目的:研究应用siRNA沉默Livin基因表达后肝癌细胞HepG2的生物学功能变化.方法:用脂质体lipofectamineTM2000将携带Livin基因的siRNA载体转染至HepG2细胞内,RT-PCR、Western blot检测转染前后Livin基因mRNA和蛋白质的表达改变；流式细胞技术(FCM)和TUNEL检测转染前后肝癌细胞凋亡变化.结果:RT-PCR及Western blot检测发现转染siRNA后Livin基因的mRNA和蛋白质表达均明显下降(P＜0.05)；FCM检测发现实验组、阴性对照组和空白组肝癌细胞HepG2在G1期所占比例分别是(58.99±1.173)％、(41.85±2.82)％、(41.25±1.24)％,实验组与其他两组比较差异均有统计学意义(P＜0.05)；TUNEL技术检测表明实验组肝癌细胞HepG2凋亡数目明显增加,细胞凋亡率为(67.56±2.56)％,凋亡率明显高于阴性对照组(27.21±12.58)％和空白组(14.09±5.16)％(P＜0.05).结论:在肝癌细胞HepG2中,Livin基因沉默具有诱导肝癌细胞凋亡、抑制肝癌细胞生长的作用.%Objective: To explore the biological significance of silencing Livin gene expression with siRNA in hepatocellular carcinoma HepG2 cells. Methods: HepG2 cells were transfected with synthetic small interfering RNA (siRNA) targeting Livin. Expression of Livin mRNA and protein were respectively measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Flow cytometry (FCM) and TUNEL assay were used to examine cell cycle and apoptosis in transfected cells. Results: The result of RT-PCR and Western blot showed that the mRNA and protein levels of Livin declined markedly in transfected cells (P＜0.05). FCM demonstrated that the cell cycle was arrested in the G1 phase, the apoptotic rate in those groups were (58.99 ± 1.173)%, (41.85 ±2.82)%, (41.25 ±1.24)%, as the transfected group, there were significant differences compared with other two groups (P＜0.05). The result of TUNEL assay indicated that the number of apoptotic cells in transfected group were obviously increase, the apoptotic rate was (67.56 ± 2.56)%, there were significant differences compared with negative control group (27.21 ± 12.58)% and mock group (14.09±5.16)%(P＜0.05). Conclusion: siRNA-mediated downregulation of Livin expression can induce apoptosis and diminish the growth, proliferation in hepatocellular carcinoma HepG2 cells.
展开▼
