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MicroRNA-29 family, a crucial therapeutic target for fibrosis diseases
Biochimie xxx (2013) 1e5
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Review
MicroRNA-29 family, a crucial therapeutic target for fibrosis diseases Yong He a, b, Cheng Huang a, b, Xiang Lin a, b, Jun Li a, b, * a b
School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei 230032, China Institute for Liver Diseases of Anhui Medical University(AMU), Anhui Medical University, Hefei 230032, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 February 2013 Accepted 19 March 2013 Available online xxx
MicroRNAs (miRNAs) are a class of approximately 20-nucleotides single-stranded endogenous RNAs that regulate gene expression at the post-transcriptional level. miRNAs have recently been known to regulate cell proliferation, differentiation, and apoptosis. Fibrosis is the leading cause of organ dysfunction in diseases and results from an imbalance in the turnover of extracellular matrix components. Accumulating studies have demonstrated that miR-29 family participates in the development of liver fibrosis, renal fibrosis, pulmonary fibrosis, cardiac fibrosis. In this review, we are discussing the comprehensive role of miR-29 family in moderating profibrotic effect and its potential as therapeutic approach to fibrosis diseases. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: miR-29 family Extracellular matrix Fibrosis diseases Review
1. Introduction Fibrosis is a common feature of organ dysfunction in diseases, and is associated with an excessive accumulation of extracellular matrix (ECM), such as collagens and a-smooth muscle actin (a-SMA), while other ECM proteins such as fibronectins, elastin and fibrillins also play an important role in the development of fibrosis [1e3]. Both an increased synthesis and decreased degradation of ECM components can lead to fibrosis. In particular, matrix metalloproteinases (MMPs) that degrade the ECM may be upregulated, whereas their inhibitors, tissue inhibitor of metalloproteinases (TIMPs), may be downregulated [4e6]. Following tissue injury and profibrotic mediators, such as transforming growth factor-b (TGF-b) [7e10], platelet-derived growth factor (PDGF) [11e13], fibroblasts differentiate into myofibroblasts and the activation and/or recruitment of fibroblasts with a resistance to apoptosis result in the development of fibrosis. Although some progress has been made to understand the mechanism of the development of fibrosis, current therapeutic options are limited to emerge as an effective antifibrotic agent. Therefore, it is necessary to provide an update on
Abbreviations: miRNA, microRNA; ECM, extracellular matrix; MMPs, matrix metalloproteinases; HSC, hepatic stellate cell; COL, collagens; a-SMA, a-smooth muscle actin; TGF-b, transforming growth factor-b; 30 UTR, 30 untranslated region; PDGF, platelet-derived growth factor; SMAD3, signaling effectors (mothers against decapentaplegic protein)3; LPS, lipopolysaccharide; NF-kB, nuclear factor kappa B; FXR, nuclear receptor farnesoid X receptor; IGF, insulin-like growth factor; HGF, hepatocyte growth factor; CTGF, connective tissue growth factor; FBN1, fibrillins. * Corresponding author. School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei 230032, Anhui Province, China. Tel./fax: þ86 551 65161001. E-mail addresses: [email protected], [email protected] (J. Li).
our understanding of fibrosis for presenting novel therapeutic strategies against tissue fibrogenesis. MicroRNAs (miRNAs), which are endogenous small non-coding RNAs (18e25 nucleotides) that work as post-transcriptional regulators of gene expression by binding to the 30 untranslated region (30 UTR) of target mRNAs [14e17]. Most miRNA genes are located in introns, exons and UTRs of protein coding genes [18]. miRNAs are transcribed as long polyadenylated RNAs, the pri-miRNAs molecules are transcribed by RNA-Polymerase II and cut by the endonuclease Drosha, releasing intermediate approximately 60-nucleotide-long hairpin structures called pre-miRNAs [19]. The pre-miRNAs are exported to the cytoplasm by exportin 5 and further processed into approximately 22-nucleotide-long mature miRNA by another endonucleases, Dicer, and RNase III. Each mature miRNA is then integrated into the RNA-induced silencing complex (RISC), this miRNA/RISC complex mediates gene expression by causing translational repression or mRNA degradation [14,16]. One miRNA might bind to a number of mRNAs transcripts and in turn one mRNA can be targeted by a widespread panel of miRNA species. Recently, multiple lines of evidence suggests that aberrant miRNA expression has been shown to be related with the development of various tissue fibrosis diseases, for example, miR-146a inhibited TGF-b1-induced HSC proliferation and a-SMA expression, at least in part, via inhibiting Smad4 [20], miR-21 mediated fibrogenic activation of pulmonary fibroblasts and lung fibrosis through regulating the expression of an inhibitory Smad, Smad7 [21], miR-126 was downregulated in cystic fibrosis airway epithelial cells and altered expression of miR-126 was a negative regulator of TOM1 [22]. The miR-29 family is composed of miR-29a, miR-29b, and miR29c, differing only in two or three bases, miR-29a and miR-29b1 as
