微小RNA-195调控转化生长因子-β/Smad信号通路影响肝纤维化机制研究

Study on the role of microRNA-195 in transforming growth factor-β/Smad signal pathway and the fibrosis mechanism

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 explore the effect of miRNA - 195 （miR- 195） on Smad7 expression and rat hepatic stellate cells line （HSC - T6 ） activation incuced by transforming growth factor - β1 （TGF- β1）- Methods HSC -T6 were cultured in vitro, with 5 ng · mL-1 TGF - β1as an injury factor simulating the rats liver fibrosis models. The cells divided into six 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. After the HSC - T6 treated with 5 ng ·mL-1 TGF- β1for 24,48,72 h,the mRNA expression of miR- 195, SmadT, and a -SMA was detected by Real -time PCR. The protein expression of Smad7 was detected by Western blot. Results Under the induced by TGF -β1, the expression of miR - 195 increased gradually （ P 〈 0. 01 ） , the expression of Smad7 showed a decreasing trend （ P 〈 0. 05 ） , and the expression of ct - SMA was gradually upregulated （ P 〈 0. 01 ） with time. Compared with the modelgroup, miR - 195 mimic could promote activation of HSC - T6 induced by TGF - β1, and increase miR - 195, ct - SMA mRNA expression （ all P 〈 0. 01 ）, as well as reduce Smad7 mRNA and protein expression （ all P 〈 0. 01 ） ; while miR - 195 inhibitor could inhibit activation of HSC -β1 induced by TGF - β1, decrease miR - 195, a - SMA mRNA expression （ all P 〈 0. 01 ）, as well as upregulate Smad7 mRNA and protein expression （ all P 〈 0. 01 ）. Conclusion miR - 195 is involved in pro - activation of HSC - T6 by inhibiting Smad7 expression.