TGF-β is a central mediator of fibrosis [1
] and it is ubiquitously expressed in tissues [85
]. TGF-β also induces expression of CTGF, and CTGF can in turn enhance TGF-β signaling, along with a number of other pro-fibrotic factors including vascular endothelial growth factor (VEGF), endothelin-1 (ET), insulin-like growth factor-1 (IGF-1), integrins, and Wnts [86
]. Therefore, miRNA regulation of TGF-β and CTGF signaling is of paramount interest. Up-to-date, 10 miRNAs showed consistent experimental evidence of being TGF-β/CTGF signaling regulators. Six of them were dysregulated in more than one fibrotic disorder (see Table
miR-17-92 is a proto-oncogenic cluster (also called oncomir-1) consisting of six miRNAs. Very recently, miR-18a and miR-19a/b from this cluster have been shown to regulate CTGF and thrombospondin-1 (TSP-1) in the context of liver and cardiac fibrosis [60
]. In a previous report, CTGF and TSP-1 were validated as direct miR-18a/miR-19a/b targets in cancer [87
]. Kodama et al
] reported down-regulation of the miR-17-92 cluster in liver fibrosis in a p53-dependent manner and an associated up-regulation of CTGF. Similarly, van Almen et al
] showed down-regulation of the miR-17-92 cluster and spontaneous upregulation of CTGF and TSP-1 in a mouse model of age-related heart failure, which is characterized by widespread interstitial EMC accumulation. Down-regulation of the miR-17-92 cluster paralleled by CTGF and TSP-1 gene up-regulation was confirmed in human idiopathic cardiomyopathy specimens. Transfection experiments in aged neonatal rat cardiomyocytes were used to confirm CTGF and TSP-1 modulation by miR-18a and 19a/b. Down-regulation of the miR-17-92 cluster has also been reported in human IPF lung tissue [48
miR-133, miR-30 and miR-590.
Two other major regulators of CTGF expression in cardiac fibrosis are miR-133 and miR-30. miR-133 is a cardiac specific miRNA and its expression is limited to cardiomyocytes, while miR-30 is expressed in both cardiac fibroblasts and cardiomyocytes as well as in other tissues. Duisters et al
] reported miR-133- and miR-30c-mediated repression of CTGF in two animal models of overloaded heart disease: a rat renin-dependent hypertensive failing heart model (Ren2 rats) and a mouse mechanical stress-induced model (mice undergone transverse aortic constriction). They first showed down-regulation of the two miRNA families and up-regulation of CTGF in vivo
, then confirmed the direct interaction of miR-133 and miR-30c with CTGF mRNA in vitro
using cultured cardiomyocytes and cardiac fibroblasts, and finally proved the functional effects of this interaction on collagen synthesis. Interestingly, the authors also showed that miR-133 and miR-30c do not exert additive effects on CTGF regulation, which might be explained by the overlapping seed regions of these miRs in the 3’UTR of the CTGF gene. Similar down-regulation of miR-133 and miR-30c and increased levels of CTGF were observed in human hypertrophic heart.
The importance of miR-133 down-regulation in cardiac fibrosis is further supported by the findings of Shan and colleagues [53
], who investigated the expression of miR-133 in the heart of smokers with chronic atrial fibrillation (AF), and in a canine model of nicotine-induced AF. Fibrotic structural remodeling was strongly induced by nicotine in both human and canine heart tissue. This was associated with an increase in TGF-β1 and the TGF-β pathway downstream targets CTGF and collagens both in vivo
and in vitro
. By in silico analysis, the authors selected miR-133 and miR-590 as potential regulators of TGF-β1 and TGF-βRII, respectively. miR-133 and miR-590 were strongly down-regulated in dog atrial fibroblasts treated with nicotine, and the direct interaction of the two miRNAs with their predicted targets could be confirmed by reporter gene assay. miR-30c down-regulation has also been reported in human IPF lung tissue [48
Most data link the five miR-200 family members to EMT [54
], thus indicating an indirect role in fibrogenesis. Recent data from Wang et al
] showed in addition that the miR-200 family contributes directly to fibrosis by regulation of TGF-β2. Expression of miR-141 and miR-200a has been investigated in mouse models of early (ApoE knockout mice injected with streptozotocin) and advanced (C57BL6 mice treated with adenine) diabetic nephropathy, as well as in rat proximal tubular epithelial cells (NRK52E). Both miRNAs were down-regulated in mice, but only miR-200a was sensitive to TGF-β stimulation in NRK52E cells. At the same time, TGF-β stimulation induced transformation of NRK52E cells towards a mesenchymal phenotype as indicated by reduced E-cadherin expression and increased vimentin and α-SMA expression along with enhanced ECM synthesis. In turn, forced up-regulation of both miR-141 and miR-200a in NRK52E cells reduced TGF-β2, Smad3 activity and ECM proteins, and also prevented EMT, suggesting a miR-200a/TGF-β feedback loop in the pathogenesis of diabetic CKD. In addition, reporter gene assays confirmed TGF-β2 as a direct target of miR-141/miR-200a.