0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.03.010
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well as miR-29c and miR-29b2. MiR-29 family are encoded and transcribed in tandem by two genes located on chromosome7 or chromosome1, respectively [23]. The members of the miR-29 family are of special interest, because miR-29 family has been shown to be involved in inhibiting extracellular matrix proteins (ECM) synthesis indicating an antifibrotic function. To our surprise, the Targetscan prediction program indicates that miR-29 family has potential to repress many kinds of collagens (COL1A1, COL 2A1, COL3A1, COL4A1, COL 4A3, COL 4A5, COL 5A1, COL 5A2, COL 6A3, et al) synthesis via binding to its 30 UTR (http://www.targetscan. org/cgi-bin/target-scan/vert_50/targetscan.cgi?mirg¼rno-miR29a). Recently, there is overwhelming evidence that aberrant miR29 family expression was involved in the development of tissue fibrosis, such as liver fibrosis, renal fibrosis, pulmonary fibrosis, cardiac fibrosis. In this review, we make a summary on the functional roles of miR-29 family members in the fibrotic process of several organs and its potential as novel therapeutic target. 2. miR-29 family and liver fibrosis Liver fibrosis occurs in many types of chronic liver diseases and is characterized by an excessive accumulation of ECM [24e26]. Hepatic stellate cells (HSCs) are considered to contribute to fibrogenic reactions and the activation of HSCs has been shown to play a central role in the development of liver fibrosis [27e29]. Following exposure to inflammatory cytokines, activated HSCs lose their lipid droplets, migrate to injured sites and are transformed into myofibroblast-like cells that express a-SMA, secrete profibrogenic mediators finally leading to liver fibrosis [30]. In these inflammatory cytokines, transforming growth factor beta 1 (TGF-b1) is believed to be recognized as the main profibrogenic mediator [31,32]. Recently, the microarray analysis revealed that the members of miR-29 family (miR-29a, 29c) were identified to be downregulated in activated HSCs as compared to that in quiescent HSCs [33,34]. Some reports demonstrated that all three members of the miR-29 family were significantly downregulated in livers of carbon tetrachloride (CCl4)-treated mice as well as in mice that underwent bile duct ligation by using real-time qPCR [35]. Next, the authors went on finding that TGF-b1 as well as inflammatory signal molecules, namely, lipopolysaccharide (LPS) and nuclear factor kappa B (NFkB) could downregulate the expression of miR-29 in murine HSC. Interestingly, TGF-b stimulation led to decreased miR-29 levels, while hepatocyte growth factor (HGF) stimulation resulted in elevated miR-29 expression in HSC and miR-29a/b overexpression caused a significantly reduction of collagen-I and -IV synthesis by targeting the 30 UTR of the collagen-1 and -4 subtypes [36]. Ogawa et al. [37] also found that miR-29b was the most effective suppressor of type I collagen at the mRNA and protein level via its direct binding to Col1A1 30 UTR. In primary HSCs, the expression of miR-29 family was also downregulated under TGF-b1 stimulation [35]. During HSC activation in primary culture, miR-29b overexpression markedly attenuated Col1a1 and Col1a2 mRNAs expression and additionally inhibited the increased expression of aSMA, DDR2, FN1, ITGB1, and PDGFR-b, which were key genes involved in the activation of HSCs [38]. Additionally, miR-29 acted as an antifibrogenic mediator by interfering with profibrogenic cell communication via platelet-derived growth factor (PDGF)-C and insulin-like growth factor (IGF)-I [39]. Recently, the nuclear receptor farnesoid X receptor (FXR) was reported to have potent antifibrotic activity in HSCs [40,41]. Interestingly, miR-29a was a nuclear receptor farnesoid X receptor (FXR) target gene because a genetic activation of FXR enhanced miR-29a promoter activity, therefore, miR-29a was involved in the antifibrotic effect of FXR in HSC [42]. In line with these early observations suggested that
miR-29 family may function as a negative regulator in HSC activation, further functional analysis to determine the precise roles of miR-29 family in liver fibrosis provides a novel therapeutic approach for treating liver fibrosis (Fig. 1). 3. miR-29 family and renal fibrosis Renal fibrosis characterized by accumulation of fibroblasts and excessive matrix proteins is a hallmark of end-stage renal disease and is a major cause of therapeutic failure [43,44]. TGF-b1 has been considered as a key mediator in renal fibrosis and mainly causes renal fibrosis by activating its downstream Smad signaling pathway [45]. Smads can be divided into three distinct subclasses: receptoractivated Smads (R-Smads), common-partner Smads (Co-Smads) and inhibitory Smads (anti-Smads). In TGF-b1/Smad signaling pathway, Smad2 and Smad3 function as R-Smads, Smad4 functions as Co-Smad, and Smad7 functions as an anti-Smad. Recent studies revealed that Smad3, but not Smad2, was a key signaling pathway of fibrogenesis in response to TGF-b1 and