MicroRNA-34a-5p在肝纤维化中的表达及作用

Expression and role of MicroRNA-34a-5p in liver fibrosis

目的:探讨大鼠肝纤维化组织及活化肝星状细胞中MicroRNA-34a-5p水平及其在肝纤维化中的作用。方法:建立肝纤维化大鼠模型,转化生长因子-β1(TGF-β1)活化肝星状细胞,采用HE染色观察肝脏组织形态学变化,采用RT-PCR测定大鼠肝组织和活化肝星状细胞中α-平滑肌肌动蛋白(α-SMA)和MicroRNA-34a-5p水平。将肝纤维化模型大鼠根据随机数字表法分为空白对照组、阴性转染对照组和MicroRNA-34a-5p组;将TGF-β1活化的HSC-T6细胞分为空白对照组、阴性转染对照组和MicroRNA-34a-5p组。采用RT-PCR和蛋白质印迹法测定3组大鼠肝组织和肝星状细胞中α-SMA、I型胶原蛋白(collagenI)、沉默调节蛋白1(SIRT1)、分泌型糖蛋白-3α(Wnt-3α)、分泌型糖蛋白-5α(Wnt-5α)和β-波链蛋白(β-catenin)的水平。结果:与对照组比较,肝纤维化组大鼠肝组织和活化肝星状细胞中α-SMA水平升高(P<0.05),MicroRNA-34a-5p水平降低(P<0.05)。与空白对照组和阴性转染对照组比较,MicroRNA-34a-5p转染组大鼠肝组织和活化肝星状细胞中MicroRNA-34a-5p水平升高(P<0.05),而α-SMA、collagenI、SIRT1、Wnt-3α、Wnt-5α和β-catenin的mRNA和蛋白水平均下降(P<0.05),空白对照组和阴性转染对照组间大鼠肝组织和活化肝星状细胞中各指标差异无统计学意义(P>0.05)。结论:肝纤维化组织及活化肝星状细胞中MicroRNA-34a-5p水平降低,可能通过靶向SIRT1而抑制Wnt/β-catenin信号通路,从而发挥抗肝纤维化的作用。

Objective: To investigate the level of MicroRNA-34 a-5 p in rat liver fibrosis and activated hepatic stellate cells,and to clarify its role in hepatic fibrosis. Methods: The rats model of hepatic fibrosis were established.The hepatic stellate cells were activated by transforming growth factor-β1(TGF-β1). Morphological changes of liver tissue were observed by hematoxylin-eosin(HE) staining.The levels of α-smooth muscle actin(α-SMA) and MicroRNA-34 a-5 p in rat liver tissue and activated hepatic stellate cells were determined by reverse transcription-polymerase chain reaction(RT-PCR).Liver fibrosis model rats were divided into blank control group, negative transfection control group(transfected with microRNA-34 a-5 p mimetic negative control) and MicroRNA-34 a-5 p group(transfected with microRNA-34 a-5 p simulated matter) according to random number method;The TGF-β1 activated HSC-T6 cells were divided into blank control group, negative transfection control group and MicroRNA-34 a-5 p group.The α-SMA, collagen I, silencing regulator 1(SIRT1), secreted glycoprotein-3α(Wnt-3α), secreted glycoprotein-5α(Wnt-5α) and β-catenin levels of rat liver tissue and hepatic stellate cells were determined by RT-PCR and Western blotting. Results: Compared with the control group, the levels of α-SMA in the liver fibrosis tissue and activated hepatic stellate cells were increased(P<0.05), and the levels of MicroRNA-34 a-5 p were decreased(P<0.05).Compared with the blank control group and negative transfection control group, the levels of MicroRNA-34 a-5 p in the liver tissue and activated hepatic stellate cells of the microRNA-34 a-5 p group were increased(P<0.05), the α-SMA, collagen I, SIRT1, Wnt-3α, Wnt-5α, β-catenin levels decreased(P<0.05).There was no significant difference in each indicators in the liver tissue and activated hepatic stellate cells between the blank control group and negative transfection control group(P>0.05). Conclusion: MicroRNA-34 a-5 p level is decreased in hepatic fibrosis and activated hepatic stellate cells. MicroRNA-34 a-5 p may inhibit Wnt/β-catenin signaling pathway by targeting SIRT1, thereby exerts anti-fibrosis effect.