miR‐193a/b‐3p relieves hepatic fibrosis and restrains proliferation and activation of hepatic stellate cells

Abstract MicroRNAs (miRNAs) have been confirmed to participate in liver fibrosis progression and activation of hepatic stellate cells (HSCs). In this study, the role of miR‐193a/b‐3p in concanavalin A (ConA)‐induced liver fibrosis in mice was evaluated. According to the results, the expression of miR‐193a/b‐3p was down‐regulated in liver tissues after exposure to ConA. Lentivirus‐mediated overexpression of miR‐193a/b‐3p reduced ConA‐induced liver injury as demonstrated by decreasing ALT and AST levels. Moreover, ConA‐induced liver fibrosis was restrained by the up‐regulation of miR‐193a/b‐3 through inhibiting collagen deposition, decreasing desmin and proliferating cell nuclear antigen (PCNA) expression and lessening the content of hydroxyproline, transforming growth factor‐β1 (TGF‐β1) and activin A in liver tissues. Furthermore, miR‐193a/b‐3p mimics suppressed the proliferation of human HSCs LX‐2 via inducing the apoptosis of LX‐2 cells and lowering the levels of cell cycle‐related proteins Cyclin D1, Cyclin E1, p‐Rb and CAPRIN1. Finally, TGF‐β1 and activin A‐mediated activation of LX‐2 cells was reversed by miR‐193a/b‐3p mimics via repressing COL1A1 and α‐SMA expression, and restraining the activation of TGF‐β/Smad2/3 signalling pathway. CAPRIN1 and TGF‐β2 were demonstrated to be the direct target genes of miR‐193a/b‐3p. We conclude that miR‐193a/b‐3p overexpression attenuates liver fibrosis through suppressing the proliferation and activation of HSCs. Our data suggest that miR‐193a‐3p and miR‐193b‐3p may be new therapeutic targets for liver fibrosis.

effective pro-fibrotic cytokine, which promotes the synthesis of ECM proteins. 9 Blocking of TGF-β signalling could partly alleviate liver fibrosis. 10 MicroRNAs (miRNAs) are a kind of small non-coding singlestrand RNAs that repress protein translation via pairing to the 3′ untranslated regions of their target mRNAs. 11 Increasing evidence has demonstrated the pivotal roles of miRNAs in the pathogenesis of liver fibrosis. [12][13][14] A research by Ma et al reported that miR-214 promoted the development of liver fibrosis via inducing the activation of HSCs. 15 In addition, miR-873-5p has been demonstrated to be a marker for liver fibrosis and regulate the early stages of liver fibrosis. 16 MiR-351 has been confirmed to promote liver fibrosis via targeting the vitamin D receptor, and miR-351 inhibition is recognized as a therapeutic intervention for fibrotic diseases. 12 MiR-193a and miR-193b are members of miR-193 family, which are well conserved in different species. MiR-193a-3p/miR-193a-5p and miR-193b-3p/ miR-193b-5p are mature miRNAs produced by miR-193a/miR-193b.
Roy et al found that the expression level of miR-193a-3p was decreased not only in the liver tissues of patients with liver cirrhosis but also in CCL 4 -induced liver fibrosis in mice. 17 In addition, a previous study showed that the level of miR-193b was down-regulated in human liver fibrosis samples, as compared with normal liver samples. 18 So miR-193 family may participate in the regulation of liver fibrosis, whereas its potential mechanisms are unclear and worthy of further investigation.
Concanavalin A (ConA) is a common inducer of immune-mediated liver injury. After intravenous injection of ConA, an immunerelated liver fibrosis could be detected in mice. 19 In this study, the effect of miR-193a/b-3p on ConA-induced liver fibrosis was evaluated in mice. Moreover, whether miR-193a/b-3p affected the viability and activation of HSCs was determined in human LX-2 cells.

| Animals and induction of liver fibrosis in mice
Male BALB/c mice at 8-10 weeks of age were obtained from Liaoning changsheng biotechnology co., Ltd. The mice were fed adaptively for 1 week. To induce liver fibrosis, the mice were injected with ConA
One week after the sixth ConA injection, the mice were killed by intraperitoneal injection of pentobarbital sodium (200 mg/kg). The serum samples and liver tissues were obtained for subsequent tests.

