Inhibition of miR‐188‐5p alleviates hepatic fibrosis by significantly reducing the activation and proliferation of HSCs through PTEN/PI3K/AKT pathway

Abstract Persistent hepatic damage and chronic inflammation in liver activate the quiescent hepatic stellate cells (HSCs) and cause hepatic fibrosis (HF). Several microRNAs regulate the activation and proliferation of HSCs, thereby playing a critical role in HF progression. Previous studies have reported that miR‐188‐5p is dysregulated during the process of HF. However, the role of miR‐188‐5p in HF remains unclear. This study investigated the potential role of miR‐188‐5p in HSCs and HF. Firstly, we validated the miR‐188‐5p expression in primary cells isolated from liver of carbon tetrachloride (CCl4)‐induced mice, TGF‐β1‐induced LX‐2 cells, livers from 6‐month high‐fat diet (HFD)‐induced rat and 4‐month HFD‐induced mice NASH models, and human non‐alcoholic fatty liver disease (NAFLD) patients. Furthermore, we used miR‐188‐5p inhibitors to investigate the therapeutic effects of miR‐188‐5p inhibition in the HFD + CCl4 induced in vivo model and the potential role of miR‐188‐5p in the activation and proliferation of HSCs. This present study reported that miR‐188‐5p expression is significantly increased in the human NAFLD, HSCs isolated from liver of CCl4 induced mice, and in vitro and in vivo models of HF. Mimicking the miR‐188‐5p resulted in the up‐regulation of HSC activation and proliferation by directly targeting the phosphatase and tensin homolog (PTEN). Moreover, inhibition of miR‐188‐5p reduced the activation and proliferation markers of HSCs through PTEN/AKT pathway. Additionally, in vivo inhibition of miR‐188‐5p suppressed the HF parameters, pro‐fibrotic and pro‐inflammatory genes, and fibrosis. Collectively, our results uncover the pro‐fibrotic role of miR‐188‐5p. Furthermore, we demonstrated that miR‐188‐5p inhibition decreases the severity of HF by reducing the activation and proliferation of HSCs through PTEN/AKT pathway.


| INTRODUC TI ON
Hepatic fibrosis (HF) and scarring, a serious global health problem, are defined as the common pathological process associated with end-stage liver cirrhosis and carcinoma. 1 This process of fibrosis is labelled with persistent hepatic damage, chronic inflammation and wound healing reactions. 2 During the process of fibrogenesis, activation of several mediators causes the excessive production and agglomeration of extracellular matrix (ECM) components and interstitial collagens lead to HF and scar deposition. 3,4 Hepatic stellate cells (HSCs) are characterized as the major mesenchymal cells in the liver which play key roles in several cellular processes during HF. 5 HSCs remain quiescent in healthy individuals; however, following persistent hepatic injury, quiescent HSCs will be activated by trans-differentiating into fibrogenic myofibroblast-like cells.
This trans-differentiation promotes the massive accumulation of ECM components, expression of α-SMA and cell proliferation in response to pro-inflammatory signals originated from impaired parenchymal cells. 2,[4][5][6] Activated HSCs respond and secrete numerous pro-fibrogenic cytokines. Among these pro-fibrotic cytokines, transforming growth factor β (TGFβ) is considered a potent cytokine resulting in HF. 7 Thus, inactivation and proliferation inhibition of HSCs have been widely accepted to obstruct the HF. 8 Non-coding RNAs (ncRNA), group of evolutionary conserved endogenous RNAs, influence gene expression and may regulate initiation and progression of NASH (non-alcoholic steatohepatitis) and HF. 9 Among these regulatory molecules, microRNAs (miRNAs), short ≈ 22 nucleotides in size, negatively regulate expressions of target genes at the post-transcriptional level by targeting 3′ untranslated regions (3′ UTR) of their target mRNAs. 10,11 Several studies have explored the fundamental roles of miRNAs in the progression of HF by regulating several cellular processes including HSC activation, proliferation and collagen production. 9,12 For example, overexpression of miR-193a/b-3p inhibited TGF-β1-induced activation and proliferation of HSCs by suppressing HSC activation genes COL1A1 and α-SMA, and attenuated HF. 13 Likewise, miR-146a-5p inhibited the secretion of pro-inflammatory factors and activation of HSCs suggesting the therapeutic role of miR-146a-5p in HF. 14 Hyun et al investigated that the expression of miR-188-5p is significantly up-regulated in carbon tetrachloride (CCl 4 )-induced HF model. 15,16 However, the functional importance of miR-188-5p in HF is still unclear. This study aimed to determine the expression of miR-188-5p in the TGF-β1-induced HSCs and the fibrotic livers, and investigated the role of miR-188-5p in modulating the activation, proliferation and fibrogenesis of HSCs, both in vitro and in vivo.  Table 1.

