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目的筛选强效抑制生存素Survivin基因表达的小干扰核糖核酸(siRNA),探讨Sur- vivin基因抑制对小鼠结肠癌细胞凋亡的影响。方法根据siRNA的设计规则和Survivin序列,设计3段侯选siRNA,聚合酶链反应(PCR)法制备表达框；将siRNA表达框与Survivin-绿色荧光蛋白(EGFP)融合基因表达质粒(pSurvivin-EGFP)共转染293T细胞,24～48 h后根据荧光强度筛选强效siRNA；再制备其表达质粒转染小鼠结肠癌CT26细胞,应用实时荧光定量(FQ)-聚合酶链反应和蛋白印迹(Western blot)法分析Survivin基因的表达；DNA末端标记(TUNEL)法和流式细胞仪(FCM)Annexin V/丙化碘锭(PI)双染色法检测细胞凋亡。结果筛选出1段高效抑制Survivin表达的siRNA,可使CT26细胞内Survivin mRNA拷贝数明显减少,抑制率为86．9%；蛋白表达水平亦显著降低；TUNEL检测显示转染48 h后RNA干扰组和对照组凋亡细胞数分别为(60．13±5．71)个和(5．47±0．63)个,两组差异有统计学意义(P＜0．01)；FCM检测转染后1、2、3、4 d凋亡细胞比率分别为2．1%、4．9%、11．7%和32．6%,对照组4个时间点凋亡率均低于1．5%,两组差异有统计学意义(P＜0．01)。结论siRNA能有效抑制小鼠结肠癌CT26细胞内Survivin基因表达并诱导结肠癌细胞凋亡。
Objective To screen small interfering RNA (siRNA) that inhibits Survivin gene expression in survivin and to investigate the effect of Survivin gene silencing on the apoptosis of mouse colon cancer cells. Methods According to the design rules of siRNA and Survivin sequence, three candidate siRNAs were designed and the expression cassette was prepared by polymerase chain reaction (PCR). The siRNA expression cassette and Survivin-EGFP fusion gene expression plasmid (pSurvivin-EGFP ) Were cotransfected into 293T cells and screened for potent siRNA 24 h to 48 h according to the fluorescence intensity. The recombinant plasmids were transfected into CT26 cells of mice colon carcinoma. The expression of p27 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) and Western blot Survivin gene expression was analyzed by Western blotting. Apoptosis was detected by TUNEL method and flow cytometry (FCM) Annexin V / PI staining. Results One-stage siRNA targeting Survivin was screened out. The number of Survivin mRNA was significantly decreased in CT26 cells with the inhibition rate of 86.9% and the expression of Survivin protein significantly decreased. The TUNEL assay showed that RNA interference group (60.13 ± 5.71) and (5.47 ± 0.63), respectively, the difference between the two groups was statistically significant (P <0.01). FCM After 1, 2, 3, 4 d apoptotic cells were 2.1%, 4.9%, 11.7% and 32.6% respectively, and the apoptotic rates in control group at 4 time points were all lower than 1.5 %, The difference between the two groups was statistically significant (P <0.01). Conclusion siRNA can effectively inhibit Survivin gene expression in colon cancer CT26 cells and induce apoptosis in colon cancer cells.