MicroRNA-582-5p在HBV感染中的作用及分子机制研究

目的探究微小RNA-582-5p(microRNA-582-5p,miR-582-5p)在HBV感染中的作用及分子机制。方法通过miRNA芯片筛选出miR-582-5p作为研究对象。分别通过过表达和抑制内源性miR-582-5p表达的方法,研究其对HBV DNA、HBV RNA、HBeAg、HBsAg等HBV复制和表达相关指标的影响。在分子机制研究中,双荧光素酶报告基因检测miR-582-5p对HBV启动子和增强子的影响,生物信息学预测miR-582-5p的靶基因,分别使用实时定量PCR(quantitative real-time PCR,qRT-PCR)和Western blot检测miR-582-5p对靶基因NOTCH1 mRNA和蛋白表达的影响。最后检测HBV相关蛋白表达对miR-582-5p的影响。结果qRT-PCR结果表明miR-582-5p随着HBV复制水平升高而逐渐降低。miR-582-5p抑制HBV DNA、HBV RNA、HBeAg和HBsAg的表达。miR-582-5p能抑制HBV S2 promoter和HBV EnhancerⅠ的活性,miR-582-5p能直接靶向HBV促进因子NOTCH1,抑制其mRNA和蛋白表达。HBx蛋白抑制miR-582-5p的表达。结论miR-582-5p通过抑制HBV S2 promoter和HBV EnhancerⅠ的活性或靶向抑制HBV促进因子NOTCH1而抑制HBV复制和表达;同时,HBV的复制与表达抑制miR-582-5p的表达。

Function and molecular mechanism of microRNA-582-5p in HBV infection

Objective To explore the role and molecular mechanism of microRNA-582-5p(miR-582-5p)in HBV infection.Methods miR-582-5p was selected as the research object by miRNA chip.The effects of miR-582-5p on the replication and expression related indicators of HBV,such as HBV DNA,HBV RNA,HBeAg and HBsAg,were studied by overexpression and inhibition of endogenous miR-582-5p expression.In the molecular mechanism study,the effect of miR-582-5p on HBV promoter and enhancer was detected by dual-luciferase reporter assays,and the target gene of miR-582-5p was predicted by bioinformatics.The effects of miR-582-5p on target gene NOTCH1 mRNA and protein expression were detected by quantitative real-time PCR and Western blot assay,respectively.Finally,the effect of HBV-related protein expression on miR-582-5p was detected.Results The quantitative real-time PCR results showed that miR-582-5p decreased gradually with the increasing HBV replication level.miR-582-5p inhibited the expression of HBV DNA,HBV RNA,HBeAg and HBsAg.miR-582-5p could inhibit the activity of HBV S2 promoter and HBV EnhancerⅠ,and miR-582-5p could directly target HBV promoting factor NOTCH1,to inhibit the expression of mRNA and protein.HBx protein inhibited the expression of miR-582-5p.Conclusions miR-582-5p inhibits the replication and expression of HBV by inhibiting the activity of HBV S2 promoter and HBV EnhancerⅠ,or inhibiting HBV promoting factor NOTCH1 by targeting,while the replication and expression of HBV inhibit the expression of miR-582-5p.