核糖核酸干扰抑制生存素表达诱导小鼠结肠癌细胞凋亡  

Induction of murine colon carcinoma apoptosis by small interference RNA targeted to Survivin

作　　者：李喆[1] 赵平[2] 王建军[3] 盛援[1] 廖小玲[2] 戚中田[2] 

机构地区：[1]第二军医大学长海医院普通外科,上海200433 [2]第二军医大学长海医院,基础部微生物学教研室,上海200433 [3]华中科技大学同济医学院附属同济医院妇产科

出　　处：《中华实验外科杂志》2006年第7期792-794,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(30500606)

摘　　要：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 interference RNAs that could inhibit Survivin expression and to explore the effect of RNA interference of Survivin on apoptosis of murine colon carcinoma. Methods Three siRNA target sequences for Survivin were designed and the siRNA expression cassettes were prepared by PCR. The siRNA expression cassettes were transfected into 293T cells along with Survivin and green fluorescent protein fusion gene expression plasmid to select more effective siRNA. The expression vector of an effective siRNA was constructed and transfected into murine colon carcinoma CT26 cells. The expression of Survivin was detected by real-time fluorescent quantitative polymerase chain reaction and Western blot. The apoptosis of CT26 cells was assayed by TUNEL and flow cytometry through FITC-Annexin V/PI dying methods. Results A candidate siRNA that down-regulated the expression of Survivin in 293T cells was selected, which could efficiently inhibitthe expression of Survivin mRNA and protein in CT26 cells. The apoptosis of CT26 cells was significantly increased. TUNEL revealed that amount of apoptosis cells was （60.13 ± 5.71） in siRNA groups and （5.47 ± 0.63） in controls at 48th h after transfection. FCM demonstrated that the apoptosis rate was 2.1%, 4.9 %, 11.7 % and 32.6 % at 24th, 48th, 72nd and 96th h after transfection respectively, which of controls were lower than 1.5 %. Conclusion SiRNA could suppress the expression of Survivin and induce CT26 cell apoptosis effectively, which lay a foundation for further research on treatment of malignant tumor targeting to Survivin.

关 键 词：SURVIVIN RNA干扰 结肠癌 脱噬作用 

分 类 号：R735.35[医药卫生—肿瘤]