MicroRNA-302c通过结缔组织生长因子调控腹膜透析相关腹膜纤维化
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摘要
目的通过观察microRNA-302c(miR-302c)对腹膜间皮细胞结缔组织生长因子(connect tissue growth factor,CTGF)表达及上皮细胞-间充质细胞转分化(epithelial-to-mesenchymal transition,EMT)的影响,探讨miR-302c对腹膜透析(peritoneal dialysis,PD)相关腹膜纤维化的作用及机制。方法收集PD患者腹膜透析流出液中腹膜间皮细胞,检测miR-302c、CTGF、EMT及纤维化相关指标的表达,并分析其相关性;体外培养人腹膜间皮细胞株,通过转染慢病毒过表达miR-302c,检测腹膜间皮细胞CTGF、EMT及纤维化相关指标的变化。结果 PD患者腹膜透析流出液中miR-302c水平降低(F=443.165,P<0.001),与波形蛋白(Vimentin)(r=-0.887,P=0.001)和CTGF(r=-0.840,P=0.002)水平呈负相关,与紧密连接蛋白-1(Zo-1)水平呈正相关(r=0.873,P=0.001)。过表达miR-302c抑制转化生长因子β1(transforming growth factor-β1,TGF-β1)诱导的人腹膜间皮细胞株E-钙黏蛋白(E-cadherin)(F=13.910,P=0.043)表达下调,α-平滑肌肌动蛋白(α-SMA)(F=11.833,P=0.026)及Ⅰ型胶原蛋白(Collagen Ⅰ)(F=10.673,P=0.031)表达上调,同时也抑制了TGF-β1诱导的CTGF表达上调(F=8.340,P=0.044)。结论 miR-302c可能通过CTGF调控PD过程中腹膜间皮细胞EMT及纤维化。
Object To explore the effect and mechanisms of miRNA-302c on the expression of connective tissue growth factor(CTGF) and epithelial-to-mesenchymal transition(EMT) in peritoneal mesothelial cells from peritoneal dialysis(PD) patients. Methods Human peritoneal mesothelial cells were isolated by collection of overnight peritoneal lavage from PD patients and centrifugation of the lavage. The expression of miR-302c, CTGF and EMT related factors were assayed. Human peritoneal mesothelial cells were transfected with miR-302c and cultured containing 5 ng/ml transforming growth factor-β1(TGF-β1)for 48 h, and the expression of CTGF and EMT related factors were then measured by western blot. Results There was a marked reduction of miR-302c level in human peritoneal mesothelial cells from prolonged PD patients(F=443.165, P<0.001). miR-302c level was negatively correlated with Vimentin(r=-0.887) and CTGF(r=-0.840), and was positively correlated with Zonula occludens-1(Zo-1)(r=0.873).Over-expression of miR-302c inhibited the down-regulation of E-cadherin(F=13.910,P=0.043) and upregulation of anti-α smooth muscle actin(α-SMA)(F=11.833, P=0.026), collagen Ⅰ(F=10.673, P=0.031)and CTGF(F=8.34, P=0.044) induced by TGF-β1 in cultured human peritoneal mesothelial cells. Conclusion MiR-302c is a vital factor protecting peritoneal mesothelial cells from undergoing EMT and fibrosis in PD patients, probably through inhibition of CTGF expression.
Object To explore the effect and mechanisms of miRNA-302c on the expression of connective tissue growth factor(CTGF) and epithelial-to-mesenchymal transition(EMT) in peritoneal mesothelial cells from peritoneal dialysis(PD) patients. Methods Human peritoneal mesothelial cells were isolated by collection of overnight peritoneal lavage from PD patients and centrifugation of the lavage. The expression of miR-302c, CTGF and EMT related factors were assayed. Human peritoneal mesothelial cells were transfected with miR-302c and cultured containing 5 ng/ml transforming growth factor-β1(TGF-β1)for 48 h, and the expression of CTGF and EMT related factors were then measured by western blot. Results There was a marked reduction of miR-302c level in human peritoneal mesothelial cells from prolonged PD patients(F=443.165, P<0.001). miR-302c level was negatively correlated with Vimentin(r=-0.887) and CTGF(r=-0.840), and was positively correlated with Zonula occludens-1(Zo-1)(r=0.873).Over-expression of miR-302c inhibited the down-regulation of E-cadherin(F=13.910,P=0.043) and upregulation of anti-α smooth muscle actin(α-SMA)(F=11.833, P=0.026), collagen Ⅰ(F=10.673, P=0.031)and CTGF(F=8.34, P=0.044) induced by TGF-β1 in cultured human peritoneal mesothelial cells. Conclusion MiR-302c is a vital factor protecting peritoneal mesothelial cells from undergoing EMT and fibrosis in PD patients, probably through inhibition of CTGF expression.
引文
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[2] Liu Y,Dong Z,Liu H,et al. Transition of mesothelial cell to fibroblast in peritoneal dialysis:EMT, stem cell or bystander?[J].Perit Dial Int,2015,35(1):14-25.
[3] Liu H,Zhang N,Tian D. MiR-30b is involved in methylglyoxal-induced epithelial-mesenchymal transition of peritonealmesothelial cells in rats[J]. Cell Mol Biol Lett,2014,19(2):315-329.
[4] Morishita Y,Yoshizawa H,Watanabe M,et al. MicroRNA expression profiling in peritoneal fibrosis[J]. Transl Res,2016,169:47-66.
[5] Xiao L,Zhou X,Liu FY,et al. MicroRNA-129-5p modulates epithelial-to-mesenchymal transition by targeting SIP1and SOX4 during peritoneal dialysis[J]. Lab Invest,2015,95(7):817–832.
[6] Yoshimoto N,Toyama T,Takahashi S,et al. Distinct expressions of microRNAs that directly target estrogen receptor alpha in human breast cancer[J]. Breast Cancer Res Treat,2011,130(1):331–339.
[7] Kai Zhu,Qi Pan,Luo-qi Jia,et al. MiR-302c inhibits tumor growth of hepatocellular carcinoma by suppressing the endothelial-mesenchymal transition of endothelial cells[J]. Sci Rep,2014,4:5524.
[8] Oak SR,Murray L,Herath A,et al. A micro RNA processing defect in rapidly progressing idiopathic pulmonary fibrosis[J]. PLoS One,2011,6(6):e21253.
[9] Xiao L,Peng X,Liu FY,et al. AKT regulation of mesothelial-to-mesenchymal transition in peritoneal dialysis is modulated by Smurf2 and deubiquitinating enzyme USP4[J]. BMC Cell Biol,2015,16:7.
[10] Yu JW, Duan WJ, Huang XR, et al. MicroRNA-29b inhibits peritoneal fibrosis in a mouse model of peritoneal dialysis[J]. Lab Invest,2014,94(9):978-990.
[11] Zhang K, Zhang H, Zhou X, et al. MiRNA589 regulates epithelial-mesenchymal transition in human peritoneal mesothelial cells[J]. J Biomed Biotechnol,2012,2012:673096.
[12] Huang JW, Yen CJ, Wu HY, et al. Tamoxifen downregulates connective tissue growth factor to ameliorate fibrosis[J]. Blood Purif,2011,31(4):252-258.
[13] Chunming J, Miao Z, Cheng S, et al. TanshinoneⅡA attenuates peritoneal fibrosis through inhibition of fibrogenic growth factors expression in peritoneum in a peritoneal dialysis rat model[J]. Ren Fail,2011,33(3):355-362.