TGFb1/Smad3 signaling could promote renal fibrosis [46,47], but the downstream fibrosis-specific genes of TGF-b1/Smad3-mediated fibrosis remain unclear. However, Qin et al. [48] have indicated that wild-type mice had reduced expression of miR-29 along with the development of progressive renal fibrosis in obstructive nephropathy by using a microRNA microarray and real-time PCR. The authors finally revealed that miR-29 was lost with progressive renal fibrosis in Smad3 wild-type mice, but increased with protection against renal fibrosis in Smad3 knockout mice in UUO Nephropathy. Moreover, in cultured fibroblasts and tubular epithelial cells, TGF-b1 downregulated miR-29b expression, while upregulated collagen-I and -III expression via the Smad3-dependent, not Smad2-dependent mechanism and Smad3-mediated TGF-b1induced downregulation of miR-29 by binding to the promoter of miR-29. In vitro, overexpression of miR-29b inhibited, but knockdown of miR-29 increased, TGF-b1-induced expression of collagens I and III, but not a-SMA expression in renal tubular cells. Interestingly, gene transfer of miR-29 prevented renal fibrosis in a mouse model of obstructive nephropathy by transfecting Dox-inducible miR-29b-expressing plasmid system into left kidney of mouse [48]. In addition, Wang et al. [49] have reported that the expression of the miR-29 family which targeted collagen gene expression was reduced and the expression of ECM proteins was increased by exposing proximal tubular cells, primary mesangial cells, and podocytes to TGF-b1. Furthermore, in both resting and TGF-b1treated cells, ectopic expression of miR-29 repressed the
Fig. 1. The role of miR-29 family in liver fibrosis. TGF-b, LPS and NF-kB stimulation could lead to decreased miR-29 levels, while HGF stimulation could result in elevated miR-29 expression in HSCs. miR-29a/b overexpression in HSC caused a significantly reduction of collagen synthesis by targeting the 30 UTR of the collagen and interfering with profibrogenic cell communication via PDGF-C and IGF-I.
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expression of collagens I and IV at both the mRNA and protein levels by targeting the 30 UTR of these genes. Interestingly, administration of the Rho-associated kinase inhibitor fasudil prevented renal fibrosis and restored expression of miR-29 [49]. Therefore, it is important to further investigate the roles of miR-29 in renal fibrosis, which may have therapeutic potential for progressive renal fibrosis (Fig. 2). 4. miR-29 family and pulmonary fibrosis Pulmonary fibrosis is a chronic fibrosing interstitial lung disease of unknown etiology, which leads to the thickening of alveolar walls and scarring of the lung by exposure to chronic inflammatory responses, including persistent infections, autoimmune reactions, allergic responses, and tissue injury [50,51]. Recently, Cushing et al. [52] have found that all the members of miR-29 family were downregulated by TGF-b1 in IMR-90 cells and in bleomycin-treated lungs. Additionally, miR-29 downregulation was also observed in mesenchymal cells at the entrance of the alveolar duct and in the subepithelial mesenchyme of terminal bronchioles. Moreover, all reported miR-29 targets were significantly upregulated in miR-29 knockdown cells, including Col1A1, Col1A2, Col3A1, Col4A1, Col4A2, and Col15A1 by performing genome-wide array analysis of IMR-90 cells 48 h after transfection of miR-29 antisense or control oligos, and ITGA11, ADAM12, ADAMTS9, and NID1 were shown to be direct targets of miR-29. In addition, many fibrosis-associated genes upregulated by TGF-b1 were suppressed by miR-29 knockdown. Interestingly, a comparison of TGF-b1 and miR-29 targets revealed that miR-29 controlled an additional subset of fibrosisrelated genes, including laminins and integrins, independent of TGF-b1 [52]. Furthermore, Yang et al. [53] indicated that miR-29 mediated TGF-b1-induced extracellular matrix synthesis through activation of PI3K-AKT pathway in human lung fibroblasts. To our surprise, miR-29 was also found to be a downstream target gene of Smad3 and negatively regulated by TGF-b/Smad signaling in pulmonary fibrosis [54]. Moreover, mice or pulmonary fibroblasts null for Smad3 were shown to protect against bleomycin or TGF-b1-induced loss of miR-29 along with fibrosis in vivo and in vitro. Interestingly, overexpression of miR-29 could in turn negatively regulate TGF-b and connective tissue growth factor (CTGF) expression and Smad3 signaling. Sleeping Beauty (SB)mediated miR-29 gene transferred into normal and diseased lung tissues was able to prevent and treat pulmonary fibrosis including inflammatory macrophage infiltration induced by bleomycin in mice [54]. Idiopathic pulmonary fibrosis (IPF) is a chronic progressive and lethal fibrotic lung diseases [55,56]. The role of miR-29 in IPF lungs remain still unclear, but it has been reported that a significant decrease in miR-29 in the lungs of IPF patients [57]. So,
Fig. 2. The role of miR-29 family in renal fibrosis. TGF-b1 acted by stimulating Smad3 to negatively regulate the miR-29 families, which inhibited TGF-b1-induced expression of collagens I and III, to mediate renal fibrosis.
