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目的研究微小RNA-195(miR-195)调节转化生长因子-β1(TGF-β1)诱导的大鼠肝星状细胞(HSC-T6)活化与Smad7表达的关系。方法体外培养HSC-T6,用5 ng·mL~(-1)TGF-β1作为刺激因子,模拟大鼠肝纤维化模型。将细胞分为6组:对照组、模型组(TGF-β1组)、miR-195 mimic实验组、miR-195 mimic NC实验组、miR-195 inhibitor实验组及miR-195 inhibitor NC实验组。分别用5 ng·mL~(-1)TGF-β1处理HSC-T6细胞24,48,72 h后,以实时荧光定量PCR法检测miR-195、Smad7及α-SMA mRNA表达,以免疫印迹法检测Smad7蛋白表达。结果在TGF-β1诱导下,miR-195的表达随时间逐渐升高、α-SMA表达逐步上调而Smad7表达呈降低趋势,差异均有统计学意义(P<0.01,P<0.01;P<0.05)。与模型组比较,miR-195 mimic能促进TGF-β1诱导的HSC-T6活化,升高miR-195、α-SMA mRNA表达而降低Smad7mRNA和蛋白表达,差异均有统计学意义(均P<0.01)。同时,miR-195inhibitor可逆转TGF-β1诱导的HSC-T6活化,降低miR-195、α-SMA mRNA表达而升高Smad7 mRNA和蛋白表达,差异均有统计学意义(均P<0.01)。结论 miR-195促进TGF-β1诱导的HSC-T6活化与Smad7表达相关。
Objective To investigate the relationship between miR-195 and TGF-β1-induced activation of rat hepatic stellate cells (HSC-T6) and Smad7 expression. Methods HSC-T6 was cultured in vitro, and 5 ng · mL -1 TGF-β1 was used as stimulator to simulate rat hepatic fibrosis model. The cells were divided into 6 groups: control group, model group (TGF-β1 group), miR-195 mimic experimental group, miR-195 mimic NC experimental group, miR-195 inhibitor experimental group and miR-195 inhibitor NC experimental group. The expressions of miR-195, Smad7 and α-SMA mRNA in HSC-T6 cells were detected by real-time fluorescence quantitative PCR after treated with 5 ng · mL -1 TGF-β1 for 24,48 and 72 h, Smad7 protein expression was detected. Results The expression of miR-195 gradually increased along with the time and the expression of α-SMA was up-regulated while the expression of Smad7 was decreased with the increase of TGF-β1 (P <0.01, P <0.01) ). Compared with the model group, miR-195 mimic could promote the activation of HSC-T6 induced by TGF-β1 and increase the expression of miR-195 and α-SMA mRNA and Smad7 mRNA and protein (all P <0.01 ). At the same time, miR-195inhibitor could reverse the activation of HSC-T6 induced by TGF-β1, decrease the expression of miR-195 and α-SMA mRNA and increase the expression of Smad7 mRNA and protein (all P <0.01). Conclusion miR-195 can promote TGF-β1-induced HSC-T6 activation and Smad7 expression.