Survivin启动子调控的hTERT基因RNAi载体的构建及其肿瘤靶向干扰作用研究

Construction of Survivin promoter regulated hTERT gene-targeted RNAi vector and research of its tumor-targeting silencing effect

目的:构建带有肿瘤特异性Survivin启动子、hTERT基因靶向的shRNA表达载体pYr-Survivinp-miR30-hTERT,探讨该载体的肿瘤靶向性及其RNAi作用效率,为RNAi技术在肿瘤基因治疗中的进一步优化进行有益的探索。方法:化学合成hTERT基因shRNA寡核苷酸序列,克隆至质粒pYr-CMV-Mir30-shRNA,构建重组质粒pYr-CMV-Mir30-hTERT;克隆Survivin启动子序列,取代该质粒原有的CMV启动子,构建重组质粒pYr-Survivinp-miR30-hTERT,同时构建阴性对照质粒pYr-CMV-Mir30-NC,以上质粒均进行酶切及测序鉴定。将以上载体分别转染人乳腺癌MCF-7细胞、人宫颈癌Hela细胞及293T细胞,荧光显微镜下观察细胞转染情况,并采用半定量RT-PCR、western blot技术检测MCF-7细胞和Hela细胞中hTERT mRNA和蛋白的表达。结果:重组质粒经酶切及测序鉴定证实均符合设计要求,构建成功;荧光检测结果表明,CMV组在三种细胞中均有荧光蛋白的表达,而Survivin组质粒仅在MCF-7细胞和Hela细胞中表达绿色荧光,转染效率均能达到70%以上,而在293T细胞中则未见荧光;RT-PCR和Western blot的结果均显示MCF-7细胞和Hela细胞的Survivin组和CMV组中hTERT mRNA和蛋白的表达均受到明显抑制(P<0.05),两组之间无明显差异(P>0.05)。结论:Survivin启动子调控的hTERT基因RNAi载体能有效抑制Survivin阳性肿瘤细胞中hTERT的表达,而对正常细胞不产生影响。

Objectives： To construct tumor specific Survivin promoter regulated hTERT gene-targeted RNAi expression vector （pYr-Survivinp-miR30-hTERT） and investigate its tumor-targeting gene suppression effects and efficiency for the improvement of RNAi technology. Methods： The hTERT gene-targeted RNAi oligonucleotides sequence was synthesized and cloned to the plasmid pYr-CMV-Mir30-shRNA to construct the recombinant plasmid pYr-CMV-Mir30-hTERT; the CMV promoter in the plasmid was then replaced by the Survivin promoter, to construct the plasmid pYr-Survivinp-miR30-hTERT. The negative control plasmid pYr-CMV-Mir30-NC was constructed by the same method and these new recombinant vectors were confirmed by enzyme digestion and sequencing. Next,they were transfected into three kinds of cells （human breast cancer MCF-7 cells, human cervical carcinoma Hela cells,and human embryo renal cell-293T cells） and the fluorescent microscope was used to observe the cell transfection situation. Finally, semi-quantitative RT-PCR and western blot were performed to evaluate the expression of hTERT gene in MCF-7 and Hela cells. Results： Enzyme digestion and sequencing confirm that the recombinant plasmid was constructed successfully; the results of fluorescence microscopy showed that in CMV group all three kinds of cells had fluorescent protein expression, while in Survivin group green fluorescent only in MCF-7 and Hela cells , the transfection efficiency were both more than 70%;in 293T cells were not seen fluorescent cells. The results of RT-PCR and western blot showed that the expression hTERT mRNA and protein were significantly inhibited in MCF-7 and Hela cells of both Survivin and CMV groups of （P〈0.05）, while there was no significant difference in these two groups （P〉0.05）. Conclusions：The hTERT gene-targeted RNAi expression vector mediated by Survivin promoter can inhibit expression of hTERT gene in Survivin positive tumor cells effectively , but did not affect the normal cells.