慢病毒介导的microRNA-21-siRNA对肝癌细胞系HepG2生物学行为的影响

目的 通过构建慢病毒介导( lentivirus,LV)的microRNA-21-siRNA,探讨下调microRNA-21对肝癌细胞系HepG2生物学行为的影响.方法 针对目标序列合成特异性siRNA干扰片段并克隆入慢病毒载体pGCSIL-GFP,将重组microRNA-21-RNAi-LV转染肝细胞癌HepG2.体外设为干扰组(microRNA-21 -siRNA,SI组)、阴性对照组(negative control,NC组)和正常组(normal,N组)；分别采用聚合酶链反应方法检测microRNA-21目的基因的表达的情况；用噻唑蓝比色法检测增殖情况、transwell小室迁移实验检测迁移情况、Hochest33258凋亡实验检测凋亡情况.体内实验设为SI组和NC组,观察反义microRNA-21对裸鼠移植瘤生长的影响.结果 (1)micmRNA-21 -RNAi-LV可显著降低microRNA-21的表达.(2) MTT实验96h后SI组增殖能力显著低于NC组和N组(P=0.0031,P＜0.05).(3)迁移能力减弱(P =0.0004,P＜0.05).(4)SI组细胞凋亡明显增加,促凋亡基因caspase3基因表达上调(P =0.0002,P＜0.05).(5)各组裸鼠皮下肿瘤生长比较差异无统计学意义(P=0.0624,P＞0.05).结论 microRNA-21 -siRNA通过抑制microRNA-21的表达抑制细胞增殖,促进细胞凋亡,降低其迁移能力.

The effect of microRNA-21-siRNA-lentivirus on biological behaviors in human hepatic cancer cell line HepG2

Objective To study the effect of lentivirus-mediated microRNA-21 RNAi on biological behaviors in human hepatic cancer cells.Methods MicroRNA-21 specific siRNA gene was synthesized and cloned into the recombinant lentiviral vector,pGCSIL-GFP.HepG2 cells were infected by microRNA-21 siRNA recombinant lentivirus (miR-21-siRNA-Ⅳ).The HepG2 cells were devided into SI group,NC group and N group in vitro.The expression of the targets of miR-21 was detected by RT-PCR.Cell growth was analyzed by MTT assay.The invasion was dectected by tmnswell method.Apoptosis was detected by Hoechst33258.BALB/c nude mice were randomly divided into SI group and NC group.The growth of transplant tumors in BALB/c nude mice were observed.Results ( 1 ) The expression level of miR-21 was inhibited significantly by miR-21-siRNA-lⅣ.(2) The proliferation of HepG2 was also markedly suppressed in MTT at the 96 h point ( P =0.0031,P ＜ 0.05 ).(3) The number of cells that migrated through the chamber of SI group decreased ( P =0.0004,P ＜ 0.05 ).(4) The cell apopotosis in SI group increased markedly.In addition,the caspase 3 mRNA significantly increased ( P =0.0002,P ＜ 0.05 ).( 5 ) Tumor growth curve was not statistically different between groups ( P =0.0002,P ＜ 0.05 ).Conclusions MicroRNA-21 specific siRNA suppresses the proliferation and migration of HepG2 cells and induces tumor cell apoptosis inhibiting the growth of transplanted tumor in Balb/c nude mice.