摘要目的 研究微小RNA (miRNA)在HBx缺失突变体致人肝细胞异常增殖中的作用及其相关机制. 方法 利用miRNA芯片技术检测稳定表达HBx-d382及HBx的L02(即分别含HBx基因缺失突变体HBx-d382及野生型HBx基因)细胞系中miRNA的表达,实时荧光定量PCR对上述miRNA进行验证.选取在L02/HBx-d382和L02/HBx细胞中均表达明显下调的两个miRNA:miR-338-3P、miR-551b进行功能研究,分为实验组(转染miRNA模拟物组)、阴性对照组(NC,转染miRNA阴性对照)及脂质体组(lipo,单加转染试剂),通过脂质体转染到细胞中,四甲基偶氮唑盐法检测细胞存活率,流式细胞仪检测细胞周期,实时荧光定量PCR和Wstern blot检测细胞周期蛋白D1、细胞周期蛋白G1和E2F转录因子的改变.采用SPSS 16.0统计软件,数据均进行了正态检验并符合正态性,组间均数比较采用成组t检验. 结果 (1)与L02/pcDNA3.0细胞比较,L02/HBx-d382细胞有6个miRNA表达上调,5个miRNA表达下调; L02/HBx细胞有4个miRNA表达上调,12个miRNA表达下调.实时荧光定量PCR验证的结果与芯片相一致.(2)转染了miR-338-31p-模拟物和miR-551b-模拟物后,L02/HBx-d382及L02/HBx细胞增殖均受抑制,细胞周期均阻滞在G1期,细胞增殖能力减弱.L02/HBx-d382细胞中,细胞增殖能力:lipo组、NC组、miR-338-3p组和miR-551b组分别为90.0％±1.3％、88.0％±1.6％、56.0％±6.1％和62.0％±6.4％,miR-338-3p组与:lipo组、NC组比较,t值分别为10.402、9.133 ;miR-551b组和与lipo组、NC组比较,t值分别为8.763、7.403,P值均＜0.01.L02/HBx细胞中,细胞增殖能力:lipo组、NC组、miR-338-3p组和miR-551b组分别为91.0％±1.7％、89.0％±2.1％、60.0％±7.7％和66.0％±9.3％,miR-338-3p组与lipo组、NC组比较,t值分别为9.105、8.074;miR-551b组与lipo组、NC组比较,t值分别为7.673、7.52,P值均＜0.01.实时荧光定量PCR检测结果显示细胞周期蛋白D1、细胞周期蛋白G1和E2F转录因子的mRNA水平均无明显改变,但Western blot法发现上述基因的蛋白表达均明显下调,差异有统计学意义. 结论 ①HBx及其缺失突变体能影响L02细胞的miRNA表达谱;②在L02/HBx-d382细胞中下调更明显的miR-338-3p和miR-551b可能通过调控细胞周期蛋白D1、细胞周期蛋白G1和E2F转录因子的表达而调节了细胞的生长.

abstractsObjective To study the effect of micro (mi)RNA on cellular proliferation induced by hepatitis B x protein,HBx,in human liver cells and to investigate the underlying molecular mechanism of this cancer-related effect.Methods The human L02 hepatocyte cell line was stably transfected with HBx (L02/HBx) or an HBx mutant (L02/HBx-d382) that induces higher levels of cellular proliferation.The differential miRNA expression profiles were determined by microarray analysis and confirmed by realtime PCR.Two miRNAs,miR-338-3p and miR-551b,that were found to be significantly down-regulated in the L02/HBx-d382 cells were selected for further study and transfected individually into cells using the lipofectamine procedure.The cell survival rate was analyzed by MTT assay,and cell cycles were assessed by flow cytometry.Expressions of cyclinD1,cyclinG1,and E2F1 were assessed by real-time PCR and Western blotting.Results Compared with the microarray miRNA profile of L02/pcDNA3.0 cells,six miRNAs were up-regulated and five miRNAs were down-regulated in the L02/HBx-d382 cells,while four miRNAs were upregulated and 12 were down-regulated in the L02/HBx cells.The microarray results were consistent with realtime PCR results.Transfection of miR-338-3p and miR-551b significantly inhibited the cell survival rates (P＜0.001) and induced G0/G1 phase cycle arrest According to MTT results:for L02/HBx-d382 cells,compared with lipofectamine or non-transfected (NC) controls,the t value of miR-338-3p was 10.402,9.133 and the t value of miR-551b was 8.763,7.403; for L02/HBx cells,compared with lipofectamine or NC controls,the t value of miR-338-3p was 9.105,8.074 and the t value of miR-551b was 7.673,7.52.According to flow cytometry results:for L02/HBx-d382 cells,compared with lipofectamine or NC controls,the t value of miR-338-3p was 12.173,11.107and the t value of miR-55 1b was 15.364,13.377; for L02/HBx cells,compared with lipofectamine or NC controls,the t value of miR-338-3p was 15.416,13.378,and the t value of miR-551 b was 13.276,13.109.The protein levels of cyclinD1,cyclinG1,and E2F1 were significantly reduced by both miR-338-3p and miR-551b (P＜0.001).For L02/HBx-d382 cells,compared with lipofectamine or NC controls:E2F1 had t=11.132,10.031 and 12.017,10.973,respectively; cyclinD1 had t=15.654,15.013 and 15.447,14.733,respectively; cyclinG1 had t=8.017,7.661 and 7.402,7A17,respectively.For L02/HBx cells,compared with lipofectamine or NC controls:E2F1 had t=14.244,13.331 and 15,022,14.468,respectively; cyclinD1 had t=8.695,8.137 and 7.877,7.503,respectively;cyclinG1 had t=7.73,7.471 and 7.596,7.41,respectively.In contrast,the mRNA levels for E2F1,cyclinD1,and cylcinG1 showed no significant differences between the miRNA transfected cells and controls.Conclusion Wild-type HBx and the high proliferation-inducing mutant HBx can influence the miRNA expression profile of L02 cells.HBx down-regulates miR-338-3p and miR-55 1b in L02 cells,and the high proliferation-inducing mutant has a more robust effect.The mechanism of miR-338-3p- or miR-55 1b-mediated cell growth inhibition appears to be related to the direct modulation ofcyclinD1,cyclinG1,and E2F1.