Fig. 3. The role of miR-29 family in pulmonary fibrosis. All miR-29 family members were downregulated by TGF-b1 stimulation. MiR-29 mediated TGFb1-induced collagens synthesis through activation of PI3K-AKT pathway and could negatively regulate TGF-b1 and CTGF expression and was also found to be a downstream target gene of Smad3 in human lung fibroblasts.
further studies to clarify the precise roles of miR-29 family in IPF could provide a potential therapeutic agents for treating pulmonary fibrosis (Fig. 3). 5. miR-29 family and cardiac fibrosis Cardiac fibrosis is an important pathological feature of structural remodeling which results in ventricular stiffness, diminished contractility, and abnormalities in cardiac conductance [58,59]. Recently, van Rooij et al. [60] have found that miR-29 was dramatically downregulated in the region of the fibrotic scar after myocardial infarction (MI) and the miR-29 family targeted many mRNAs that encoded proteins involved in fibrosis, including multiple collagens(COL1A1, COL1A2, COL3A1), fibrillins (FBN1), and elastin. Furthermore, downregulation of miR-29 with anti-miRs in vitro and in vivo was sufficient to increase the expression of collagens, whereas overexpression of miR-29 in fibroblasts inhibited collagens expression. Recently, miR-29 family includes miR-29s, which was reported to directly target at least 16 extracellular matrix genes. Furthermore, miR-29s had strong antifibrotic effects on heart, kidney, and other organs and miR-29s was also demonstrated to be proapoptotic and involved in the regulation of cell differentiation [61]. Unfortunately, the specific mechanism of miR29 family in cardiac fibrosis is not very clear and the downregulation of miR-29 provides a novel idea for cardiac fibrosis, which suggests that strategies to increase miR-29 expression may have therapeutic value in treating post-MI cardiac remodeling (Fig. 4).
Fig. 4. The role of miR-29 family in cardiac fibrosis. TGF-b1 could trigger the downregulation of miR-29 in cardiac fibroblasts and then upregulate the expression of ECM (COL1A1, COL1A2, COL3A1, and FBN1) involved in cardiac fibrosis.
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6. Conclusion and prospective On the basis of other findings, we make a summary on the comprehensive knowledge of the biological roles of miR-29 family in various fibrosis diseases, including in the fibrosing pathogenesis of liver, kidney, lung and heart. Recently, research focusing on small molecules such as miRNAs has been aroused to understand their roles in diseases. In particular, miR-29 family was considered to be a very important and common regulator of tissue fibrosis, because many reports have revealed that miR-29 family could repress collagen synthesis via direct binding to its 30 UTR in fibroblasts. To our excitement, an artificial intronic miRNA expression system had been established, and activated-HSC-cell-specific gene silencing could be induced well by the artificial intronic miRNA expression system to realize antifibrosis in vitro [62]. It is increasingly evident that miRNAs antagonists or agonist had been proposed as a potentially novel therapeutic tools [21,63]. Recent studies showed that miR-29 family also connected with DNA methylation was involved in many diseases [64e66]. So, the effect of miR-29 family on diseases was very extensive and indepth understanding of miR29 family with their cell-specific roles may provide a rationale for the development of miRNA-based strategies for the treatment of fibrosis diseases.