Like the miR-17-92 cluster, miR-21 is traditionally regarded as an oncomir, and due to its ubiquitous expression it is one of the most widely investigated miRs [89
]. Evidence for an important role of miR-21 in fibrosis comes from studies on heart, lung and kidney [68
]. Thum and colleagues [69
] described miR-21 up-regulation in three mouse models of heart failure: β1
-adrenergic receptor transgenic mice, C57BL/6 mice undergone transverse aortic constriction, and C57BL/6 mice treated with isopropanol. In different cell types isolated from failing hearts, miR-21 was predominantly up-regulated in cardiac fibroblasts, and mediated protection from apoptosis. Anti-miR-21 treatment and extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK) blockade induced apoptosis to a similar extent, suggesting a possible regulation of ERK/MAPK signaling by miR-21. Accordingly, overexpression of miR-21 in both mouse and rat fibroblasts led to increased expression and activation of ERK/MAPK proteins. The authors identified sprouty1, a negative regulator of ERK/MAPK, as the direct target of miR-21 mediating the effects on this pathway. Nevertheless, more recently, Patrick et al
] demonstrated in mir21-/-
mice, which were exposed to different stimuli resulting in cardiac overload, that miR-21 contributes to, but is not essential for ERK/MAPK activation in stress-dependent cardiac remodeling.
Liu et al.
] confirmed the pro-fibrotic activity of miR-21 in human IPF and murine bleomycin-induced lung fibrosis. Like in heart failure, miR-21 up-regulation was proportional to the severity of lung fibrosis in the animal model. TGF-β1 expression showed the same trend. Therefore, the authors elucidated the causal relation between miR-21 and TGF-β1, showing that miR-21 expression was reduced in TGFβRII dominant negative mice injected with bleomycin. Consistently, TGF-β1 treatment of cultured pulmonary fibroblasts induced miR-21 up-regulation and, in turn, miR-21 overexpression induced TGF-β1 expression, Smad2 activation, and ECM synthesis. These effects were mediated by direct repression of Smad7, the inhibitory Smad which counter-regulates Smad2/3 activation. Finally, administration of miR-21 antisense oligonucleotides in vivo
attenuated bleomycin-induced lung fibrosis.
Studies on the role of miR-21 in renal fibrosis further elucidated the interaction of this miRNA with TGF-β/Smad signaling [70
]. In these experiments, TGF-β-mediated upregulation of miR-21 in tubular epithelial cells was prevented by knockdown of Smad3, but not by knockdown of Smad2. Consistently, Smad3 deficient mice, but not Smad2 deficient mice, exposed to kidney injury by unilateral urethral obstruction were protected from miR-21 up-regulation and fibrosis.
miR-216, miR-217 and miR-192.
miR-216 and miR-217 are two clustered miRNAs encoded in the second intron of the RP23 gene. They showed pro-fibrotic effects in murine mesangial cells (MCs) [71
]. Kato et al
. reported upregulation of miR-216/217 in mouse MCs following TGF-β treatment, with consequent amplification of TGF-β signaling. miR-216 and miR-217 directly target phosphatase and tensin homolog (PTEN), which inhibits the phosphoinositide-3-kinase pathway (PI3K), thereby preventing activation of Akt. Thus, down-regulation of PTEN de-represses Akt signaling in MCs leading to prolonged cell survival, hypertrophy and increased collagen synthesis. In addition, since the promoter region of the RP23 gene contains multiple Smad binding elements and E-boxes, the authors could demonstrate that the expression of miR-216 and miR-217 in MCs was induced by TGF-β and zinc finger E-box-binding homebox (Zeb) 1 and 2. In turn, Zeb 1 and 2 are regulated by miR-192 [62