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J Cell Mol Med
2020 Jul 01;2413:7127-7140. doi: 10.1111/jcmm.15119.
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MicroRNA-708 modulates Hepatic Stellate Cells activation and enhances extracellular matrix accumulation via direct targeting TMEM88.
Xu T, Pan L, Li L, Hu S, Zhou H, Yang C, Yang J, Li H, Liu Y, Meng X, Li J.
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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.
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32463570 ???displayArticle.pmcLink???PMC7339227 ???displayArticle.link???J Cell Mol Med ???displayArticle.grants???[+]
1607a0202062 The fund of Anhui Science and Technology Department Soft Science Project, 81700522 National Natural Science Foundation of China, 1704a0802161 Anhui Provincial Natural Science Foundation, 1808085MH235 Anhui Provincial Natural Science Foundation, 0601067101 the Fund of Anhui Medical University Doctoral Start Research, 2018xkj021 Anhui Medical University Scientific Research Foundation
Figure 2. TMEM88 was decreased in TGFâ€Î²1â€stimulated LXâ€2 cells. A, The kinetic profiles of TMEM88 expression were observed in LXâ€2 cells during activation induced by TGFâ€Î²1 in different times and concentrations. B, The slides of LXâ€2 cells transfected with pEGFPâ€C2â€TMEM88 were taken out to immunofluorescence, and the result showed that the TMEM88 protein was mainly localized in cytoplasm. C, D, RTâ€qPCR and Western blotting detected the expression level of TMEM88 both at mRNA and protein levels when transfected with pEGFPâ€C2â€TMEM88 and pEGFPâ€C2, respectively (GFP fluorescence proteinâ€positive cells were sorted after 48 h using MoFlo XDP cell Sorter of Center For Scientific Research, Beckman Coulter). The results were expressed as the mean ± standard of three different experiments. *P < .05 compared with the control group, #
PÂ <Â .05 compared with the control group
Figure 3. TMEM88 inhibited HSCs activation in TGFâ€Î²1â€stimulated LXâ€2 cells. A, The mRNA expression levels of αâ€SMA and Co1.I were analysed with RTâ€qPCR in activated LXâ€2 cells transfected with pEGFPâ€C2â€TMEM88 and TMEM88â€siRNA, respectively. The results showed that TMEM88 could inhibit the mRNA expression levels of αâ€SMA and Co1.I. B, The protein expression levels of αâ€SMA and Co1.I were measured by Western blotting analysis in activated LXâ€2 cells transfected with pEGFPâ€C2â€TMEM88 and TMEM88â€siRNA. The results showed that TMEM88 could inhibit the protein expression levels of αâ€SMA and Co1.I. The results were expressed as the mean ± standard of three different experiments. *P < .05 compared with the normal group, #
PÂ <Â .05 compared with the control group
Figure 4. TMEM88 alleviated ECM accumulation in TGFâ€Î²1â€stimulated LXâ€2 cells. A, The mRNA expression levels of MMP2 and TIMP1 were analysed with RTâ€qPCR in activated LXâ€2 cells transfected with pEGFPâ€C2â€TMEM88 and TMEM88â€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 LXâ€2 cells transfected with pEGFPâ€C2â€TMEM88 and TMEM88â€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, #
PÂ <Â .05 compared with the control group
Figure 5. TMEM88 inhibited cell proliferation and promoted cell apoptosis in TGFâ€Î²1â€stimulated LXâ€2 cells. A, Cell apoptosis of LXâ€2 cells was measured by flow cytometry analysis. B, Proliferation of LXâ€2 cells was determined by EDU DNA incorporation assay. Fluorescence microscope was used for the imaging. C, The TMEM88â€3′â€UTR constructs or blank plasmid were transfected into LXâ€2 cells with control or miRâ€708 mimics, followed by dualâ€luciferase assays. D, The mutant 3′UTRâ€TMEM88 constructs or blank plasmid were transfected into LXâ€2 cells with control or miRâ€708 mimics, followed by dualâ€luciferase assays. E, The mRNA expression level of miRâ€708 was analysed with RTâ€qPCR in human fibrotic liver tissues and normal tissues. F, The mRNA expression level of miRâ€708 was analysed with RTâ€qPCR in TGFâ€Î²1â€stimulated LXâ€2 cells. The results were expressed as the mean ± standard of three different experiments. *P < .05 compared with the normal group, #
PÂ <Â .05 compared with the control group
Figure 6. MiRâ€708 promoted cell activation by targeting TMEM88 in TGFâ€Î²1â€stimulated LXâ€2 cells. A, The mRNA and protein expression level of TMEM88 was measured by Western blotting and RTâ€qPCR analysis respectively in activated LXâ€2 cells transfected with miRâ€708 mimics and miRâ€708 inhibitor, respectively. B, The mRNA expression levels of αâ€SMA and Co1.I were analysed with RTâ€qPCR in activated LXâ€2 cells transfected with miRâ€708 mimics and miRâ€708 inhibitor, respectively. The results showed that miRâ€708 could increase the mRNA expression levels of αâ€SMA and Co1.I. C, The protein expression levels of αâ€SMA and Co1.I were measured by Western blotting analysis in activated LXâ€2 cells transfected with miRâ€708 mimics and miRâ€708 inhibitor, respectively. The results showed that miRâ€708 could increase the protein expression levels of αâ€SMA and Co1.I. The results were expressed as the mean ± standard of three different experiments. *P < .05 compared with the normal group, #
PÂ <Â .05 compared with the control group
Figure 7. MiRâ€708 enhanced ECM accumulation on Wnt/βâ€catenin signalling pathway in TGFâ€Î²1â€stimulated LXâ€2 cells. A, The mRNA expression levels of MMP2 and TIMP1 were analysed with RTâ€qPCR in activated LXâ€2 cells transfected with miRâ€708 mimics and miRâ€708 inhibitor, respectively. The results showed that miRâ€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 LXâ€2 cells transfected with miRâ€708 mimics and miRâ€708 inhibitor, respectively. The results were expressed as the mean ± SD of three different experiments. The results showed that miRâ€708 could increase the protein expression level of TIMP1, whereas decrease the protein expression level of MMP2. C, The protein expression level of βâ€catenin, Wnt3a and Wnt2b was performed in activated LXâ€2 cells transfected with pEGFPâ€C2â€TMEM88 and TMEM88â€siRNA, respectively. The results were expressed as the mean ± standard of three different experiments. *P < .05 compared with the control group, #
PÂ <Â .05 compared with the control group
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