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Conflict of interest None declared. Acknowledgments This project was supported by the National Science Foundation of China (No 30873081, 81072686). References
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Review
MicroRNA-29 family, a crucial therapeutic target for fibrosis diseases Yong He a, b, Cheng Huang a, b, Xiang Lin a, b, Jun Li a, b, * a b
School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei 230032, China Institute for Liver Diseases of Anhui Medical University(AMU), Anhui Medical University, Hefei 230032, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 February 2013 Accepted 19 March 2013 Available online xxx
MicroRNAs (miRNAs) are a class of approximately 20-nucleotides single-stranded endogenous RNAs that regulate gene expression at the post-transcriptional level. miRNAs have recently been known to regulate cell proliferation, differentiation, and apoptosis. Fibrosis is the leading cause of organ dysfunction in diseases and results from an imbalance in the turnover of extracellular matrix components. Accumulating studies have demonstrated that miR-29 family participates in the development of liver fibrosis, renal fibrosis, pulmonary fibrosis, cardiac fibrosis. In this review, we are discussing the comprehensive role of miR-29 family in moderating profibrotic effect and its potential as therapeutic approach to fibrosis diseases. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: miR-29 family Extracellular matrix Fibrosis diseases Review
1. Introduction Fibrosis is a common feature of organ dysfunction in diseases, and is associated with an excessive accumulation of extracellular matrix (ECM), such as collagens and a-smooth muscle actin (a-SMA), while other ECM proteins such as fibronectins, elastin and fibrillins also play an important role in the development of fibrosis [1e3]. Both an increased synthesis and decreased degradation of ECM components can lead to fibrosis. In particular, matrix metalloproteinases (MMPs) that degrade the ECM may be upregulated, whereas their inhibitors, tissue inhibitor of metalloproteinases (TIMPs), may be downregulated [4e6]. Following tissue injury and profibrotic mediators, such as transforming growth factor-b (TGF-b) [7e10], platelet-derived growth factor (PDGF) [11e13], fibroblasts differentiate into myofibroblasts and the activation and/or recruitment of fibroblasts with a resistance to apoptosis result in the development of fibrosis. Although some progress has been made to understand the mechanism of the development of fibrosis, current therapeutic options are limited to emerge as an effective antifibrotic agent. Therefore, it is necessary to provide an update on
Abbreviations: miRNA, microRNA; ECM, extracellular matrix; MMPs, matrix metalloproteinases; HSC, hepatic stellate cell; COL, collagens; a-SMA, a-smooth muscle actin; TGF-b, transforming growth factor-b; 30 UTR, 30 untranslated region; PDGF, platelet-derived growth factor; SMAD3, signaling effectors (mothers against decapentaplegic protein)3; LPS, lipopolysaccharide; NF-kB, nuclear factor kappa B; FXR, nuclear receptor farnesoid X receptor; IGF, insulin-like growth factor; HGF, hepatocyte growth factor; CTGF, connective tissue growth factor; FBN1, fibrillins. * Corresponding author. School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei 230032, Anhui Province, China. Tel./fax: þ86 551 65161001. E-mail addresses: [email protected], [email protected] (J. Li).
our understanding of fibrosis for presenting novel therapeutic strategies against tissue fibrogenesis. MicroRNAs (miRNAs), which are endogenous small non-coding RNAs (18e25 nucleotides) that work as post-transcriptional regulators of gene expression by binding to the 30 untranslated region (30 UTR) of target mRNAs [14e17]. Most miRNA genes are located in introns, exons and UTRs of protein coding genes [18]. miRNAs are transcribed as long polyadenylated RNAs, the pri-miRNAs molecules are transcribed by RNA-Polymerase II and cut by the endonuclease Drosha, releasing intermediate approximately 60-nucleotide-long hairpin structures called pre-miRNAs [19]. The pre-miRNAs are exported to the cytoplasm by exportin 5 and further processed into approximately 22-nucleotide-long mature miRNA by another endonucleases, Dicer, and RNase III. Each mature miRNA is then integrated into the RNA-induced silencing complex (RISC), this miRNA/RISC complex mediates gene expression by causing translational repression or mRNA degradation [14,16]. One miRNA might bind to a number of mRNAs transcripts and in turn one mRNA can be targeted by a widespread panel of miRNA species. Recently, multiple lines of evidence suggests that aberrant miRNA expression has been shown to be related with the development of various tissue fibrosis diseases, for example, miR-146a inhibited TGF-b1-induced HSC proliferation and a-SMA expression, at least in part, via inhibiting Smad4 [20], miR-21 mediated fibrogenic activation of pulmonary fibroblasts and lung fibrosis through regulating the expression of an inhibitory Smad, Smad7 [21], miR-126 was downregulated in cystic fibrosis airway epithelial cells and altered expression of miR-126 was a negative regulator of TOM1 [22]. The miR-29 family is composed of miR-29a, miR-29b, and miR29c, differing only in two or three bases, miR-29a and miR-29b1 as