| RNA isolation and quantitative real-time PCR
Total RNAs were extracted using TRIpure reagent (BioTeke, Beijing, China) from liver tissues and LX-2 cells. Then total RNAs were reversely transcribed into first-strand cDNA using Super M-MLV (BioTeke). Quantitative real-time PCR was carried out using 2 × Power Taq PCR MasterMix (BioTeke) and SYBR Green (Solarbio, Beijing, China) on an Exicycler™ 96 real-time quantitative thermal block (Bioneer, Daejeon, Korea). The primers used for PCR amplification were listed in Table 1. Expression values of mRNAs and miRNAs were normalized with β-actin and U6 respectively. The relative expression levels were calculated by the 2 −ΔΔCT method.

| Assessment of serum hepatotoxicity indices
The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were used to assess hepatotoxicity and detected by commercial detection kits (Sigma, Santa clara, CA), following the manufacturer's protocols.

| Sirius red staining
The liver tissues were fixed with 10% formaldehyde, embedded in paraffin and cut into 5-μm sections. Then the sections were stained TA B L E 1 Oligonucleotide primer sets for real-time PCR

| Terminal deoxynucleotidyl transferasemediated nick end labelling
The apoptosis of LX-2 cells was detected using one step terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) Apoptosis Assay Kit (Beyotime). Briefly, LX-2 cells were placed on slides and permeabilized in 0.1% Triton X-100 (Beyotime) for 5 minutes at room temperature. Then the slides were incubated with TUNEL reaction mixture at 37 °C for 60 minutes. The nuclei were stained with DAPI for 5 minutes. The slides were observed under a fluorescence microscope and images were taken.

| Western blot
Total protein was isolated from LX-2 cells using RIPA (Solarbio) and quantified using a BCA Protein Assay Kit (Solarbio

| Luciferase reporter assay
The

| Statistical analysis
All experimental data are expressed as mean ± SD. One-way ANOVA was performed to analyse data among multiple groups using GraphPad Prism 5 (GraphPad Software Inc, La Jolla, CA). A P value of less than 0.05 was considered statistically significant.

| Expression of miR-193a/b-3p in liver tissues of mice after exposure to ConA
Firstly, the altered expression of miR-193a/b-3p in liver tissues of ConA-

| Overexpression of miR-193a/b-3p attenuated ConA-induced liver injury
To enhance the expression of miR-193a/b-3p in liver tissues, the mice were injected with lentiviral particles containing miR-193a/b-3p. As presented in Figure 2A and B, ConA-induced decreased expression of miR-193a/b-3p in liver tissues was significantly upregulated after infection of lentivirus. In kidney and heart tissues, the expression of miR-193a/b-3p was declined slightly after exposure to ConA, but the difference was not statistically significant ( Figure S1). Lentivirus-mediated overexpression of miR-193a/b-3p in kidney and heart tissues was also confirmed. Moreover, the serum levels of ALT and AST were increased by about 9.2 and 4.0 folds, respectively, after exposure to ConA for 6 weeks. Whereas, overexpression of miR-193a/b-3p could effectively reduce the ALT and AST levels ( Figure 2C & D), suggesting that ConA-induced liver injury was alleviated by enhancing miR-193a/b-3p expression.

| Overexpression of miR-193a/b-3p relieved ConA-induced liver fibrosis
Marked fibrosis was observed by Sirius red staining in liver tissues after treatment with ConA for 6 weeks, which could be obviously restrained by lentivirus-mediated overexpression of miR-193a/b-3p ( Figure 3A). In addition, the expressions of desmin, a marker for HSCs, and PCNA were determined by double immunofluorescence staining. As shown in Figure 3

| Effect of miR-193a/b-3p overexpression on apoptosis and cell cycle of human HSCs LX-2
As illustrated in Figure 4A Figure 4G-I, the ability of miR-193a-3p or miR-193b-3p in binding with CAPRIN1 was evaluated by dual-luciferase report system. The results showed that in WT CAPRIN1+miR-193a-3p or miR-193b-3p group, the relative luciferase activity was significantly decreased.

