miR-16-5p通过调控PD-L1表达影响肝星状细胞炎症反应的研究

MiR-16-5p inhibits inflammatory response of hepatic stellate cells by regulating PD-L1 expression

目的分析miR-16-5p在肝星状细胞中对程序性死亡受体-1(PD-1)表达的影响,并探讨其在肝纤维化及炎症反应中的作用机制。方法以大鼠肝星状细胞HSC-T6为研究对象,用5 ng/ml转化生长因子β1(TGF-β1)处理细胞0、24、48、72 h后,光镜下观察细胞形态,RT-qPCR法检测细胞TNF-α和IFN-γ表达水平,筛选出TGF-β1最佳作用时间。将细胞分为6组:对照组、模型组、miR-16-5p超表达组、miR-16-5p抑制组、超表达-空载组、抑制-空载组,采用RT-qPCR法检测miR-16-5p表达水平以验证转染效果;光镜下观察细胞形态变化;采用CCK-8试剂盒检测细胞增殖能力;采用Western blot法检测细胞PD-1配体(PD-L1)、NF-κB、p-NF-κB蛋白表达水平;采用RT-qPCR法检测细胞miR-16-5p、PD-L1以及炎症因子TNF-α、IFN-γ、IL-6和IL-10 mRNA表达水平。结果5 ng/ml TGF-β1处理细胞0、24、48、72 h后,细胞增殖且形态发生改变;炎症因子TNF-α、IFN-γ在TGF-β1处理48 h时后表达水平均明显增高(均P<0.05)。与模型组相比,对照组和miR-16-5p超表达组细胞miR-16-5p的mRNA表达水平以及PD-L1和IL-10蛋白表达水平均明显升高(均P<0.05),p-NF-κB/NF-κB的蛋白表达水平以及TNF-α、IFN-γ和IL-6的mRNA表达水平均降低(均P<0.05),miR-16-5p超表达组细胞增殖能力降低(P<0.05);miR-16-5p抑制组miR-16-5p、PD-L1、IL-10的mRNA表达水平均降低(均P<0.05),p-NF-κB/NF-κB的蛋白表达水平以及TNF-α、IFN-γ、IL-6的mRNA表达水平均升高(均P<0.05),细胞增殖能力增强(P<0.05)。结论miR-16-5p在肝星状细胞中通过调控PD-L1表达降低肝细胞炎症水平,抑制细胞增殖,其机制可能参与抑制肝纤维化发生。

Objective To investigate the effect of miR-16-5p on the inflammatory response of hepatic stellate cells and related mechanisms.Methods Rat hepatic stellate HSC-T6 cells were treated with 5 ng/ml TGB-β1 for 0,24,48 and 72 h.The morphology of HSC-T6 cells was observed with light microscope,and the expression of TNFαand IFNγwere detected by RT-qPCR to screen out the optimal action time of TGFβ1.HSC-T6 cells were divided into six groups:control group,model group,miR-16-5p mimics group,miR-16-5p inhibitor group,mimics NC group and inhibitor NC group.The expression of miR 16-5p was detected by RT-qPCR to verify the transfection effect.The morphological changes of cells were observed with light microscope;the proliferation activity of cells was detected by CCK 8 kit;the expression of PD-L1 and NFκB and p-NFκB were detected by Western blot.The mRNA expression levels of miR 16-5p,PD-L1,TNF-α,IFN-γ,IL-6 and IL-10 were detected by RT-qPCR.Results Cell proliferation and morphological changes were observed after treatment with 5 ng/ml TGFβ1 for 0,24,48 and 72 h,and the expression levels of TNFαand IFNγincreased significantly 48 h after treatment with TGFβ1(P<0.05).Compared with the model group,the mRNA expression levels of miR-16-5p and the protein expression levels of PD-L1 and IL-10 were significantly higher in the control and miR 16-5p mimic group cells(all P<0.05),and the protein expression levels of p-NFκB/NFκB and the mRNA expression levels of TNFα,IFNγand IL 6 were lower(all P<0.05),and the proliferation ability of cells in the miR 16-5p mimic group was reduced(P<0.05).The mRNA expression levels of miR 16-5p,PD-L1,and IL 10 were decreased in the miR 16-5p inhibitor group(all P<0.05),and the protein expression levels of p-NFκB/NF-κB and the mRNA expression levels of TNFα,IFNγ,and IL 6 were increased(all P<0.05),and the cell proliferation activity was increased(all P<0.05).Conclusion miR-16-5p can inhibit the inflammation response and proliferation of HSC cells by regulating the expression of PD-L1,which may be involved in the inhibition of liver fibrosis.