MicroRNA‐708 modulates Hepatic Stellate Cells activation and enhances extracellular matrix accumulation via direct targeting TMEM88

Abstract Transmembrane protein 88 (TMEM88) is a potential 2‐transmembrane‐type protein that interacts with the PDZ domain of Dishevelled‐1 (DVL‐1), a crucial component of Wnt signalling pathway through its C‐terminal Val‐Trp‐Val (VWV) motif in Xenopus embryo cells. Since the significant function of β‐catenin in liver fibrosis, it is urgent to study the TMEM88 mechanism in liver fibrosis. The current research was for evaluating the function of TMEM88 in the process of the liver fibrosis and clarifying the inherent mechanism. The study found that TMEM88 is decreased in human fibrotic liver tissues. Functionally, TMEM88 significantly reduced the expression levels of α‐smooth muscle actin (α‐SMA) and collagen type I (Col.I) and repressed extracellular matrix (ECM) accumulation by restoring the balance between matrix metalloproteinases (MMPs) and TIMPs (tissue inhibitor of metalloproteinases). TMEM88 inhibited HSCs proliferation and evaluated the apoptosis of activated LX‐2 cells by regulating Wnt3a, Wnt2b and β‐catenin of Wnt/β‐catenin signalling pathway. Moreover, we demonstrated that miR‐708 particularly targeted TMEM88 3′‐UTR regions and down‐regulated the expression level of TMEM88 in TGF‐β1‐stimulated LX‐2 cells. MiR‐708 promoted the generation of ECM and cell activation in activated LX‐2 cells. These results determined that miR‐708 could promote HSCs activation and enhance ECM accumulation via direct targeting TMEM88 by Wnt/β‐catenin signalling pathway. This will provide a potential target for future research in the process of liver fibrosis.


| INTRODUC TI ON
Liver fibrosis, the ultimate common pathway for chronic or iterative liver damage, is characterized by the accumulation of extracellular matrix (ECM). 1,2 Hepatic stellate cells (HSCs) are the main cell in liver fibrosis. 3 Of note, activated HSCs with transforming growth factor-β1 (TGF-β1) are important in the excessive production of ECM proteins, more importantly, alpha-smooth muscle actin (α-SMA) and type I collagen (Col.I) which are recognized as fibrosis markers and are participated in a series of fibrotic processes. [4][5][6] In addition, there is increasing evidence that matrix metalloproteinases (MMPs)/metalloproteinase tissue inhibitors (TIMPs) systems control the relationship between ECM synthesis and degradation. 7,8 The up-regulated expression level of TIMP1 and the suppressed expression level of MMP2 resulted in subsequent accumulation of ECM. 9 Therefore, finding an intrinsic target for inhibiting ECM accumulation from HSCs will be a new direction for studying the progression of liver fibrosis.
Transmembrane protein 88 (TMEM88) is transmembrane protein found on the cell membrane via blocking Wnt/β-catenin signalling pathway. 10,11 Moreover, growing evidence reported that Wnt/βcatenin signalling pathway participates in liver fibrosis progression. 12 TMEM88 could mediate inflammatory cytokines secretion by Wnt/ β-catenin signalling pathway. 13 Furthermore, chronic liver inflammation could lead to liver fibrosis. 14,15 Hereof, it is central to investigate the function of TMEM88 in liver fibrosis progression. LX-2 cells are stabilizing and infinite source of human HSCs, retaining the key features of activated HSCs. 16 The results showed that TMEM88 may be related to HSCs activation and the generation of ECM and proliferation by regulating Wnt/β-catenin signalling pathway in activated LX-2 cells. Furthermore, the expression level of TMEM88 was subsequently examined both in the human fibrotic liver tissues and activated LX-2 cells.
MicroRNAs, short 20-22 nucleotides, are recognized to hold essential parts in liver fibrosis progression by binding to the 3′-untranslated region (3′-UTR) of the target mRNAs. [17][18][19] Analysis of mi-croRNAs expression has identified a group of dysregulated miRNAs in liver fibrosis. 20,21 According to the bioinformatics tools, Mirtarbase and Mirbase, the upstream gene of TMEM88 is miR-708. The role of miR-708 has been found in hepatocellular carcinoma (HCC), 22,23 the expression level of miR-708 was lower in HCC. 24 Moreover, a unique character of HCC is its close association with liver fibrosis. More than 80% of HCC develop in liver fibrosis. 25 Therefore, we assumed that miR-708 is a target for liver fibrosis. However, the underlying mechanism of how miR-708 affects the characters of liver fibrosis remains unclear. MiR-708 was supposed to regulate the cell activation in activated HSCs by targeting TMEM88. In addition, we found that miR-708 dramatically enhanced the Wnt/β-catenin signalling pathway in LX-2 cells and thereby aggravated liver fibrosis both in vivo and in vitro. These results determined that miR-708 could promote HSCs activation and enhance ECM accumulation via direct targeting TMEM88 by Wnt/β-catenin signalling pathway. This will provide a potential target for future research in the process of liver fibrosis.

