009a ; ji et al . , 2009 ) ( table 1 ) .
the members of the mir-29 family are of particular interest because they are shown to inhibit ecm synthesis indicating an antifibrotic function .
the mir-29 family consists of mir-29a , mir-29b , and mir-29c , differing only in two or three bases . mir-29a and mir-29b1 as well as mir-29c and mir-29b2 , are encoded and transcribed in tandem by two genes located on chromosome 7 or chromosome 1 , respectively ( mott et al .
the authors proved mir-29 mediated repression of elastin , collagen i and iii synthesis in cardiac fibroblasts and its regulation by tgf-. accordingly , downregulation of mir-29 was suggested to enhance the fibrotic response after myocardial infarction , whereas overexpression of mir-29 in cardiac fibroblasts reduced collagen expression .
similarly , ogawa et al . ( 2010 ) demonstrated that mir-29 inhibits the production of fibrillar collagen in hsc , suggesting also a function of mir-29 in liver fibrosis ( roderburg et al . , 2011 ) .
furthermore , highly conserved binding sites for the mir-29a and mir-29b are found in many 3-utr sequences of the various subunits of ecm proteins like collagen type v and xv subunits , laminin  1 , and nidogen .
( 2011 ) suggest that mir-29a and mir-29b are also involved in a widespread panel of other fibrosis associated genes including adam metalloproteinases mainly type 12 and 9 , ecm formatting components such as fibrillin-1 and follistatin-1 , and different integrin chains .
( in review ) showed that mir-29 not only targets gene expression of ecm associated proteins , but also the expression of profibrogenic growth factors ( manuscript submitted ) .
they found putative mir-29a and mir-29b binding sites in mrna of igf - i but most notably also in members of pdgf linked signaling pathways such as pdgf- receptor , pdgf - c , and vegf - a .
accordingly , mir-29 mediated inhibition of igf - i and pdgf - c was proven in hsc .
( 2011 ) collect evidence that also the pdgf- receptor is targeted by mir-29 regulation . as a consequence , mir-29 acts as antifibrogenic mirna by two pathways ( 1 ) by inhibition of ecm formation and ( 2 ) interfering with the profibrogenic cell communication pathways via pdgf - b and pdgf - c signaling ( figure 1b ) .
interestingly , mir-29 expression is in turn highly regulated by pdgf - bb ( ogawa et al . , 2010 ) .
thus , the loss of mir-29 in myofibroblastic hsc during liver fibrosis is mainly due to profibrogenic stimulation by tgf- and pdgf - bb ( ogawa et al . , 2010 ; kwiecinski et al . , 2011 )
the members of the mir-29 family are encoded by two genes , located on chromosome 7 or 1 , respectively .
the pri - mir-29a and b1 as well as the mir-29c and b2 are each transcribed in tandem .
the promoter region of the mir-29a / b1 gene contains several putative binding sites for the profibrogenic repression of mir-29 : three ap1 consensus sequences ( ap1-re ) , three tgf- inhibitory elements ( tie ) and in addition the three putative smad binding elements ( sbe ) .
these transcriptional binding sites and their interaction are suggested to mediate profibrogenic stimulation of pdgf - bb ( i.p . of the ap1 re sites ) and of tgf- by smad and ap1 signaling .
( b ) mir-29 signaling in the healthy and the fibrotic liver . in the healthy liver
, mir-29 is highly expressed in hepatic stellate cells ( 1 ) and is responsible for repression of extracellular matrix proteins ( ecm ) in particular collagens ( 2 ) , but also of expression of growth factors such as pdgf - c and igf - i . during fibrogenesis ,
profibrogenic growth factors are increased and stimulate hepatic stellate cells in a paracrine and autocrine manner ( 3 ) .
profibrogenic stimulation of tgf- and pdgf - bb results in mir-29 repression ( 4 ) . the loss of mir-29 by tgf- and pdgf - bb results in the abolished repression of profibrogenic expression of ecm , pdgf - c or igf - i ( 5 ) .
the enhanced secretion of pdgf - c and igf - i stimulates autocrinely stellate cells , leading to increased proliferation and ecm production ( 6 ) .