0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.03.010
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well as miR-29c and miR-29b2. MiR-29 family are encoded and transcribed in tandem by two genes located on chromosome7 or chromosome1, respectively [23]. The members of the miR-29 family are of special interest, because miR-29 family has been shown to be involved in inhibiting extracellular matrix proteins (ECM) synthesis indicating an antifibrotic function. To our surprise, the Targetscan prediction program indicates that miR-29 family has potential to repress many kinds of collagens (COL1A1, COL 2A1, COL3A1, COL4A1, COL 4A3, COL 4A5, COL 5A1, COL 5A2, COL 6A3, et al) synthesis via binding to its 30 UTR (http://www.targetscan. org/cgi-bin/target-scan/vert_50/targetscan.cgi?mirg¼rno-miR29a). Recently, there is overwhelming evidence that aberrant miR29 family expression was involved in the development of tissue fibrosis, such as liver fibrosis, renal fibrosis, pulmonary fibrosis, cardiac fibrosis. In this review, we make a summary on the functional roles of miR-29 family members in the fibrotic process of several organs and its potential as novel therapeutic target. 2. miR-29 family and liver fibrosis Liver fibrosis occurs in many types of chronic liver diseases and is characterized by an excessive accumulation of ECM [24e26]. Hepatic stellate cells (HSCs) are considered to contribute to fibrogenic reactions and the activation of HSCs has been shown to play a central role in the development of liver fibrosis [27e29]. Following exposure to inflammatory cytokines, activated HSCs lose their lipid droplets, migrate to injured sites and are transformed into myofibroblast-like cells that express a-SMA, secrete profibrogenic mediators finally leading to liver fibrosis [30]. In these inflammatory cytokines, transforming growth factor beta 1 (TGF-b1) is believed to be recognized as the main profibrogenic mediator [31,32]. Recently, the microarray analysis revealed that the members of miR-29 family (miR-29a, 29c) were identified to be downregulated in activated HSCs as compared to that in quiescent HSCs [33,34]. Some reports demonstrated that all three members of the miR-29 family were significantly downregulated in livers of carbon tetrachloride (CCl4)-treated mice as well as in mice that underwent bile duct ligation by using real-time qPCR [35]. Next, the authors went on finding that TGF-b1 as well as inflammatory signal molecules, namely, lipopolysaccharide (LPS) and nuclear factor kappa B (NFkB) could downregulate the expression of miR-29 in murine HSC. Interestingly, TGF-b stimulation led to decreased miR-29 levels, while hepatocyte growth factor (HGF) stimulation resulted in elevated miR-29 expression in HSC and miR-29a/b overexpression caused a significantly reduction of collagen-I and -IV synthesis by targeting the 30 UTR of the collagen-1 and -4 subtypes [36]. Ogawa et al. [37] also found that miR-29b was the most effective suppressor of type I collagen at the mRNA and protein level via its direct binding to Col1A1 30 UTR. In primary HSCs, the expression of miR-29 family was also downregulated under TGF-b1 stimulation [35]. During HSC activation in primary culture, miR-29b overexpression markedly attenuated Col1a1 and Col1a2 mRNAs expression and additionally inhibited the increased expression of aSMA, DDR2, FN1, ITGB1, and PDGFR-b, which were key genes involved in the activation of HSCs [38]. Additionally, miR-29 acted as an antifibrogenic mediator by interfering with profibrogenic cell communication via platelet-derived growth factor (PDGF)-C and insulin-like growth factor (IGF)-I [39]. Recently, the nuclear receptor farnesoid X receptor (FXR) was reported to have potent antifibrotic activity in HSCs [40,41]. Interestingly, miR-29a was a nuclear receptor farnesoid X receptor (FXR) target gene because a genetic activation of FXR enhanced miR-29a promoter activity, therefore, miR-29a was involved in the antifibrotic effect of FXR in HSC [42]. In line with these early observations suggested that
miR-29 family may function as a negative regulator in HSC activation, further functional analysis to determine the precise roles of miR-29 family in liver fibrosis provides a novel therapeutic approach for treating liver fibrosis (Fig. 1). 