| Animals and liver specimens
Adult male C57BL/6J mice and E3 rats were obtained from the Experimental Animal Center situated in Xi'an Jiaotong University. All the animals were housed in a specific pathogen-free animal facility centre with controlled temperature and humidity, and maintained under 12-h light/dark cycle with free access to water and feed. All To determine the expression of miR-188-5p in high-fat diet (HFD)-induced liver fibrosis models, livers were obtained from 8 control and 8 4-month HFD-induced C57BL/6J mice (Research Diet, D12492), and 8 control and 8 6-month HFD-induced E3 rats (described in our previous experiments 17 ).

| Isolation of primary cells
We used 8-week-old male C57BL/6J mice to isolate primary cells.
Briefly, we randomly divided mice into control group and CCl 4 (2 mL/ kg body weight)-induced chronic liver injury model group (n = 6 each group). As described earlier, 18 hepatocytes and non-parenchymal cells (NPC) were isolated using differential centrifugation to evaluate the expression of miR-188-5p.   Figure S2. At the 14 th week of experiment, all mice were sacrificed. Liver tissues were collected and divided into two parts.

| Inhibition of miR-188-5p in vivo
One part was immediately snap-frozen in liquid nitrogen and then stored at −80°C for further experiments, while another section was fixed in formalin and embedded in paraffin, for histological analysis.

| Statistical analysis
Quantitative data were presented as means ± SEM. Statistical analysis between different groups was assessed by GraphPad Prism 6.02 (GraphPad Software) Software. Student's t test and two-way ANOVA with Tukey's multiple comparison tests were performed to compare two groups or multiple groups, respectively.
Detailed materials and methods are described in Supporting information.

| Expression of miR-188-5p is up-regulated in human liver fibrosis biopsy sample and in vivo and in vitro models for liver fibrosis
Several investigations have reported that miR-188-5p plays critical role in numerous diseases. [21][22][23][24][25][26][27][28] Recent miRNA signature showed that miR-188-5p is up-regulated in the serum from alcoholinduced liver cirrhosis and toxic doses of acetaminophen (APAP) liver injury patients. However, the expression of miR-188-5p was down-regulated in the type 2 diabetes mellitus patients, while no change was observed in the expression of miR-188-5p from HBV patients. 29 Moreover, it has been identified that miR-188-5p is significantly up-regulated in the HSCs associated with portal hypertension and CCl 4 -induced liver fibrosis model. 15,30 However, it is still unclear how miR-188-5p takes part in the progression of liver fibrosis. In order to explore the role of miR-188-5p in liver fibrosis, we first determined the relative expression of miR-188-5p in human liver fibrosis biopsy sample from 5 CHB + NAFLD and 4 CHB patients and in vivo and in vitro models for liver fibrosis.
We used miR-188-5p-fluorescence in-situ hybridization (FISH) to determine the expression of miR-188-5p in the human HF tissue specimens. We observed that the miR-188-5p expression was upregulated in the human liver biopsy sample from CHB + NAFLD patients ( Figure 1A). To further determine the expression of miR-188-5p in human HSCs, we induced LX-2 cells with TGF-β1, which is one of the most important inflammatory mediator and activator of HSCs. 31 We observed that the expression levels of  Figure 1C) and 6-month HFD-induced E3 rats ( Figure 1F). Similarly, the results obtained from RT-qPCR analysis showed that miR-188-5p expression was significantly up-regulated among HFD groups as compared to their respective control groups ( Figure 1D,G). Furthermore, RT-qPCR analysis showed that miR-188-5p expression was significantly upregulated in the NPC cells isolated from liver of CCl 4 -induced mice as compared to control, whereas no significant change was observed in primary hepatocytes isolated from liver of CCl 4 -induced mice ( Figure 1E). These data indicated that miR-188-5p is highly expressed in activated HSCs and in vivo models of liver fibrosis.

| Up-regulation of miR-188-5p promotes HSC activation and proliferation in LX2 cells
To explore the functional relevance of miR-188-5p in liver fibrosis, we modulated the expression of miR-188-5p using mimics or inhibitors in LX-2 cells. Firstly, the overexpression of miR-188-5p via mimics up-regulated the expression level of miR-188-5p ( Figure 2A). This overexpression of miR-188-5p promoted the expression of HSC activation markers α-SMA and Col1α2 at protein level ( Figure

| Modulation in miR-188-5p expression regulates the PTEN-dependent proliferation and activation of LX-2 cells by targeting PI3K/AKT pathway
It is known that miRNAs regulate protein expressions through mRNA These results indicate that PTEN is a direct target of miR-188-5p.
The above findings brought us that miR-188-5p

| Inhibition of miR-188-5p reduces the severity of hepatic damage in HFD + CCl 4 -induced liver fibrosis mice
To determine the biological roles of miR-188-5p in liver fibrosis, we subjected C57BL/6J mice (n = 8 each group) to HFD feeding with weekly CCl 4 i.p. injections for 14 weeks and transduced with miR-188-5p inhibitors or NC via tail vein at different intervals ( Figure S2).

