慢病毒介导shRNA特异性沉默livin基因促进SPC-A1细胞凋亡

Lentivirus-mediated shRNA silencing of livin gene promotes apoptosis of SPC-A1 cells

目的:建立慢病毒介导的livin基因沉默系统,探讨其对肺癌细胞凋亡的影响。方法:Livin shRNA慢病毒感染肺腺癌细胞株SPC-A1沉默livin基因表达。应用PI染色经荧光镜下观察SPC-A1细胞凋亡形态,流式细胞术检测SPC-A1细胞凋亡率及亚二倍体峰形成,Real-timePCR及Western blotting方法检测livin和caspase3表达的改变。结果:livin基因在肺腺癌细胞株SPC-A1中持续高表达。经慢病毒介导shRNA使livin基因表达沉默后,镜下可见肺腺癌细胞出现典型凋亡形态特征,流式细胞术检测出现亚二倍体峰,细胞凋亡率较空白对照及阴性病毒对照细胞明显增加(8.21%vs0.08%,0.13%;P<0.05),RT-PCR及Western blotting检测结果显示,caspase3mRNA表达无改变,但cleaved-caspase3蛋白表达上调。结论:慢病毒载体介导的shRNA能抑制肺腺癌细胞株SPC-A1中livin基因的表达,从而促进SPC-A1细胞凋亡。

Objective：To construct a lentiviral livin shRNA vector to silence livin gene expression, and to study its effect on apoptosis of lung carcinoma cells. Methods： Livin expression in human lung adenoearcina SPC-A1 cells was silenced by lentiviral livin shRNA infection. The morphology of apoptotic ceils was observed by propidine iodide staining and fluoroscope; apoptosis rate and sub-lipliod apoptotic peak of SPC-A1 cells were assessed by flow cytometry; expression of livin and easpase 3 in SPC-A1 cells was examined by real-time PCR and Western blotting analysis. Results： Livin was constitutively expressed in SPC-A1 cells. After livin expression was silenced by lentiviral livin shRNA infection, SPC-A1 cells showed the characteristic morphology of apoptosis under fluoroscope, and the sub-lipliod apoptotic peak was identified by flow cytometry. Apoptosis rate in livin shRNA infected SPC-A1 cells was significantly higher than that in blank and negative control groups （8.3% vs 0. 08% and 0.13% , P 〈 0.05）. caspase 3 mRNA expression in SPC-A1 cells had no change but the expression of cleaved-easpase 3 was greatly upregulated after lentiviral livin shRNA infection as showed by RT-PCR and Western blotting analysis. Conclusion： Lentiviral livin shRNA can inhibit livin expression in human lung adenocarcina SPC-A1 cells and induce cell apopotosis.