3. miR-29 family and renal fibrosis Renal fibrosis characterized by accumulation of fibroblasts and excessive matrix proteins is a hallmark of end-stage renal disease and is a major cause of therapeutic failure [43,44]. TGF-b1 has been considered as a key mediator in renal fibrosis and mainly causes renal fibrosis by activating its downstream Smad signaling pathway [45]. Smads can be divided into three distinct subclasses: receptoractivated Smads (R-Smads), common-partner Smads (Co-Smads) and inhibitory Smads (anti-Smads). In TGF-b1/Smad signaling pathway, Smad2 and Smad3 function as R-Smads, Smad4 functions as Co-Smad, and Smad7 functions as an anti-Smad. Recent studies revealed that Smad3, but not Smad2, was a key signaling pathway of fibrogenesis in response to TGF-b1 and TGFb1/Smad3 signaling could promote renal fibrosis [46,47], but the downstream fibrosis-specific genes of TGF-b1/Smad3-mediated fibrosis remain unclear. However, Qin et al. [48] have indicated that wild-type mice had reduced expression of miR-29 along with the development of progressive renal fibrosis in obstructive nephropathy by using a microRNA microarray and real-time PCR. The authors finally revealed that miR-29 was lost with progressive renal fibrosis in Smad3 wild-type mice, but increased with protection against renal fibrosis in Smad3 knockout mice in UUO Nephropathy. Moreover, in cultured fibroblasts and tubular epithelial cells, TGF-b1 downregulated miR-29b expression, while upregulated collagen-I and -III expression via the Smad3-dependent, not Smad2-dependent mechanism and Smad3-mediated TGF-b1induced downregulation of miR-29 by binding to the promoter of miR-29. In vitro, overexpression of miR-29b inhibited, but knockdown of miR-29 increased, TGF-b1-induced expression of collagens I and III, but not a-SMA expression in renal tubular cells. Interestingly, gene transfer of miR-29 prevented renal fibrosis in a mouse model of obstructive nephropathy by transfecting Dox-inducible miR-29b-expressing plasmid system into left kidney of mouse [48]. In addition, Wang et al. [49] have reported that the expression of the miR-29 family which targeted collagen gene expression was reduced and the expression of ECM proteins was increased by exposing proximal tubular cells, primary mesangial cells, and podocytes to TGF-b1. Furthermore, in both resting and TGF-b1treated cells, ectopic expression of miR-29 repressed the
Fig. 1. The role of miR-29 family in liver fibrosis. TGF-b, LPS and NF-kB stimulation could lead to decreased miR-29 levels, while HGF stimulation could result in elevated miR-29 expression in HSCs. miR-29a/b overexpression in HSC caused a significantly reduction of collagen synthesis by targeting the 30 UTR of the collagen and interfering with profibrogenic cell communication via PDGF-C and IGF-I.
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expression of collagens I and IV at both the mRNA and protein levels by targeting the 30 UTR of these genes. Interestingly, administration of the Rho-associated kinase inhibitor fasudil prevented renal fibrosis and restored expression of miR-29 [49]. Therefore, it is important to further investigate the roles of miR-29 in renal fibrosis, which may have therapeutic potential for progressive renal fibrosis (Fig. 2). 4. miR-29 family and pulmonary fibrosis Pulmonary fibrosis is a chronic fibrosing interstitial lung disease of unknown etiology, which leads to the thickening of alveolar walls and scarring of the lung by exposure to chronic inflammatory responses, including persistent infections, autoimmune reactions, allergic responses, and tissue injury [50,51]. Recently, Cushing et al. [52] have found that all the members of miR-29 family were downregulated by TGF-b1 in IMR-90 cells and in bleomycin-treated lungs. Additionally, miR-29 downregulation was also observed in mesenchymal cells at the entrance of the alveolar duct and in the subepithelial mesenchyme of terminal bronchioles. Moreover, all reported miR-29 targets were significantly upregulated in miR-29 knockdown cells, including Col1A1, Col1A2, Col3A1, Col4A1, Col4A2, and Col15A1 by performing genome-wide array analysis of IMR-90 cells 48 h after transfection of miR-29 antisense or control oligos, and ITGA11, ADAM12, ADAMTS9, and NID1 were shown to be direct targets of miR-29. In addition, many fibrosis-associated genes upregulated by TGF-b1 were suppressed by miR-29 knockdown. Interestingly, a comparison of TGF-b1 and miR-29 targets revealed that miR-29 controlled an additional subset of fibrosisrelated genes, including laminins and integrins, independent of TGF-b1 [52]. Furthermore, Yang et al. [53] indicated that miR-29 mediated TGF-b1-induced extracellular matrix synthesis through activation of PI3K-AKT pathway in human lung fibroblasts. To our surprise, miR-29 was also found to be a downstream target gene of Smad3 and negatively regulated by TGF-b/Smad signaling in pulmonary fibrosis [54]. Moreover, mice or pulmonary fibroblasts null for Smad3 were shown to protect against bleomycin or TGF-b1-induced loss of miR-29 along with fibrosis in vivo and in vitro. Interestingly, overexpression of miR-29 could in turn negatively regulate TGF-b and connective tissue growth factor (CTGF) expression and Smad3 signaling. Sleeping Beauty (SB)mediated miR-29 gene transferred into normal and diseased lung tissues was able to prevent and treat pulmonary fibrosis including inflammatory macrophage infiltration induced by bleomycin in mice [54]. Idiopathic pulmonary fibrosis (IPF) is a chronic progressive and lethal fibrotic lung diseases [55,56]. The role of miR-29 in IPF lungs remain still unclear, but it has been reported that a significant decrease in miR-29 in the lungs of IPF patients [57]. So,
Fig. 2. The role of miR-29 family in renal fibrosis. TGF-b1 acted by stimulating Smad3 to negatively regulate the miR-29 families, which inhibited TGF-b1-induced expression of collagens I and III, to mediate renal fibrosis.