| Inhibition of miR-188-5p expression reduces the HFD + CCL 4 -induced NASH-associated pro-fibrotic factors and pro-inflammatory cytokines in liver tissue
We next followed up on elevated fibrosis markers and hepatic damage examined in the HFD + CCl 4 -induced liver injury during serum and liver histopathology analysis, respectively, by examining the expression profile of liver inflammatory cytokines and pro-fibrogenic genes. The present study determined that HFD + CCl 4 enhanced the inflammatory infiltrates and degree of fibrosis in the liver. However, it was significantly decreased after the inhibition of miR-188-5p.
Therefore, we probed into the regulatory role of miR-188-5p during the process of fibrogenesis by modulating pro-inflammatory and pro-fibrotic genes.
In miR-188-5p inhibitor group, the relative expression of profibrotic genes tnfα, tgfβ, timp2 and fn significantly decreased compared to the inhibitors-NC control group ( Figure 6A-D). Moreover, ECM remodelling gene mmp2 which was significantly increased in HFD + CCl 4 group was also significantly reduced after the inhibition of miR-188-5p ( Figure 6E). Similarly, HFD + CCl 4 significantly increased the expression level of pro-inflammatory mcp1, IL-1β and il-6. However, inhibition of miR-188-5p significantly reduced the expression of these pro-inflammatory genes ( Figure 6F-H). We further validated the expression level of anti-inflammatory il-10 that was significantly decreased in the HFD + CCl 4 , but inhibition of miR-188-5p significantly increased its expression ( Figure 6I). Together, these findings show that inhibition of miR-188-5p significantly reduces the severity of HF in HFD + CCl 4 -induced mice showing severe NASH in term of higher pro-fibrotic and pro-inflammatory changes in gene expression as compared to HFD. Furthermore, immunoblotting analysis showed that HSC activation markers α-SMA and COL1A2 were significantly down-regulated in liver samples after the inhibition of miR-188-5p ( Figure 7J). A similar trend was observed for the HSC proliferation marker PCNA that was also significantly decreased after the inhibition of miR-188-5p. Remarkably, inhibition of miR-188-5p led to a significant increase in expression of its target gene PTEN at both transcriptional and translational level ( Figure 7J, Figure S3B,C). This increase in the PTEN expression after the inhibition of miR-188-5p led to the down-regulation of AKT pathway ( Figure 7J). Thus, we observed a significant decrease in the activation and proliferation markers after the inhibition of miR-188-5p.

| Inhibition of miR-188-5p expression alleviates the HF by down-regulating the HSC activation and proliferation markers in HFD + CCl 4induced liver fibrosis through PTEN/AKT pathway
Collectively, these results revealed that inhibition of miR-188-5p led to the down-regulation of HSC activation and proliferation markers and severity of HF through PTEN/AKT pathway.

| D ISCUSS I ON
HF characterizes a major health problem worldwide. It is considered as a scarring process that is associated with excessive deposition of ECM in liver. HSCs are the primary effector cells for the deposition of ECM in fibrotic liver. 33 This progressive fibrotic response is characterized by activation and proliferation of HSCs, and aberrant expression of cellular transcriptional factors. 34 Ample evidence has revealed that ncRNAs, especially numerous miRNAs, function as critical regulators in activation and proliferation of HSCs, and regulate liver fibrosis. 9,35-37 However, the function and underlying mechanism of miR-188-5p in HSC activation, proliferation and liver fibrosis remain unknown. In the current study, we investigated the potential role of miR-188-5p in activation and proliferation of HSC and NASH-associated liver fibrosis.
Our results demonstrated that miR-188-5p is highly expressed in HFD-induced murine models of NASH and TGF-β1-induced human LX-2 cells compared to normal liver tissues and LX-2 cell line, respectively. However, it has not been studied how TGF-β1 induces the maturation and the expression of miR-188-5p and several other dysregulated miRNAs. Some studies have probed that TGFβ signalling links with Drosha/DGCR8 complex in SMAD-dependent manner and modulates the maturation of several miRNAs. 38  It has been well documented that PTEN negatively regulates the process of liver fibrosis. 43 Several miRNAs have been reported to directly bind with PTEN to regulate its expression and play critical roles in the activation and proliferation of HSCs. 44,45 We investigation. However, we also observed the suppression of miR-188-5p expression after the transfection of miR-188-5p inhibitors, which might result from miRNA sequestering and degradation. [46][47][48] Overall, our study identified miR-188-5p as an important regulator of HSC cell activation and proliferation, and emphasized a critical role of this miRNA in mediating HF, at least in part, via PTEN/ PI3K/AKT.
Previous studies have demonstrated that altered miRNA expression is closely associated with liver fibrosis. 12 However, the functions of miRNA may vary. Studies have shown that several miRNAs can function as pro-fibrotic, while other function as anti-fibrotic. 31,[49][50][51] In the current study, miR-188-5p was identified to up-regulate in HF.
Furthermore, its up-regulation is associated with high expression of pro-fibrotic and pro-inflammatory genes, which strongly suggests a potential role of miR-188-5p in the progression of liver fibrosis. Hyun

CO N FLI C T O F I NTE R E S T S
The authors declare that there is no conflict of interests.