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

The effect of microRNA-21-siRNA-lentivirus on biological behaviors in human hepatic cancer cell lineHepG2

目的通过构建慢病毒介导（1entivirus，LV）的microRNA一21一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-21一RNAi—LV转染肝细胞癌HepG2。体外设为干扰组（microRNA-21一siRNA，SI组）、阴性对照组（negativecontrol，NC组）和正常组（normal，N组）；分别采用聚合酶链反应方法检测microRNA一21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）microRNA一21一RNAi．LV可显著降低microRNA一21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-21一siRNA通过抑制microRNA-21的表达抑制细胞增殖．促讲细朐凋f．降低其迁移能力。

ObjectiveL 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-1V）. 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 MT＇F assay. The invasion was dectected by transwell 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/e nude mice were observed. Results （ 1 ） The expression level of miR-21 was inhibited significantly by miR-21-siRNA-1V. （2） The proliferation of HepG2 was also markedly suppressed in MTF 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 ）. Condusions 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.