| Specimen collection
Normal liver tissues (the control group) and human fibrotic liver tissues were taken from patients undergoing partial hepatectomy who undergone liver biopsy for staging and grading of liver fibro-  Table 1. Opti-MEM were used to detect results. LX-2 cells were harvested after 48 hours of transfection and then lysed. Dual-luciferase reporter assay was used to detect the luciferase activities.

| Cell culture
LX-2 cells were donated by Scott L. Friedman and were cultured with DMEM supplemented with 10% FBS and 1% penicillin and streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO 2 and 95% air.

| EDU DNA incorporation assay
Cell proliferation was detected by standard EDU DNA incorporation assay. Exponentially growing LX-2 cells plated on 13 mm glass coverslips. Then, pEGFP-C2-TMEM88, TMEM88-siRNA and NC were transfected into cells with Lipofectamine™2000, respectively. After culture for 24 hours, the cells were labelled with 50 μmol/L EDU for 2 hours, and then rinsed twice with ice-cold phosphate-buffered saline (PBS). After labelling, LX-2 cells were fixed in 4% paraformaldehyde for 30 minutes and rinsed with 2 mg/mL glycine for 5 minutes, then permeabilized with 1% Triton X-100 for 10 minutes, rinsed with PBS for 5 minutes. Apollo staining was performed for 30 minutes and then Hoechst staining for 30 minutes. The images were taken by fluorescence microscopy (Olympus).

| Flow cytometry
Apoptosis of LX-2 cell was analysed by FITC Annexin V apoptosis detection kit I. The cells were seeded in a 6-well plate at a density of 1 × 10 5 cells per well and transfected with TMEM88 (pEGFP-C2-TMEM88, TMEM88-siRNA) for 24 hours at 37°C in a CO 2 incubator. After a wash with cold PBS, LX-2 cells were re-suspended in 1 × binding buffer, then stained with 5 μL FITC Annexin V and 5 μL Propidium iodide (PI). The flow cytometer was used to detect the apoptosis.

| Quantitative Real-Time PCR
The cell culture medium was abandoned from the 6-well plate and cleaned 3 times by PBS. LX-2 cells were cytolysis to total RNA by TRIzol. Then, RNA was transcripted reverse to generate cDNA.
Relative levels of specific mRNA were determined using the RTqPCR Detection System with TB Green ® supermix according to the manufacturer's instructions. The β-actin gene was used as an internal control for normalization. The primers used for PCR amplification are shown in Table 2.

| Immunohistochemistry
Human fibrotic liver tissues and normal liver tissues were fixed in paraffin after fixation in 10% neutral paraformaldehyde and stained for routine histology. Liver sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol, and anti- The slides were counterstained with haematoxylin before dehydration and installation.

| Double immunofluorescence staining
The liver tissues were permeabilized with 0.2% Triton X-100 containing 1% BSA for 10 minutes, and blocked with 5% BSA for 1 hours at room temperature. To determine the co-localization of TMEM88 and α-SMA, FITC-conjugated TMEM88 probes in combination with Cy3-conjugated anti-α-SMA antibody (1:50) were used in the hybridization assays. The cells were mounted with SlowFade Gold antifade reagent with DAPI, and images were taken using fluorescence microscopy. TMEM88 was shown as green fluorescence and α-SMA as red fluorescence.

| Statistical analysis
Statistical data analysis was performed by SPSS ver.18.0. The differences in groups were checked by one-way ANOVA. The data were presented as the mean ± standard error at least three times experiments independently. If the P value < .05, the data were considered significant difference, and if the P value < .01, the data were considered strongly significant difference.

