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目的:构建靶向hTERT、hTR的新型RNAi表达框架,探讨单独及联合干扰hTERT、hTR基因对肿瘤细胞端粒酶活性、细胞凋亡及周期的影响,以期找到肿瘤基因治疗的新策略。方法:融合PCR构建靶向hTERT、hTR的RNAi表达框架。各表达框架经鉴定后分别或联合转染A549细胞,TRAP-银染法及TRAP-q PCR法检测端粒酶活性,流式细胞仪检测细胞凋亡及周期。结果:靶向hTERT、hTR的新型RNAi表达框架构建成功。与空白对照组或阴性对照组比较,无论转染靶向hTERT或靶向hTR的RNAi表达框架后,A549细胞的端粒酶活性均明显降低,细胞凋亡率均明显增加,细胞G1阻滞明显增加,而联合转染靶向hTERT与靶向hTR的RNAi表达框架后,A549细胞的上述改变较各自单独转染更为明显(均P<0.05)。结论:靶向hTERT、hTR的新型RNAi表达框架能够有效抑制A549细胞的端粒酶活性,诱导细胞凋亡,改变细胞周期。以人工mi RNA表达框架为基础的新型RNAi技术有望成为肿瘤基因治疗的新工具。
OBJECTIVE: To construct a novel RNAi expression system targeting hTERT and hTR, and to explore the influence of hTR and hTR gene on the telomerase activity, apoptosis and cycle of tumor cells by hTERT and hTR gene alone and in combination, in order to find a new strategy for gene therapy of tumor. Methods: The RNAi expression system targeting hTERT and hTR was constructed by fusion PCR. A549 cells were transfected with or without A549 cells respectively. Telomerase activity was detected by TRAP-silver staining and TRAP-q PCR. Cell apoptosis and cell cycle were detected by flow cytometry. Results: A novel RNAi expression vector targeting hTERT and hTR was successfully constructed. Compared with the blank control group or the negative control group, the telomerase activity of A549 cells was significantly decreased, the apoptosis rate was significantly increased, and the cell G1 arrest was significantly higher than that of the transfected hTERT or hTR- However, the co-transfection of RNAi expression vector targeting hTERT and hTR targeting A549 cells was more obvious than that of transfection alone (all P <0.05). CONCLUSION: The novel RNAi expression system targeting hTERT and hTR can effectively inhibit telomerase activity, induce apoptosis and change cell cycle in A549 cells. The new RNAi technology based on the artificial miRNA expression framework is expected to become a new tool for gene therapy of tumors.