c-FLIP调节TRAIL诱导胃癌细胞凋亡敏感性的研究

Study on the regulation of c- FLIP for the sensitivity of TRAIL- induced apoptosis of gastric cancer cells

目的探讨凋亡抑制蛋白c-FLIP对肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导胃癌细胞SGC7901凋亡的影响。方法采用RNAi技术干扰c-FLIP的表达,MTT法测定细胞活力、采用Hoechst 33258染色及流式细胞仪检测细胞凋亡,定量PCR和Western blot法检测c-FLIP,半胱氨酸天冬氨酸蛋白酶-8(caspase-8)的表达。结果特异性的RNAi能够显著抑制c-FLIP mRNA和蛋白的表达。单独使用不同浓度TRAIL处理SGC7901细胞24 h后,发现细胞的存活率有所降低,但没有显著差异。在干扰c-FLIP表达的同时用TRAIL 100 ng/ml作用胃癌细胞SGC7901不同时间(6 h,12 h,24 h),发现作用24 h后,细胞的存活率为对照组的62.8%,显著降低(P<0.05),而处理后不同时间的对照组细胞存活率无显著差异(P>0.05)。凋亡实验显示在二者共同作用SGC7901细胞24 h后,能够促进细胞的凋亡。Western blot结果显示二者共同作用24 h后,c-FLIP显著降低、caspase-8及激活型caspase-8(p-18)的表达均显著升高。结论抑制c-FLIP的表达增强了TRAIL诱导胃癌细胞SGC7901的凋亡。

Objective To explore the role of c - FLIP in TRAIL induced cell apoptosis in gastric carcinoma cells. Methods The surviv- al rate of cells was detected by MTT. Two SiRNAs targeting c - FLIP genes were transfected into SGC -7901 cells by lipofectamine 2000 reagent. The effect of decrease of expression of c - FLIP was detected by qPCR and Western blot. The apoptotic rate of SGC - 7901 cells after treatment with TRAIL was analyzed by Hoechst 33258 staining and flow cytometry. Caspase - 8 and activated caspase - 8 were detected by Western blot. Results c -FLIP mRNA and protein were significantly decreased by c -FLIP siRNAs, especially the second siRNA. At 24 h after exposure of gastric carcinoma cells in TRAIL with different dosages of 1, 10, 100, and 1 000 ng/ml, the cell survival rates were 98.9%, 94.8%, 91.7% and 83.7% respectively. There was no significant difference between control group and TRAIL group. After treatment with 100 ng,/ml TRAIL for 24 h, cell survival rate was significantly declined when the c - FLIP gene was knocked down, and the survival rate was 62.8% in control group. After treatment with 100 ng/ml TRAIL for 24 h, the rates of apoptotic ceils detected by Hoechst 33258 staining were 12.5% in TRAIL treatment group, 17.4% in TRAIL combined with c -FLIP- Si -NC treatment group, and 48.7 % in TRAIL combined with c -FLIP- SiRNA treatment group. The results of FCM showed that the rate of apoptotic cells was 14.76%, it was higher than that of other two groups （ P 〈0.05 ）. Western blotting showed that expressions of caspase - 8 and activated caspase - 8 （ p - 18 ） treated with TRAIL and c - FLIP - SiRNA were higher than those of control groups. Conclusion Inhibition of c - FLIP contributes to the anti - tumor sensitivity of TRAIL - induced apoptosis in gastric car- cinoma cells.