| TMEM88 was decreased in human fibrotic liver tissues and TGF-β1-stimulated LX-2 cells
To determine whether TMEM88 was participated in liver fibrosis, the human fibrotic liver tissues were obtained for the study.  Pannoramic SCAN 150 (3DHISTECH, Budapest, Hungary) was used for the imaging. D, The protein expression level of TMEM88 was measured by Western blotting in human fibrotic liver tissues compared with normal group. E, ISH with anti-TMEM88 probe and IHC with α-SMA were performed to determine the co-localization of TMEM88 (green) and α-SMA (red) in human fibrotic liver tissues. Fluorescence microscope was used for the imaging (Olympus). Representative images from control and human fibrotic liver tissues are presented (×100) *P < .05 compared with the normal group

| TMEM88 alleviated MMPs/TIMPs system in TGF-β1-stimulated LX-2 cells
To further observe the role of TMEM88 in the MMPs

| TMEM88 is a direct target of miR-708 in LX-2 cells
Mirtarbase, a bioinformatics tool was used to predict target genes for miR-708 to understand the underlying mechanisms by which miR-708 regulates LX-2 cells activation. The 3′-UTR miRNA of TMEM88 contains putative miR-708 binding sites predicted by prediction algorithm ( Figure S1). To confirm miR-708 regulates TMEM88 by binding to the corresponding 3′-UTRs, the 3′-UTR of TMEM88 was cloned from into the pmirGLO luciferase reporter vector. Then, the vectors of miR-708 or control mimics were cotransfected with into LX-2 cells.
Double-luciferase reporter assay was subsequently used to evaluate TMEM88 response to miR-708. These funding revealed that miR-708 significantly decreased TMEM88-3′UTR-WT luciferase activity in LX-2 cells. Importantly, miR-708 has no effect on mutant 3′UTR-TMEM88 luciferase activity in LX-2 cells (Figure 5C,D). RT-qPCR result showed that the expression level of miR-708 was up-regulated in human fibrotic liver tissues compared with normal tissues, and the expression level of miR-708 was up-regulated in TGF-β1-stimulated LX-2 cells (Figure 5E,F). Moreover, Western blotting result showed F I G U R E 4 TMEM88 alleviated ECM accumulation in TGF-β1-stimulated LX-2 cells. A, The mRNA expression levels of MMP2 and TIMP1 were analysed with RT-qPCR in activated LX-2 cells transfected with pEGFP-C2-TMEM88 and TMEM88-siRNA, respectively. The results showed that TMEM88 could decrease the mRNA expression level of TIMP1, whereas increase the mRNA expression level of MMP2. B, The protein expression levels of MMP2 and TIMP1 were measured by Western blotting analysis in activated LX-2 cells transfected with pEGFP-C2-TMEM88 and TMEM88-siRNA. The results showed that TMEM88 could decrease the protein expression level of TIMP1, whereas increase the protein expression level of MMP2. The results were expressed as the mean ± standard of three different experiments. *P < .05 compared with the normal group, # P < .05 compared with the control group F I G U R E 5 TMEM88 inhibited cell proliferation and promoted cell apoptosis in TGF-β1-stimulated LX-2 cells. A, Cell apoptosis of LX-2 cells was measured by flow cytometry analysis. B, Proliferation of LX-2 cells was determined by EDU DNA incorporation assay. Fluorescence microscope was used for the imaging. C, The TMEM88-3′-UTR constructs or blank plasmid were transfected into LX-2 cells with control or miR-708 mimics, followed by dual-luciferase assays. D, The mutant 3′UTR-TMEM88 constructs or blank plasmid were transfected into LX-2 cells with control or miR-708 mimics, followed by dual-luciferase assays. E, The mRNA expression level of miR-708 was analysed with RT-qPCR in human fibrotic liver tissues and normal tissues. F, The mRNA expression level of miR-708 was analysed with RT-qPCR in TGF-β1-stimulated LX-2 cells. The results were expressed as the mean ± standard of three different experiments. *P < .05 compared with the normal group, # P < .05 compared with the control group that mimics of miR-708 down-regulated the expression level of TMEM88 in LX-2 cells. Conversely, miR-708 inhibitor up-regulated the protein level of TMEM88 in LX-2 cells ( Figure 6A). Herein, TMEM88 is a direct target of miR-708 in LX-2 cells.

| MiR-708 promoted cell activation by targeting TMEM88 in TGF-β1-stimulated LX-2 cells
To determine the role of MiR-708 on HSCs activation, the mRNA and protein expression levels of α-SMA and Col.I which associated with cell activation were detected in TGF-β1-stimulated LX-2 cells.
Of note, miR-708 mimics significantly increased the expression levels of α-SMA and Col.I at mRNA and protein levels ( Figure 6B,C). And results of RT-qPCR and Western blotting analysis revealed that miR-708 siRNA down-regulated expression levels of α-SMA and Col.I ( Figure 6B,C). These results determined that miR-708 promoted HSCs activation in TGF-β1-stimulated LX-2 cells.

