Tianjin Life Science Research Center and Department of Pathogen Biology, Collaborative Innovation Center of Tianjin for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.
Tianjin Life Science Research Center and Department of Pathogen Biology, Collaborative Innovation Center of Tianjin for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.
Life Sci. 2018 Nov 15;213:9-17. doi: 10.1016/j.lfs.2018.10.016. Epub 2018 Oct 9.
To investigate the role and underlying mechanism of miR-185-5p in hepatitis B virus (HBV) expression and replication.
The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay (ELISA). The HBV DNA copies in the cultures medium were measured by RT-qPCR. The HBV large surface antigen promoter (S1p) activity was analyzed by luciferase reporter assay. The target relationship between miR-185-5p and ELK1 was identified by bioinformatics analysis and EGFP fluorescent reporter assay. The ELK1 expression was determined by RT-qPCR and Western blot.
miR-185-5p significantly decreased HBV large surface antigen promoter activity and subsequently the production of HBV proteins and HBV DNA copies in vitro. Further, we identified the ETS transcription factor ELK1 is a target of miR-185-5p. Overexpression and knockdown experiments showed overexpression of ELK1 stimulated HBV large surface antigen promoter activity and promoted the production of HBV proteins and HBV DNA copies, whereas knockdown of ELK1 has the opposite effects. Moreover, the rescue of ELK1 expression reversed the suppression of miR-185-5p on HBV replication and gene expression. Further mechanistic study showed that the ETS binding sites within the HBV large surface antigen promoter are required for the repression effect of miR-185-5p on HBV.
There are few reports about the interaction between miRNAs and the transcription from HBV S1p, we found that miR-185-5p decreases HBV S1p activity by targeting ELK1, which may provide a promising therapeutic strategy for HBV infection.
研究 miR-185-5p 在乙型肝炎病毒（HBV）表达和复制中的作用及机制。
通过酶联免疫吸附试验（ELISA）检测乙型肝炎表面抗原和乙型肝炎 e 抗原的相对水平。通过 RT-qPCR 检测培养物中的 HBV DNA 拷贝数。通过荧光素酶报告基因检测分析 HBV 大表面抗原启动子（S1p）的活性。通过生物信息学分析和 EGFP 荧光报告基因检测确定 miR-185-5p 与 ELK1 的靶关系。通过 RT-qPCR 和 Western blot 测定 ELK1 的表达。
miR-185-5p 显著降低 HBV 大表面抗原启动子活性，进而降低 HBV 蛋白和 HBV DNA 拷贝的产生。进一步发现，ETS 转录因子 ELK1 是 miR-185-5p 的靶基因。过表达和敲低实验表明，ELK1 的过表达刺激 HBV 大表面抗原启动子活性，促进 HBV 蛋白和 HBV DNA 拷贝的产生，而 ELK1 的敲低则产生相反的效果。此外，ELK1 表达的恢复逆转了 miR-185-5p 对 HBV 复制和基因表达的抑制作用。进一步的机制研究表明，HBV 大表面抗原启动子内的 ETS 结合位点是 miR-185-5p 对 HBV 抑制作用所必需的。
关于 miRNA 与 HBV S1p 转录之间相互作用的报道较少，我们发现 miR-185-5p 通过靶向 ELK1 降低 HBV S1p 活性，这可能为 HBV 感染提供一种有前景的治疗策略。
