hsacirc0054345靶向miR-206对TGF-β1诱导的大鼠肝星状细胞增殖、活化及凋亡的影响

Effect of hsairc054345 targeting miR-206 on TGF-β1-induced proliferation,activation and apoptosis of rat hepatic stellate cells

目的探讨环状RNA0054345(hsacirc0054345)是否通过靶向微小RNA-206(miR-206)调控转化生长因子β1(TGF-β1)诱导的大鼠肝星状细胞增殖、活化及凋亡。方法体外培养大鼠肝星状细胞HSC-T6,使用TGF-β1处理细胞建立模型,采用实时荧光定量聚合酶链反应(qRT-PCR)检测hsacirc0054345、miR-206的表达量;实验分组为:对照组、TGF-β1组、TGF-β1+si-con组、TGF-β1+si-circ0054345组、TGF-β1+si-circ0054345+anti-miR-con组、TGF-β1+si-circ0054345+anti-miR-206组。甲基噻唑基四唑(MTT)检测细胞增殖;流式细胞术检测细胞凋亡;双荧光素酶报告实验验证hsacirc0054345、miR-206的靶向关系;蛋白免疫印迹法(Western blot)检测α平滑肌动蛋白(α-SMA)、I型胶原蛋白(ColⅠ)、基质金属蛋白酶组织抑制剂1(TIMP-1)、B淋巴细胞瘤-2相关蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)的表达量。结果与对照组比较,TGF-β1组hsacirc0054345的表达水平明显升高(P<0.05),miR-206的表达水平明显降低(P<0.05),细胞存活率明显升高(P<0.05),细胞凋亡率明显降低(P<0.05),α-SMA、ColⅠ、TIMP-1和Bcl-2蛋白水平明显升高(P<0.05),Bax蛋白水平明显降低(P<0.05);与TGF-β1+si-con组比较,TGF-β1+si-circ0054345组细胞存活率明显降低(P<0.05),细胞凋亡率明显升高(P<0.05),α-SMA、ColⅠ、TIMP-1和Bcl-2蛋白水平明显降低(P<0.05),Bax蛋白水平明显升高(P<0.05);双荧光素酶报告实验证实hsacirc0054345可靶向结合miR-206;与TGF-β1+si-circ0054345+anti-miR-con组比较,TGF-β1+si-circ0054345+anti-miR-206组细胞存活率明显升高(P<0.05),α-SMA、ColⅠ和TIMP-1/Bcl-2蛋白水平明显升高(P<0.05),细胞凋亡率明显降低(P<0.05),Bax蛋白水平明显降低(P<0.05)。结论抑制hsacirc0054345表达可能通过靶向上调miR-206的表达从而抑制TGF-β1诱导的大鼠肝星状细胞增殖、活化及诱导细胞凋亡。

Objective To investigate whether hsacirc0054345 targeted miR-206 can regulate the proliferation,activation and apoptosis of rat hepatic stellate cells induced by TGF-β1.Methods The rat hepatic stellate cells HSC-T6 were cultured in vitro,and the cells were treated with TGF-β1 to establish a model.The expression levels of hsacirc0054345 and miR-206 were detected by qRT-PCR.The experimental groups were:control group,TGF-β1 group,TGF-β1+si-con group,TGF-β1+si-circ0054345 group,TGF-β1+si-circ0054345+anti-miR-con group and TGF-β1+si-circ0054345+anti-miR-206 group.MTT was used to detect cell proliferation.Flow cytometry was used to detect apoptosis.The dual luciferase report experiment verified the targeting relationship of hsacirc0054345 and miR-206.Western blot was used to detect the expression levels ofα-SMA,ColⅠ,TIMP-1,Bax and Bcl-2.Results Compared with the control group,the expression level of hsacirc0054345 in the TGF-β1 group was significantly increased(P<0.05),the expression level of miR-206 was significantly decreased(P<0.05),the cell survival rate was significantly increased(P<0.05),the apoptosis rate was significantly reduced(P<0.05),and the mortality rate was significantly reduced(P<0.05),the levels ofα-SMA,ColⅠ,TIMP-1 and Bcl-2 protein were significantly increased(P<0.05),the level of Bax protein was significantly reduced(P<0.05).Compared with the TGF-β1+si-con group,the cell survival rate of the TGF-β1+si-circ0054345 group was significantly reduced(P<0.05),and the apoptosis rate was significantly increased(P<0.05),the levels ofα-SMA,ColⅠ,TIMP-1 and Bcl-2 protein were significantly reduced(P<0.05),the level of Bax protein was significantly increased(P<0.05).The dual luciferase report experiment confirmed that hsacirc0054345 could target and bind to miR-206.Compared with the TGF-β1+si-circ0054345+anti-miR-con group,the cell survival rate of the TGF-β1+si-circ0054345+anti-miR-206 group was increased significantly(P<0.05),the levels ofα-SMA,ColⅠ,TIMP-1,Bcl-2 protein were significantly increased(P<0.05),cell apoptosis rate was significantly reduced(P<0.05),the level of Bax protein was significantly reduced(P<0.05).Conclusion Inhibition of hsacirc0054345 expression may inhibit the proliferation,activation and apoptosis of rat hepatic stellate cells induced by TGF-β1 through targeted up-regulation of miR-206 expression.