the decrease of mir-29 was observed after experimental fibrosis including liver intoxication in mice and cholestasis induced fibrosis after bile duct occlusion in rat .
furthermore , in liver biopsies of patients with chronic hepatitis c mir-29 levels are significantly reduced ( kwiecinski et al . , 2011 ; roderburg et al . ,
these in vivo studies suggest that the loss of mir-29 during liver fibrogenesis leads to the abolishment of ecm and profibrogenic mediator repression .
the findings on hsc provide additional evidence , that reduced mir-29 levels leading to profibrogenic gene expression are due to tgf- or pdgf - bb exposure .
the smad-2/3 proteins act as the main signal transducers of tgf- stimulation , initiating ecm synthesis and myofibroblastic transition ( dooley et al .
, 2000 , 2001a ) . in renal fibrosis , silencing of the mir-29c / b2 gene is assumed to involve smad-3 signaling ( qin et al . , 2011 ) .
however , the analysis of promoter regions of both mir-29 genes , shown in figure 1a , has just started by defining the transcriptional nf - kb and yy - nf - kb control , respectively ( wang et al .
, 2008 ; mott et al . , 2010 ) , and did not yet focus on tgf- signaling .
interestingly , the promoter sequence of the mir-29a / b1 gene harbors several caga - boxes .
caga - boxes function as smad-3/4 protein binding elements ( sbe ) and smad-3/4 binding is commonly followed by induction of gene transcription ( dennler et al . , 1998 ) . however , suppressive effects of smad proteins are also possible , depending on their interaction with other regulatory factors like histone deacetylases ( liberati et al . , 2001 ) or the activator protein 1 ( ap1 ; hall et al . , 2003 )
thus , tgf- induced smad-3/4 signaling and the regulatory binding of the sbe may contribute to the transcriptional repression of the mir-29a / b1 gene .
in addition , three consensus sequences for binding of the transcription factor ap1 are located in the upstream region of the mir-29a / b1 gene .
is highly activated by the ras / raf pathway after tgf- and pdgf - bb exposure ( angel and karin , 1992 ; zhang et al . , 1998 ) .
ap1 interaction with the ap1 responsive elements ( ap1 re ) drives transcription of a broad spectrum of genes involved in the early gene response and acute inflammatory stimulation .
however , ap1 can also function as transcriptional suppressor by binding to tgf- inhibitory elements ( tie ) , that are known to lead to gene silencing in the stromelysin ( kerr et al . ,
1990 ) , the hgf , and the c - myc gene ( matrisian et al . , 1992 ; yagi et al . , 2002 ) .
thus , in future experiments it has to be proven if profibrogenic mir-29 repression is mediated by the consensus sequences of the tie , the ap1 re , the sbe , or even by the interplay of different regulatory elements ( figure 1a ) .
mir-21 is an ubiquitously expressed mirna , which is highly upregulated in the majority of cancer types . in cancer , mir-21 acts as an oncogenic mirna ( oncomir )
targeting pten and other tumor suppressor proteins ( krichevsky and gabriely , 2009 ; qi et al . , 2009 ) .
previous reports have demonstrated that mir-21 is also enhanced after initiation of myocardial , renal , or pulmonary fibrosis ( thum et al . , 2008 ; liu et al . , 2010 ) .
mir-21 enrichment during fibrosis is assumed to be based on profibrogenic stimulation by tgf- , because zhong et al .
( 2011 ) have demonstrated that smad-3 transducing tgf- signaling , is crucial for mir-21 transcription . furthermore , the signal transducers of tgf- , the receptor - regulated smad-2 and smad-3 ( r - smad ) , are able to interact with the p68 rna helicase of the drosha / pdcd4 microprocessor complex , promoting the restriction of pri - mir-21 into the precursor pre - mir-21 ( davis et al . , 2008 ) .
thus , the upregulation of mir-21 during fibrotic processes is based on two tgf- stimulated mechanisms , that are mediated by the smad proteins : ( 1 ) by transcriptional induction and ( 2 ) by enhanced mir-21 maturation ( figure 2 ) .
