MiR-340-5p调控乙型肝炎病毒复制的机制

Regulatory mechanism of HBV replication by MiR-340-5p

目的阐释miR-340-5p对HBV复制的影响及其调控机制,并为HBV感染后的生物标志物和治疗用药提供新的策略。方法在肝癌细胞HepG2.2.15中转染miR-340-5p的模拟物(340-mimic)和抑制剂(340-inhibitor)及其对应的阴性对照。通过ELISA检测上清中HBeAg和HBsAg的水平变化。同时收取细胞,提取RNA和壳体化DNA,并采用qRT-PCR分别检测HBV总RNA和pgRNA的水平及HBV DNA拷贝数的变化;分子克隆构建STAT3的过表达质粒pEF-flag-stat3,通过qRT-PCR和western blot对STAT3的mRNA和蛋白表达加以验证;此外,共转染miR-340-5p的模拟物和pEF-flag-stat3后通过northern blot,qPCR和ELISA对HBV的复制情况进行检测。结果miR-340-5p过表达后,HBV转录生成总RNA和逆转录模板pgRNA的水平明显下降至对照组的45.89%、61.46%(P=0.001、P=0.003);壳体化DNA的合成及HBeAg和HBsAg的分泌水平分别受到72.46%、18.27%、14.42%的抑制(P<0.001、P<0.001、P=0.005);干扰内源性miR-340-5p则与对照组相比分别增加44.02%、45.56%、22.66%、11.85%、14.04%(P=0.009、P=0.01、P=0.011、P=0.013、P=0.027)。相比之下在此基础上过表达信号转导和转录激活因子(STAT)3能够减弱被miR-340-5p抑制的HBV复制。进一步的实验结果也表明STAT3对HBV的复制有促进作用。结论miR-340-5p能够通过靶向作用于STAT3抑制其转录和翻译进而抑制HBV的复制。

Objective To disclose the effect of miR-340-5p on HBV replication and its regulatory mechanism,furthermore provide new strategies for biomarkers and therapeutic drugs after HBV infection.Methods The miR-340-5p mimic(340-mimic)and inhibitor(340-inhibitor)and their corresponding negative controls were transfected into liver cancer cells HepG2.2.15.The changes of HBeAg and HBsAg levels in the supernatant were detected by ELISA.At the same time,the RNA and the encapsidated DNA were extracted in collected cells,and qRT-PCR was used to detect the levels of HBV total RNA and pgRNA and the changes in HBV DNA copy numbers;qRT-PCR and Western blotting were used to verify the mRNA and protein expression of STAT3;in addition,after co-transformation of miR-340-5p mimic(340-mimic)and pEF-flag-stat3,the replication of HBV was detected by Northern blotting,qPCR and ELISA.Results When miR-340-5p is overexpressed,the levels of total RNA produced by HBV transcription and the reverse transcription template pgRNA decreased significantly to 45.89%and 61.46%of the control group(P=0.001,P=0.003);the synthesis of encapsidated DNA and the secretion levels of HBeAg and HBsAg were inhibited by 72.46%,18.27%and 14.42%respectively(P<0.001,P<0.001,P=0.005),while interference with endogenous miR-340-5p increased 44.02%,45.56%,22.66%,11.85%,and 14.04%respectively compared with the control group(P=0.009,P=0.080,P=0.011,P=0.013,P=0.027).In contrast,overexpression of signal transducer and activator of transcription(STAT)3 on this basis can attenuate the inhibition of HBV replication by miR-340-5p.The data also shows that STAT3 promotes HBV replication.Further experimental result also showed that STAT3 could promote the replication of HBV.Conclusions miR-340-5p can suppress the transcription and translation of STAT3 by targeting it,thereby inhibiting the replication of HBV.