乙型肝炎病毒表面抗原在HepG2细胞中调节微小RNA-31的表达

HBsAg regulation of miR-31 expression in HepG2 cells

目的研究HBV及HBsAg对肝癌细胞HepG2中微小RNA-31（miR-31）表达的调节作用。方法构建哺乳动物HBsAg表达载体并转染HepG2细胞，筛选稳定过表达HBsAg的肝癌细胞系，用酶联免疫吸附法及细胞免疫组织化学法鉴定克隆，并用实时荧光定量聚合酶链反应检测miR-31在肝癌细胞系中的表达水平。结果成功构建哺乳动物HBsAg表达载体，建立稳定表达HBsAg的肝癌细胞系HepG2-H2。整合HBV基因组的HepG2．2．15细胞中miR-31的相对表达量明显下调为0．003±0．001，与普通HepG2细胞的1．0±0．023相比，t=583j8，P〈0．001，差异有统计学意义ImiR-31在稳定过表达HBsAg的HepG2-H2细胞中的相对表达量明显高于空白对照细胞HepG2组及阴性对照细胞HepG2-H0组，F=24．922，P〈0．05，差异有统计学意义I而HepG2组与HepG2-H0组比较，P〉O．05，差异无统计学意义。结论HBV能够抑制miR-31的表达，而HBsAg则促进miR-31的表达。

Objective To investigate the effect of hepatitis B virus （HBV） and hepatitis B virus surface antigen （HBsAg） on microRNA-31 （miR-31） expression in HBV-related hepatocellular carcinoma using HepG2 hepatoma cells. Methods Stable HBsAg-overexpressing cell lines were established by transfecting HepG2 cells with an HBsAg-bearing mammalian expression vector, and the clones （designated as HepG2-H2 cells） were validated by enzyme-linked immunosorbent assay and immunohistochemistry. Effects on expression of miR-31 were determined by measuring the mRNA level by real-time PCR and performing statistical comparisons with levels detected in HepG2-H0 cells （stably transfected with empty vector） and HepG2.2.15 cells （stably transfectexi with the HBV genome）. Results The HepG2-H2 HBsAg- overexpressing transfectant cell line was successfully established. The expression level of miR-31 was significantly higher in the HepG2-H0 cells than in the HepG2.2.15 cells （t = 583.8, P 〈 0.001）. In contrast, the expression level of miR-31 was significantly higher in the HBsAg-overexpressing HepG2-H2 cells than in the HcpG2-H0 cells （F = 24.9, P 〈 0.05）. Conclusion Intact HBV leads to down-regulation of miR-31 expression and HBsAg overexpression leads to up-regulation ofmiR-31 expression in hepatocarcinoma cells.