RNA干扰对子宫内膜癌HEC-1A细胞PTTG影响的研究  

Experimental studies of inhibiting effect of RNA interference on pituitary tumor transforming gene in endometrial carcinoma

作　　者：张曙萱[1] 刘永利[1] 袁丽萍[1] 

机构地区：[1]徐州市第一人民医院妇产科,江苏徐州221000

出　　处：《中国妇幼健康研究》2011年第6期760-764,共5页Chinese Journal of Woman and Child Health Research

基　　金：徐州市科技发展基金资助项目（XM09B054）

摘　　要：目的 建立靶向垂体瘤转化基因（PTTG）干扰RNA质粒表达载体,观察对子宫内膜癌HEC-1A细胞PTTG的影响.方法 根据PTTG基因设计3对干扰序列,合成特异性干扰PTTG的siRNA片段,并与pSilencer3.0-Hl载体连接,构建3个PTTG干扰载体（pSilencer3.0-Hl-PTTG-siRNA）,分别命名为PTTG siRNA-1、PTTG siRNA-2、PTTG siRNA-3.利用脂质体将其转染子宫内膜癌系HEC-1A细胞,以带有绿色荧光蛋白的真核表达质粒pSilencer3.0-Hl作为转染效率测定组,免疫荧光技术检测转染效率;应用RT-PCR 及Western blot分别检测PTTG mRNA和蛋白表达水平.结果 序列分析法证明构建pSilencer3.0-Hl-PTTG-siRNA质粒成功;RT-PCR和Western-blot分析表明PTTG siRNA-2在mRNA水平和蛋白水平特异性抑制PTTG基因的表达最强.结果 显示HEC-1A细胞阴性对照组荧光表达百分率约为5.3%,转染组荧光表达百分率为74.9%,瞬时转染效率约为69.6%,已经可以满足实验需要.结论 成功构建了靶向抑制PTTG基因的siRNA干扰载体,筛选了干扰效果最强的序列,为进一步实验打下了基础.Objective To construct a recombinant small interference RNA（siRNA） expression vector targeting pituitary tumor transforming gene（F1TG） and investigate its inhibiting effect on endometrial carcinoma. Methods Three pairs of interfering sequences were designed and specific PTTG siRNAs were synthesized and inserted into pSilencer3. 0-HI vector to construct three PTI＇G interference vectors （pSilencer3.0-H1-PTTG-siRNA）, which named as PTFG siRNA-1, PTFG siRNA-2, PTTG siRNA-3 respectively. The interfering plasmids were used to transfect HEC-1A cells with lipofectmine 2000 transfection reagent. Transfection efficiency was measured with pSilencer3.0- H1 plasmid with EGFP fluorescence protein. PTTG mRNA and protein expression levels were measured with RT-PCR and Western blot methods. Results Successful construction of vector PTTG was confirmed by sequencing analysis. After the HEC-1A cells were transfected, the outcomes of RT-PCR and Westren blot indicated that FTTG siRNA-2 showed the strongest inhibiting effect on FTTG mRNA and protein. Conclusion SiRNA expression vector targeting PTTG is successfully constructed, and the sequence with strongest interference effect is screened out, which provides foundation for further study.

关 键 词：RNA干扰 垂体瘤转化基因 真核表达质粒 子宫内膜癌 

分 类 号：R737.33[医药卫生—肿瘤]