Fig. 3. The role of miR-29 family in pulmonary fibrosis. All miR-29 family members were downregulated by TGF-b1 stimulation. MiR-29 mediated TGFb1-induced collagens synthesis through activation of PI3K-AKT pathway and could negatively regulate TGF-b1 and CTGF expression and was also found to be a downstream target gene of Smad3 in human lung fibroblasts.
further studies to clarify the precise roles of miR-29 family in IPF could provide a potential therapeutic agents for treating pulmonary fibrosis (Fig. 3). 5. miR-29 family and cardiac fibrosis Cardiac fibrosis is an important pathological feature of structural remodeling which results in ventricular stiffness, diminished contractility, and abnormalities in cardiac conductance [58,59]. Recently, van Rooij et al. [60] have found that miR-29 was dramatically downregulated in the region of the fibrotic scar after myocardial infarction (MI) and the miR-29 family targeted many mRNAs that encoded proteins involved in fibrosis, including multiple collagens(COL1A1, COL1A2, COL3A1), fibrillins (FBN1), and elastin. Furthermore, downregulation of miR-29 with anti-miRs in vitro and in vivo was sufficient to increase the expression of collagens, whereas overexpression of miR-29 in fibroblasts inhibited collagens expression. Recently, miR-29 family includes miR-29s, which was reported to directly target at least 16 extracellular matrix genes. Furthermore, miR-29s had strong antifibrotic effects on heart, kidney, and other organs and miR-29s was also demonstrated to be proapoptotic and involved in the regulation of cell differentiation [61]. Unfortunately, the specific mechanism of miR29 family in cardiac fibrosis is not very clear and the downregulation of miR-29 provides a novel idea for cardiac fibrosis, which suggests that strategies to increase miR-29 expression may have therapeutic value in treating post-MI cardiac remodeling (Fig. 4).
Fig. 4. The role of miR-29 family in cardiac fibrosis. TGF-b1 could trigger the downregulation of miR-29 in cardiac fibroblasts and then upregulate the expression of ECM (COL1A1, COL1A2, COL3A1, and FBN1) involved in cardiac fibrosis.
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6. Conclusion and prospective On the basis of other findings, we make a summary on the comprehensive knowledge of the biological roles of miR-29 family in various fibrosis diseases, including in the fibrosing pathogenesis of liver, kidney, lung and heart. Recently, research focusing on small molecules such as miRNAs has been aroused to understand their roles in diseases. In particular, miR-29 family was considered to be a very important and common regulator of tissue fibrosis, because many reports have revealed that miR-29 family could repress collagen synthesis via direct binding to its 30 UTR in fibroblasts. To our excitement, an artificial intronic miRNA expression system had been established, and activated-HSC-cell-specific gene silencing could be induced well by the artificial intronic miRNA expression system to realize antifibrosis in vitro [62]. It is increasingly evident that miRNAs antagonists or agonist had been proposed as a potentially novel therapeutic tools [21,63]. Recent studies showed that miR-29 family also connected with DNA methylation was involved in many diseases [64e66]. So, the effect of miR-29 family on diseases was very extensive and indepth understanding of miR29 family with their cell-specific roles may provide a rationale for the development of miRNA-based strategies for the treatment of fibrosis diseases.
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Conflict of interest None declared. Acknowledgments This project was supported by the National Science Foundation of China (No 30873081, 81072686). References
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