基础研究(Basic Research)

靶向hTERT、hTR新型RNAi表达框架的构建及应用效果观察

Published at: 2016年第25卷第12期

李亚红 1 , 赵丹丹 1 , 黄丹 1 , 刘蕾 1 , 朱文琦 1 , 彭剑雄 1
1 中南大学湘雅医学院 医学检验系,湖南 长沙 410013
通讯作者 剑雄 彭 Email: jxpeng@csu.edu.cn
DOI: 10.3978/j.issn.1005-6947.10.3978/j.issn.1005-6947.2016.12.012
基金:
中南大学研究生科研创新基金资助项目 2016zzts500
中南大学本科生自由探索基金资助项目 201610533579

摘要

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-qPCR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miRNA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。


Construction of novel RNAi expression cassettes targeting hTERT and hTR and their application effects

Abstract

Objective: To construct novel RNAi expression cassettes targeting hTERT and hTR, and then observe the effects of hTERT and hTR gene interference alone or in combination on telomerase activity, cell apoptosis and cell cycle in tumor cells, so as to find new strategies for tumor gene therapy. Methods: The RNAi expression cassettes targeting hTERT or hTR gene were synthesized by overlap extension PCR. After identification, the established cassettes were transfected into A549 cells alone or in combination, and then the telomerase activity was detected by TRAP-silver staining and TRAP-qPCR, and cell apoptosis rate and cell cycle were determined by flow cytometry. Results: The novel RNAi expression cassettes targeting hTERT and hTR were successfully constructed. Comparing with blank control group or negative control group, the telomerase activities were significantly reduced, the cell apoptosis rates were significantly increased and the G1 cell cycle arrest was significantly increased in A549 cells after transfection of the cassettes targeting either hTERT or hTR gene, and further, the above changes were more significant in A549 cells after combined transfection of the cassettes targeting hTERT and hTR gene than in those transfected with either of them alone (all P<0.05). Conclusion: The expression cassettes of novel RNAi targeting hTERT and hTR can effectively inhibit the telomerase activity, induce the cell apoptosis and change the cell cycle in A549 cells. Novel RNAi technology based on the artificial miRNA expression cassettes is expected to become a new tool in tumor gene therapy.


comments powered by Disqus

全文

引用

引用本文: 亚红 李, 丹丹 赵, 丹 黄, 蕾 刘, 文琦 朱, 剑雄 彭. 靶向hTERT、hTR新型RNAi表达框架的构建及应用效果观察 [J]. 中国普通外科杂志, 2016, 25(12): 1738-1744.
Cite this article as: LI Yahong, ZHAO Dandan, HUANG Dan, LIU Lei, ZHU Wenqi, PENG Jianxiong . Construction of novel RNAi expression cassettes targeting hTERT and hTR and their application effects[J]. Chin J Gen Surg, 2016, 25(12): 1738-1744.