乙型肝炎病毒调节Pasha启动子活性的初步研究

Preliminary study on the regulatory effect of hepatitis B virus on Pasha promoter activity

目的:研究乙型肝炎病毒(Hepatitis B virus,HBV)对microRNA形成相关因子Pasha表达的调控,并对其作用机制进行初步探讨。方法:用RT-PCR,Western blot的方法检测HepG2细胞及稳定表达HBV的HepG2-H7细胞中Pasha的表达差异。构建Pasha启动子的萤火虫荧光素酶报告质粒,分别转染HepG2细胞及HepG2-H7细胞,检测HBV对Pasha启动子的影响。将Pasha启动子质粒与HBV 4种蛋白的真核表达质粒共转染HepG2细胞,寻找对启动子影响较大的HBV蛋白。结果:RT-PCR和Western blot的结果显示Pasha在HepG2-H7细胞中的表达较HepG2细胞明显下降。萤火虫荧光素酶活性分析显示HBV能抑制Pasha启动子的活性,且HBx和HBs蛋白对其影响较大。结论:HBV蛋白可以通过抑制Pasha启动子活性调节其在HepG2-H7细胞中的表达。

Objective：To investigate the regulatory effect of hepatitis B virus（HBV） on the expression of Pahsa and to discuss its mechanism.Methods：RT-PCR and Western blot were performed to check Pasha expression in HepG2 and HepG2-H7 cell lines.Pasha promoter luciferase reporter plasmid,pGL3-Pasha-P,was constructed and transiently transfected into HepG2 and HepG2-H7 cells to anlyze the activity of Pasha promoter.Then HepG2 cells were transiently cotransfected with 0.5 μg of pGL3-Pasha-P and HBx,HBs,HBp,HBc expression plasmids,respectively and the luciferase activity was detected.Results：The results of RT-PCR and Western blot showed that the expressions of Pasha in HepG2-H7 cells were lower than those in HepG2 cells.The luciferase activity assay of Pasha promoter demonstrated that HBV could suppress Pasha promoter activity and HBx and HBs proteins may play important roles in this process.Conclusion：HBV can down-regulate Pasha expression in HepG2-H7 cells through inhibiting its promoter activity.