TGF-β is well known as a pivotal mediator in the pathogenesis of fibrosis (60,61). TGF-β upregulates numerous genes associated with fibrogenesis and promotes the development of fibrosis via its downstream mediators, including Smad2 and Smad3, while fibrosis is inhibited by Smad7 (62–64). Deletion of Smad3 protects from fibrosis in mice (65). However, deletion of Smad2 significantly enhanced Smad3 expression, thereby leading to ECM expansion and fibrosis development, which indicated that Smad2 functions as a negative regulator of TGF-β signaling (66). Activation of TGF-β receptor by TGF-β may induce the regulatory Smad3 proteins, which then initiate Smad4-dependent transcription (63). Of note, TGF-β1 is known to upregulate miR-21 expression (64). TGF-β signaling and Smad3 have important roles in miR-21 transcription, and Smad proteins control the maturation of miR-21 from pre-miR-21 (64,67). Smad2 and Smad3 have opposite roles in regulating the biogenesis of miR-21 - Smad3 promotes, while Smad2 prevents the expression of pre-miR-21 after stimulation with TGF-β (66,68). Smad7 is a direct target of miR-21 and is therefore depressed following upregulation of mature miR-21 (38,69).

TGF-β1 can induce the differentiation of fibroblasts into the more fibrogenic myofibroblasts (19). Its downstream mediator Smad3 was also shown to regulate the expression of Cols, fibronectin (FN), and α-smooth muscle actin (α-SMA) directly, by binding to their promoters (62). α-SMA has been proved to be critical for the trans-differentiation of hepatic stellate cells (HSCs) into myofibroblasts (70).

The crosstalk between miR-21 and the TGF-β/Smads signaling pathway can regulate the fibrotic procedure though downstream proteins in a variety of signaling pathways.

TGF-β can activate PI3K/AKT signaling though inhibiting phosphatase and tensin homolog (PTEN) (71,72), and it is well known that PTEN is a negative factor of the PI3K/AKT signaling pathway (73,74). The expression of fibrotic proteins mediated TGF-β, including FN and Cols, is regulated by PI3K/AKT signal transduction (72,75).

TGF-β has been demonstrated to regulate PI3K-dependent mammalian target of rapamycin (mTOR) to increase cellular hypertrophy (76–78). mTOR is a downstream protein of the PI3K/AKT signaling pathway. It has been shown that TGF-β can activate PI3K/AKT signaling with targeting of PTEN, and RhoA may be an upstream mediator of Akt activation and involved in TGF-β-induced activation of PI3K/AKT signaling in response to TGF-β1 (71). Akt activation has an important role in the EMT (79) as well as the EndMT (80), likely though the nuclear factor-κB (NF-κB) pathway (79), as AKT signaling can activate NF-κB signaling (81). PTEN has been demonstrated to be a direct target of miR-21 (34,74). In conclusion, miR-21 can regulate the process of fibrosis though PI3K/AKT signaling by targeting PTEN.

ERK signaling is known to have a significant impact in fibrogenesis (82). Suppression of ERK expression can block EMT of hepatic fibrosis in rats (82). Sprouty (Spry) is a target of miR-21 (32,83,84) as well as a potent inhibitor of the ERK/MAPK signaling pathway (74). Spry1 expression and MAPK signaling activation in the myocardium were completely reversed by treatment with an antagonist of miR-21 (32). In addition, miR-21 inhibitors have been shown to increase the mRNA transcript levels of Spry2 and significantly reduce the expression of profibrotic genes in activated HSCs, including TGF-β1, α-SMA and collagen (85). In addition, ERK signaling can activate the NF-κB signaling pathway (86). NF-κB signaling was shown to inhibit miR-21 expression by binding directly to its promoter and regulate the process of arsenite-induced cell transformation (87).

The signaling pathways of fibrosis are interconnected; for instance, the PI3K pathway positively regulates Smad3 transcription and increases Col production, suggesting that activation and interaction of diverse signaling pathways contributes to the pathogenesis of fibrosis (75) (Fig. 1). On the other hand, ERK signaling also interacts with AKT signaling through the NF-κB pathway, however, these pathways affect miR-21 production. In turn, miR-21 regulates these pathway through different target proteins. In conclusion, miR-21 may be a key factor in the interaction network.

It is worth mentioning that other targets of miR-21, including programmed cell death-4, tissue inhibitor of metalloproteinases-3 and reversion-inducing cysteine-rich protein with kazal motifs have functions in the migration and invasion of cancer cells or traumatic brain injury (88–90), and that these miR-21-mediated targets are negative regulators of matrix metalloproteinases (MMPs) (37,91–93). While the exact roles of miR-21 in these complex interactions remain to be fully elucidated, it has a key role in fibrosis through interlinking several fibrosis-associated pathways and is therefore a promising target for the treatment of fibrosis.

