丹参酮ⅡA通过调控miR-27a抑制肝星状细胞活化的机制研究

A mechanistic study of tanshinoneⅡA inhibiting activation of hepatic stellate cells by regulating miR-27a

目的探讨丹参酮ⅡA(TⅡA)通过调控微小RNA-27a(miR-27a)抑制人肝星状细胞(LX2细胞)活化的机制。方法使用10μg/L转化生长因子-β1(TGF-β1)诱导LX2细胞活化;实时荧光定量逆转录聚合酶链式反应(RT-qPCR)检测miR-27a的表达;瞬时转染miR-27a的模拟物和抑制剂增强或抑制miR-27a的表达,细胞计数(CCK-8)法检测细胞增殖情况,Western blot法检测胶原蛋白Iα2(COL1A2)、α平滑肌肌动蛋白(α-SMA)、磷酸化糖原合成酶激酶3β(P-GSK3β)、分泌型卷曲相关蛋白1(SFRP1)的表达;用不同浓度的TⅡA干预LX2细胞,CCK-8法检测细胞增殖情况,RT-qPCR检测miR-27a的表达,Western blot法检测COL1A2、α-SMA、糖原合成酶激酶3β(GSK3β)、P-GSK3β、SFRP1和细胞周期蛋白D1(CCND1)的表达。结果miR-27a在TGF-β1干预的LX2细胞中表达增加(P<0.05)。LX2细胞中miR-27a被敲低48 h和72 h后,细胞增殖显著被抑制(P<0.05),抑制miR-27a表达减少了LX2细胞中COL1A2、α-SMA和P-GSK3β蛋白的表达,却增加了SFRP1蛋白的表达(P<0.05),过表达miR-27a则呈现相反的结果(P<0.05)。TⅡA能抑制LX2细胞的增殖,其显著降低了miR-27a的表达,且下调了COL1A2、α-SMA、P-GSK3β、CCND1蛋白的表达并上调了SFRP1蛋白的表达(P<0.05)。结论miR-27a在活化的LX2细胞中表达升高,并能促进LX2细胞的增殖和活化。TⅡA通过下调miR-27a抑制Wnt/β-连环蛋白(Wnt/β-catenin)信号通路激活,进而抑制LX2细胞活化。

Objective To explore the mechanism by which tanshinoneⅡA(TⅡA)inhibits the activation of human hepatic stellate cells(LX2 cells)by regulating microRNA-27a(miR-27a).Methods Human HSCs(LX2 cells)were induced for activation with 10μg/L transforming growth factor-β1(TGF-β1).The expression of miR-27a was detected by real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR).The mimics and inhibitors of miR-27a were transiently transfected to enhance or suppress the expression of miR-27a.Cell counting kit-8(CCK-8)assay was used to detect cell proliferation.Protein expression levels of collagen typeⅠalpha 2 chain(COL1A2),alpha smooth muscle actin(α-SMA),phosphorylated glycogen synthase kinase-3 beta(P-GSK3β),and secreted frizzled-related protein 1(SFRP1)were determined via Western blot analysis.LX2 cells were treated with different concentrations of TⅡA,and cell proliferation was reassessed with the CCK-8 assay.RT-qPCR was used to evaluate miR-27a expression,while the expression of COL1A2,α-SMA,glycogen synthase kinase-3 beta(GSK3β),P-GSK3β,SFRP1,and cyclin D1(CCND1)was quantified with Western blot.Results The expression of miR-27a increased in LX2 cells treated with TGF-β1(P<0.05).Knockdown of miR-27a in LX2 cells for 48 h and 72 h significantly inhibited cell proliferation(P<0.05).Suppressing miR-27a expression resulted in decreased protein expression of COL1A2,α-SMA,and P-GSK3β,and increased protein expression of SFRP1 in LX2 cells(P<0.05).Overexpression of miR-27a showed opposite results(P<0.05).TⅡA inhibited the proliferation of LX2 cells,significantly reduced the expression of miR-27a,downregulated the protein expression of COL1A2,α-SMA,P-GSK3β,CCND1,and upregulated SFRP1 protein expression(P<0.05).Conclusions The expression of miR-27a is upregulated in activated LX2 cells and miR-27a promotes the proliferation and activation of LX2 cells.TⅡA suppresses the activation of the Wnt/β-catenin signaling pathway by downregulating miR-27a,consequently inhibiting the activation of LX2 cells.