fibrosis tgf- binds to the receptors leading to smad-2 or smad-3 phosphorylation , followed by aggregation with smad-4 ( 1 ) .
the smad-3/4 is shown to induce transcriptional induction of synthesis of pri - mir-21 ( 2 ) .
the further cleavage of pri - mirna into hairpin - loop precursor mirna is catalyzed by the pdcd4/drosha complex .
the smad proteins can interact with the rna helicase p68 , which is integrated in the microprocessing complex .
after tgf- exposure , both the smad-2 and smad-3 protein is bound by p68 of the complex resulting in stabilization and high microprocessing efficiency ( 3 ) .
high levels of mir-21 target the smad-7 mrna and repress translation of the inhibitory smad-7 ( 4 ) .
the decrease of the inhibitory smad-7 protein in turn abolishes the negative feed - back mechanisms of tgf- signaling ( 5 ) resulting in elevated profibrogenic tgf- stimulation .
a potential mechanism for the role of mir-21 in fibrosis is through regulating the protein synthesis of the inhibitory smad-7 ( figure 2 ; liu et al .
after tgf- stimulation , smad-7 transcription is driven by smad-2/3 and smad-4 ( stopa et al . , 2000 ; dooley et al . , 2001b ) .
enhanced smad-7 levels , in turn , are highly potent to antagonize tgf--mediated pathways ( 1 ) by blockade of the tgf- type i receptor for further binding of the receptor smads ( massague and chen , 2000 ) , and ( 2 ) by smurf2 interaction , to form an e3 ubiquitin ligase that targets the tgf- type i receptor for degradation ( kavsak et al . , 2000 ) .
therefore , the inhibition of smad-7 in response to mir-21 enhancement during fibrosis has to be considered as an important profibrogenic pathway , which might become an attractive target for future therapeutical approaches .
in contrast to mir-21 that promotes tgf- signaling , a recent study of lakner et al .
mir-19b represses fibrotic features after myofibroblastic activation of stellate cells by targeting the tgf- ii receptor and most notably smad-3 ( lakner et al . , 2012 ) .
hence , upcoming knowledge of mirna species , involved in profibrogenic signaling , will provide novel perspectives in understanding and probably also in treatment of chronic liver diseases .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 microrna ( mirna ) are small non - coding rna molecules that posttranscriptionally effect mrna stability and translation by targeting the 3-untranslated region ( 3-utr ) of various transcripts . 
 thus , dysregulation of mirna affects a wide range of cellular processes such as cell proliferation and differentiation involved in organ remodeling processes . 
 divergent mirna patterns were observed during chronic liver diseases of various etiologies . 
 chronic liver diseases result in uncontrolled scar formation ending up in liver fibrosis or even cirrhosis . 
 since it has been shown that mir-29 dysregulation is involved in synthesis of extracellular matrix proteins , mir-29 is of special interest . the importance of mir-29 in hepatic collagen homeostasis is underlined by in vivo data showing that experimental severe fibrosis is associated with a prominent mir-29 decrease . 
 the loss of mir-29 is due to the response of hepatic stellate cells to exposure to the profibrogenic mediators tgf- and pdgf - bb . 
 several putative binding sites for the smad proteins and the ap1 complex are located in the mir-29 promoter , which are suggested to mediate mir-29 decrease in fibrosis . 
 other mirna are highly increased after profibrogenic stimulation , such as mir-21 . 
 mir-21 is transcriptionally upregulated in response to smad-3 rather than smad-2 activation after tgf- stimulation . 
 in addition , tgf- promotes mir-21 expression by formation of a microprocessor complex containing smad proteins . 
 elevated mir-21 may then act as a profibrogenic mirna by its repression of the tgf- inhibitory smad-7 protein . 
Introduction
Dysregulation of miRNA in Liver Fibrosis
The Members of the miR-29 Family Repress Profibrogenic Gene Expression
Loss of miR-29 in the Fibrotic Liver is a Result of Profibrogenic Signaling
Enhanced miR-21 Levels in Response to TGF- by Transcriptional Induction and Promoted Microprocessing
Conflict of Interest Statement