The majority of cardiovascular diseases, including heart failure, myocardial infarction and atrial fibrillation, are associated with myocardial remodeling. Cardiac fibrosis is an important process in myocardial structural remodeling (98–100). miR-21 can control myocardial structural remodeling via several pathways. A study using a cardiovascular disease model demonstrated that cardiac stress can upregulate the expression of miR-21, which mediates the activation of ERK/MAPK signaling though targeting Spry1 to positively regulate cardiac fibroblast survival and growth factor secretion, resulting in fibrosis, remodeling and cardiac dysfunction; however, this process was inhibited by silencing of miR-21 (32). Similarly, a study on patients with atrial fibrillation showed that miR-21 was upregulated and Spry1 was decreased (95). In mice, the increased expression of miR-21 and the decreased expression of Spry1 by angiotensin II contributed to fibroblast survival and structural remodeling in the atrial myocardium (95). Studies on other pathways have reported a variety of roles for miR-21. In the pathogenic process of myocardial fibrosis, miR-21 and TGF-β1 were shown to be upregulated, while TGF-βRIII was downregulated (96). Overexpression of TGF-βRIII inhibited the expression of miR-21 in fibroblasts, probably via phosphorylated Smad3. However, overexpression of miR-21 induced by TGF-β1 led to the differentiation of fibroblasts into pathological myofibroblasts and increased the Cols content. Fibrosis is therefore regulated by a reciprocal feedback loop between TGF-βRIII and miR-21 (96). MMP-2 degrades the ECM and knocks down PTEN in cardiac fibroblasts, resulting in marked phosphorylation of AKT signaling and induction of MMP-2. In a murine model of myocardial infarction, miR-21 was shown to regulate MMP-2 expression in fibroblasts via PTEN, leading to cell hypertrophy and matrix protein expression (44). These findings may provide a direction for further studies.

miRs have been extensively studied in liver fibrosis, particularly the miR-29 family, miR-122 and miR-21 (101,102). Among them, miR-21 was found to have significant effects in the process of fibrosis development, while numerous risk factors, including hepatitis C virus (HCV), fatty liver, alcohol consumption and schistosome infection can cause fibrosis (54). The target cells in liver fibrosis are HSCs (42,43,54,85,94,103), and activated HSCs are responsible for the imbalance of secretion and degradation of the ECM (104). Livers of mice and humans with hepatic fibrosis in non-alcoholic steatohepatitis have shown increased levels of miR-21 with co-localized overexpression of TGF-β, Cols, α-SMA and Smad2/3-Smad4, while the protein levels of Smad7 were decreased (43). However, treatment with leptin-nicotinamide adenine dinucleotide phosphate oxidase reversed these effects in mice. Similar observations were made in HCV patients: miR-21 overexpression enhanced TGF-β signaling, and Smad7 was a negative regulator of TGF-β signaling in a positive feedback cycle to promote HSC survival and proliferation (54,103). The fibrogenic role of miR-21 was also found in biliary atresia patients and in an in vitro experiment, which it exerted through the PTEN/AKT signaling pathway and targeting of PTEN. miR-21 was shown to activate AKT signaling via phosphorylation, alongside decreased expression of MMP-2 and increased expression of α-SMA (42,94), which indicated that miR-21 promotes the transdifferentiation of HSCs into myofibroblasts as well as ECM formation. HSCs are likely to be activated via various routes, including the ERK signaling pathway. miR-21 directly interacts with the 3′-UTR of Spry2, resulting in enhanced ERK signaling in HSCs (85). Enhanced expression of α-SMA and vimentin showed that miR-21 can regulate the EMT as well as ECM formation through ERK signaling (85).

Renal fibrosis is characterized by an excess accumulation of ECM and myofibroblasts, as fibrosis in other organs, and features tubule atrophy. Gene therapy by Smad7 overexpression in obstructive nephropathy-induced fibrosis suppresses miR-21, Col, FN and α-SMA (69). Furthermore, TGF-β1 was shown to upregulate miR-21 expression via Smad3, but to downregulates it via Smad2 in tubular epithelial cells (TECs) (68). It is therefore indicated that miR-21 and its regulation via TGF-β/Smads signaling has a significant role in renal fibrosis though ECM formation and EMT. Interstitial fibrosis and tubular atrophy influence renal outcomes of immunoglobulin (Ig)A nephropathy (105). A recent study on both podocytes and TECs of patients with IgA nephropathy showed that miR-21 expression was upregulated (41). Inhibition of miR-21 prevented fibrogenic activation in podocytes and TECs by reversing the overexpression of FN and Col though inhibiting AKT pathway activation. Another study has revealed that miR-21 can suppress the AKT pathway via its target PTEN and activate the downstream protein mTORC1, leading to hypertrophy of mesangial cells and matrix protein expression (78), as mesangial cells also take a vital role in renal fibrosis (61).

Like in other tissues and organs, TGF-β1 is well known to be an important conditioner of lung fibrosis and can induce fibroblast-to-myofibroblast differentiation characterized by α-SMA expression and high expression of ECM (46,106). The EMT has a distinct role in lung fibrosis. In a study of idiopathic pulmonary fibrosis, miR-21 was shown to be highly expressed and primarily localized to myofibroblasts, alongside an increase in α-SMA expression (40). The increased miR-21 promotes fibrogenic activity of TGF-β1 in fibroblasts though regulating the expression of Smad7. Another study showed that miR-21 is upregulated in isolated lung epithelial cells induced by bleomycin. Furthermore, PTEN has been shown to have a negative regulatory role in experimental lung fibrosis (107).

Systemic sclerosis is known as a process of fibrosis with increased expression of miR-21 (39). TGF-β is also implicated in the pathogenesis and progression of skin fibrosis (108). Normal skin fibroblasts incubated with TGF-β1 showed overexpression of miR-21 (39). In bleomycin-treated mice, the pro-fibrogenic activity caused by α-SMA and Cols overexpression stimulated by miR-21 was identified to be mediated via the TGF-β signaling pathway by targeting Smad7 (39). Hypertrophic scarring, the most common type of skin disorder, occurs after abnormal wounding, and miR-21 has been reported to be of relevance in this process via the PI3K/AKT signaling pathway through regulating the expression of human telomerase reverse transcriptase (hTERT) (97). It was revealed that fibroblasts transfected with PTEN showed decreased hTERT mRNA and protein expression. An identical mechanism was found to promote cell survival and proliferation in keloid fibroblasts (57).