| Overexpression of miR-193a/b-3p repressed the activation of human HSCs LX-2 via targeting TGF-β2
To induce the activation of HSCs, LX-2 cells were treated with TGF-β1, or activin A, or combination of TGF-β1 and activin A. As presented in Figure 5A and B, the mRNA expressions of COL1A1 and α-SMA in LX-2 cells were raised after treatment with TGF-β1 or activin A, which were further promoted by combination of TGF-β1 and activin A. So, we chose to induce the activation of LX-2 cells by combination treatment with TGF-β1 and activin A. As shown in Figure 5C  The down-regulation of miR-193a-3p and miR-193b-3p has been reported in human liver fibrosis samples. 17,18 The present study also demonstrated that miR-193a/b-3p expression was decreased after treatment with ConA. Why miR-193a-3p and miR-193b-3p are down-regulated? One possible reason may be the phosphorylation The direct binding of miR-193a-3p (K) or miR-193b-3p (L) with TGF-β2 was determined by dual-luciferase report system. All data were expressed as mean ± SD (n = 3). **P < 0.01, ***P < 0.001 vs the indicated group of exportin-5 (XPO5) that suppresses pre-miRNA export from the nucleus. 21,22 However, the detailed mechanisms need to be further evaluated. Even so, we speculate that miR-193a/b-3p might play a major role in the regulation of liver fibrosis. To test this hypothesis, we enhanced the expression of miR-193a/b-3p using lentivirus in mice. The results showed overexpression of miR-193a/b-3p alleviated ConA-induced liver injury as confirmed by reducing ALT and AST levels. In addition, Sirius red staining indicated that ConA-induced liver fibrosis also relieved by miR-193a/b-3p overexpression.
Next, the exact mechanisms of miR-193a/b-3p to counteract liver fibrosis were investigated. Hepatic stellate cells are a kind of non-parenchymal cells in liver, which are dedicated to hepatic development and homeostasis under the physiological conditions. 23 But when liver injury occurs, activation of HSCs is a common consequence and contributes to liver fibrosis. 24 To mitigate liver fibrosis, loss of activated HSCs is an effective measure. 25 Desmin is one of the classical markers of HSCs. 26 Our results showed that  [27][28][29][30] Previous study suggested that there was a potential interaction between TGF-β and activin A, facilitating collagen production. 31 Our previous research also indicated that TGF-β1 and activin A levels were raised in ConA-treated mice. In the present study, overexpression of miR-193a/b-3p relieved ConA-induced liver fibrosis through decreasing TGF-β1 and activin A levels.
Liver fibrosis is a reversible process and apoptosis, senescence and reversion from activated to quiescent stage of HSCs may provide possibilities. Inducing apoptosis of HSCs is an effective way to reduce the number of activated HSCs during liver fibrosis reversal. 32 The apoptosis of HSCs also contributes to the degradation of ECM. 33 Our results indicated that overexpression of miR-193a/b-3p significantly induced apoptosis of HSCs. CAPRIN1 suppression has been confirmed to inhibit cell proliferation via blocking cell cycle progression. 34 CAPRIN1 is predicted to be a target gene of hsa-miR-193a/b-3p and mmu-miR-193a/b-3p.
Cyclin D1 is also predicted to be a target gene of hsa-miR-193a/ b-3p, but was not for mmu-miR-193a/b-3p (one binding site is mutated). So CAPRIN1 was focused in this study. Our results showed that miR-193a/b-3p could inhibit the mRNA and protein expression of CAPRIN1 via binding to its 3′-UTR. Since Cyclin D1, Cyclin E1 and the Retinoblastoma protein Rb are the regulatory proteins that control G1 to S-phase transition during the cell cycle, we also detected the effect of miR-193a/b-3p on the expression of these proteins. According to the results, overexpression of miR-193a/ b-3p also decreased the protein levels of Cyclin D1, Cyclin E1 and p-Rb, which suggested that the cell cycle of LX-2 was blocked.
However, whether miR-193a/b-3p regulates the expression of these proteins via direct binding to their 3′-UTR or other mechanisms needs to be investigated in the future. Nevertheless, these results provide evidence that overexpression of miR-193a/b-3p inhibits the proliferation of human HSCs via inducing apoptosis and regulating cell cycle.
Furthermore, we investigated whether miR-193a/b-3p could reverse TGF-β1 and activin A-induced activation of LX-2 cells. Our results showed that overexpression of miR-193a/b-3p significantly repressed TGF-β1 and activin A-induced increased COL1A1 and α-SMA expressions. TGF-β2 has been suggested to promote the expressions of ECM proteins. 35 We also found that miR- Although the detailed mechanisms need to be further elucidated, this study may shed light on how miR-193a/b-3p relieves ConA-induced liver fibrosis.

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural Science Foundation of China (No. 81270513 and 81700544).

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.