FLIP干扰性小RNA促进大肠癌细胞的凋亡  

Effect of FLIP small interfering RNA on apoptosis of colorectal cancer cells

作　　者：孙保存[1] 臧凤琳[1] 牛瑞芳[1] 魏熙胤[1] 赵秀兰[2] 张诗武[1] 

机构地区：[1]天津医科大学肿瘤研究所,天津市300060 [2]天津医科大学病理教研室,天津市300070

出　　处：《世界华人消化杂志》2005年第13期1519-1523,共5页World Chinese Journal of Digestology

基　　金：天津市自然科学基金资助项目;No.013611511~~

摘　　要：目的:研究对应FLIP基因的siRNA片段对大肠癌细胞株HT-29凋亡的影响,明确FLIP在Fas介导的凋亡途径中的作用. 方法:体外培养大肠癌细胞HT-29,通过电穿孔技术将特异性siRNA片段转染入细胞,半定量RT-PCR 法判断干扰前后FLIP mRNA水平的变化,分析RNA 干扰的特异性、时效性,并比较对应不同位点的两个siRNA片段对FLIP的干扰效果.经凋亡诱导型抗体激活后,以Annexin V染色法及DNA降解片段检测分析干扰前后HT-29细胞对Fas介导的凋亡敏感性的改变. 结果:特异性siRNA片段能有效降低FLIP mRNA水平,最大干扰效率达65.02%,明显高于作为对照的非相关片段;干扰作用于转染后24 h即可出现,48 h达高峰,72 h稍有降低;对应不同位点的两个siRNA片段对FLIP均可产生干扰作用,彼此间差别不大.在诱导型抗体的刺激下,与未转染细胞相比,转染siRNA 的HT-29细胞中凋亡细胞所占比例明显增加. 结论:特异性siRNA片段可显著降低FLIP基因mRNA 的表达水平,并提高HT-29细胞对Fas介导的凋亡敏感性.以FLIP为靶点的RNA干扰技术可望成为大肠癌基因治疗的新方法.AIM: To investigate the promotion effect of small interfering RNA of FADD-like IL-1β converting enzyme inhibitory protein (FLIP) on the cell apoptosis in colorectal cancer. METHODS: Human colorectal cancer cell line HT-29 was cultured in vitro and transfected with two siRNAs (different loci) of FLIP by electroporation technique. The level of FLIP mRNA expression before and after interfering was detected by semi-quantitative polymerase chain reaction (PCR). The specificity and time effect for interference and the interfering effect between the two siRNAs were compared. Then the cells were treated with agonistic anti-Fas antibody to induce apoptosis. The apoptosis before and after interfering was determined by Annexin V and DNA degration. RESULTS: The level of FLIP mRNA in HT-29 cells was inhibited by the specific siRNAs. The decrease of FLIP mRNA expression began to appear 24 hours after transfection. And the most apparent interfering efficiency was 65.02% 48 hours after transfection, which was markedly higher than that in the cells transfected with the control siRNAs. Both siRNAs (siRNA-F1 and siRNA-F2) from different loci had interfering effect on FLIP mRNA expression, but there was no significant difference between them. Compared with those in non-transfected cells (1.76%), the apoptotic rates were significantly higher in siRNA transfected cells (29.50%) after treated with agonistic anti-Fas antibody. CONCLUSION: Small interfering RNA of FLIP can markedly decrease the expression of FLIP mRNA and sensitize the Fas-mediated apoptosis of colorectal cancer cell line HT-29. The RNA interfering technique targeted on FLIP may provide a new method in the gene therapy of colorectal cancer.

关 键 词：FLIP 干扰性小RNA 大肠癌细胞株HT-29 HT-29细胞 半定量RT-PCR法 RNA片段 mRNA水平 FAS介导 Annexin DNA降解片段 RNA干扰技术 凋亡敏感性 基因mRNA 干扰作用 电穿孔技术 siRNA 癌基因治疗 特异性 体外培养 凋亡途径 抗体激活 

分 类 号：R735.34[医药卫生—肿瘤]