| MiR-708 treatment aggravated MMPs/TIMPs system by targeting TMEM88 in TGF-β1-stimulated LX-2 cells
To confirm the function of miR-708 on MMPs/TIMPs system, miR-708 mimics and miR-708 inhibitor were respectively transfected in TGF-β1-stimulated LX-2 cells. The results showed that miR-708 mimics significantly decreased the protein and mRNA expression level of MMP2, whereas increased the protein and mRNA level of TIMP1 in TGF-β1-stimulated LX-2 cells ( Figure 7A,B). These results revealed that miR-708 destroys the balance in the MMPs/TIMPs system by targeting TMEM88 in LX-2 cells.

| Effect of TMEM88 on Wnt/β-catenin signalling pathway activity in TGF-β1-stimulated LX-2 cells
Evidence indicated that the activation of Wnt/β-catenin signalling pathway was tightly related to the HSCs activation and ECM accumulation. Mechanically, we examined the associated proteins of this signalling pathway. The results revealed that the expression levels of β-catenin, Wnt3a and Wnt2b were significantly increased while transfected with TMEM88-siRNA ( Figure 7C). In addition, overexpression of TMEM88 promoted the protein expression levels of β-catenin, Wnt3a and Wnt2b ( Figure 7C). In summary, these results determined that TMEM88 might regulate the process of the liver fibrosis by Wnt/β-catenin signalling pathway.

| D ISCUSS I ON
Excessive ECM accumulation will cause liver fibrosis which mainly generated by myofibroblasts in chronic liver disease, 26-28 which could be regulated by Wnt/β-catenin signalling pathway. 29 β-catenin is a key pro-fibrosis and has been implicated in the pathogenesis of a variety of tissue fibrosis. 30 result. These results indicated that TMEM88 was a key part in aggravating the progression of the liver fibrosis by regulating Wnt/βcatenin signalling pathway negatively.
It is a promising means of regulating liver fibrosis-related pathways through miRNA therapy. 18,42 This study mainly found that miR-708 is significantly elevated in TGF-β1-stimulated LX-2 cells and human fibrotic liver tissues, while silencing of miR-708 and overexpression of TMEM88 can inhibit HSC activation and reduce ECM accumulation. Bioinformatics tools (Mirtarbase and Mirbase) were used to predict the target genes of miR-708. The 3′-UTR of TMEM88 was cloned into the pmirGLO luciferase reporter vector and then cotransfected into LX-2 with miR-708 and miR-708 NC respectively to determine if miR-708 passed the corresponding 3′-UTR combines to adjust TMEM88. The response of TMEM88 to miR-708 was assessed by dual-luciferase reporter assay. The study indicated that miR-708 meaningfully inhibited TMEM88 luciferase activity in LX-2 cells. In addition, double-luciferase reporter assay clearly revealed that TMEM88 is a direct target of miR-708 in HSCs. Functionally, the results showed that miR-708 could regulate HSCs activation by increasing the expression levels of α-SMA and Col.I. Meanwhile, miR-708 enhanced the accumulation of ECM by decreasing the expression level of MMP2, whereas increasing the expression level of TIMP1 in TGF-β1-stimulated LX-2 cells. Thence, we concluded that the miR-708 could play an important role in liver fibrosis.
To summarize, our data indicated that TMEM88 is essential for the development of liver fibrosis and HSCs activation regulated by Wnt/β-catenin signalling pathway in TGF-β1-stimulated LX-2 cells. In future research, we will focus on the more features of TMEM88 in the development of liver fibrosis. Based on the function of TMEM88, research on targeted drugs of TMEM88 for liver disease will be subsequently carried out. Understanding the effects of TMEM88 on inflammation, autophagy, oxidative stress and other functions during liver fibrosis is a meaningful work, which can provide a bright future for the treatment of liver disease.
F I G U R E 7 MiR-708 enhanced ECM accumulation on Wnt/β-catenin signalling pathway in TGF-β1-stimulated LX-2 cells. A, The mRNA expression levels of MMP2 and TIMP1 were analysed with RT-qPCR in activated LX-2 cells transfected with miR-708 mimics and miR-708 inhibitor, respectively. The results showed that miR-708 could increase the mRNA expression level of TIMP1, whereas decrease the mRNA expression level of MMP2. B, The protein expression levels of MMP2 and TIMP1 were measured by Western blotting analysis in activated LX-2 cells transfected with miR-708 mimics and miR-708 inhibitor, respectively. The results were expressed as the mean ± SD of three different experiments. The results showed that miR-708 could increase the protein expression level of TIMP1, whereas decrease the protein expression level of MMP2. C, The protein expression level of β-catenin, Wnt3a and Wnt2b was performed in activated LX-2 cells transfected with pEGFP-C2-TMEM88 and TMEM88-siRNA, respectively. The results were expressed as the mean ± standard of three different experiments. *P < .05 compared with the control group, # P < .05 compared with the control group