miR-155抑制HepG2.2.15细胞中乙型肝炎病毒复制与表达的研究

Depressant effect of micro RNA-155 on the expression and replication of Hepatitis B virus in HepG2.2.15 cell

目的研究人miR-155对HepG2.2.15细胞中乙型肝炎病毒(HBV)的抑制作用,为miRNAs治疗乙型肝炎提供理论依据。方法以HepG2.2.15细胞基因组为模板,PCR扩增人miR-155前体序列,双酶切连接到pmR-mCherry质粒,构建pmiR-155真核过表达载体,并转染到HepG2.2.15细胞。设重组组(pmiR-155质粒)、空载组(pmR-mCherry质粒)、转染试剂组和空白组。转染后24 h、48 h、72 h,应用实时荧光定量PCR法检测各组miR-155表达量和HBV DNA拷贝数,化学发光法定量检测HBs Ag和HBe Ag变化情况。结果实时荧光定量PCR结果显示,与空白组相比,重组组miR-155表达量明显提高。化学发光法结果示在转染后48 h,过表达的miR-155对上清培养液所分泌HBs Ag、HBe Ag抑制作用明显,抑制率分别为(83.96±1.52)%和(79.60±8.71)%。定量PCR检测示过表达的miR-155对HBV DNA拷贝数的抑制率分别为(51.87±0.36)%、(43.67±1.51)%和(68.21±6.02)%。结论 miR-155对HBV蛋白的抑制作用具特异性,呈负相关,在体外可抑制HepG2.2.15细胞中HBV的复制和表达。

Objective It is to investigate the depressant effect of microRNA -155（miR -155）on Hepatits B virus in HepG2. 2. 15 cell,and provide a theoretical basis for the treatment of hepatitis B. Methods The pre-miR-155 was amplified from total DNA of HepG2. 2. 14 by PCR. The target gene fragment was digested and cloned into the pmR-mCherry plasmid. The pmiR-155 was transfected into HepG2. 2. 15 cells by liposome-mediated method. The empty plasmid,the reagent group and untreated cells were set as control. The expression of miR -155 was detected by the real-time quantitive PCR. The ex-pression of HBsAg and HBeAg were detected by chemiluminescent assay,and the changes of HBV DNA were investigated by real-time quantitive PCR. Results MiR-155 level of HepG2. 2. 15 cells which were transfected with the recombinant plasmid was obviously higher than controls. The chemiluminescent assay showed that over-expression of miR-155 could inhibit the ex-pression of HBsAg and HBeAg with（69. 43 ± 0. 7）%,（45. 89 ± 0. 74）% respectively on the 2nd day post-transfection. The HBV DNA was markedly suppressed with（51. 87 ± 3. 74）%,（43. 67 ± 0. 687）% and（68. 21 ± 0. 47）% by real-time quantitive PCR. Conclusion The inhibition effect of HBV protein was specific and had a certain dependency on the expression of miR-155. MiR-155 can suppress the replication and expression of HBV in vitro.