筛选调节hbv增殖和hbsag产生的宿主细胞的mirna.docVIP

筛选HBV增殖和HBsAg产生的miRNA 章广玲1，2 ，王梅梅1，朱丽华1，2 ，熊亚南1 ，李怡璇2，刘民2 ，汤华2 ，袁丽杰1(063000 河北唐山，河北联合大学基础医学院病原生物学学科1; 300070 天津，天津医科大学天津市生命科学中心实验室2) [摘要筛选调节乙型肝炎病毒（HBV）增殖和乙型肝炎病毒表面抗原（HBsAg）产生的宿主细胞的miRNA，为研究HBV和miRNA之间的相互作用及作用机制奠定初步的实验依据。用328种miRNA的反义链转染HepG2 2.2.15细胞，72收获上清，MTS检测细胞的增殖活性，ELISA检测表面抗原和e抗原的表达水平，实时定量PCR检测其中HBV DNA的拷贝数。构建筛选到的miRNA的过表达载体，转染HepG2 2.2.15细胞，也分别用MTS，ELISA和实时定量PCR进行检测。miR-199a-3pASO， miR-210miR-185ASO促进了HBsAg产生miR-199a-3p， miR-210， miR-185HBsAg的产生和HBV的增殖(P0.05); miR-199a-3p，miR-210和miR-185的ASO转染组与对照组相比，分别降低了细胞内miR-199a-3p，miR-210和miR-185的表达水平(P0.05); miR-199a-3p，miR-210和miR-185 的过表达载体转染组与对照组相比，分别增加了细胞内miR-199a-3p，miR-210和miR-185的表达水平(P0.05)。 结论 没有对细胞的增殖活性产生影响的情况下，miR-199a-3p, miR-210和miR-185 抑制了HBV表面抗原的产生和HBV的增殖miRNA作为病毒与宿主细胞相互作用中的调节因子，在HBV的生命活动周期中具有重要作用。 miRNA 乙型肝炎病毒 乙型肝炎病毒表面抗原 [中图法分类号] R [文献标识码] A Screening the host cell miRNA regulating the proliferation of HBV and HBsAg expression ZHANG Guangling1,2,,Wang Meimei1,Zhu Lihua1,2 , et al. (1Department of Basic Medicine, Hebei united university, Tangshan, 063000; 2Tianjin Medical University, Tianjin, 300070,China) [Abstract] Objective To address which miRNAs influences HBsAg expression, HBeAg expression or the proliferation of HBV in producing HepG2 2.2.15 cells, and to provide evidence for an intricate physiological interaction between the cell’s miRNAs and HBV. Methods Antisense oligonucleotides of special miRNAs were used to reduce the expression of endogenous miRNAs. Enzyme linked immunosorbent assay (ELISA) and realtime PCR were performed on culture supernatant harvested from HepG2 2.2.15 cells 72 hours after transfection with miRNA ASOs, a CellTiter 96? Aqueous Non-Radioactive Cell Proliferation Assay (MTS) was used to detect the cell proliferation at the same time. Results　There are three miRNAs, miR-199a-3p, miR-210 and miR-185 that inhibited the expression of HBsAg. Conclusion　The work provide evidence that human miRNAs can inhibit HBV replication, and a