肿瘤细胞特异启动子hTERT介导靶向mdr1基因RNAi用于逆转卵巢癌细胞MDR研究.docVIP

肿瘤细胞特异启动子hTERT介导靶向mdr1基因RNAi用于逆转卵巢癌细胞MDR研究 　　【摘要】 目的：探索应用肿瘤细胞特异启动子hTERT介导靶向肿瘤多药耐药基因mdr1的siRNA在卵巢肿瘤细胞特异表达并逆转MDR的可行性。方法：应用携带luc报告基因的报告质粒验证了hTERT启动子在卵巢肿瘤细胞株A2780中的转录活性，构建由hTERT启动子引导的靶向mdr1基因的siRNA表达载体phTERT-siMDR1B，并与携带mdr1基因靶序列的报告质粒进行共转染抑制实验，进一步将phTERT-siMDR1B表达载体与mdr1基因表达载体共转染A2780细胞，检测细胞中mdr1基因的mRNA与P-gp蛋白的表达水平。以具有耐紫杉醇表型的A2780细胞作为靶细胞进行耐药评价实验。结果：hTERT启动子在卵巢肿瘤细胞株A2780中具有良好的转录活性，而在正常人二倍体细胞株MRC-5中无转录活性；hTERT启动子介导的siMDR1B具有良好的抑制效果与特异性；hTERT启动子介导的siMDR1B可显著抑制细胞中mdr1基因的mRNA与P-gp蛋白的表达水平；转染了hTERT启动子介导的siMDR1B表达元件的细胞对紫杉醇的耐药程度显著降低。结论：hTERT启动子介导的靶向mdr1基因的RNAi用于逆转卵巢癌细胞MDR是可行的，本研究结果可为进一步开展卵巢肿瘤MDR逆转研究提供重要基础。 　　【关键词】 卵巢癌； 多药耐药； hTERT启动子； RNA干扰 　　Study of Tumor Cell Specific Promoter hTERT Mediated RNAi Which Targeted mdr1 Gene in Reversion MDR of Ovarian Cancer Cells/LIN Yan-zhen，CHENG Tong，ZHANG Ya-li，et al.//Medical Innovation of China，2015，12（34）：008-012 　　【Abstract】 Objective：To discuss the feasibility of tumor cell specific promoter hTERT mediated RNAi which targeted mdr1 gene in reversion multidrug resistance（MDR） of ovarian cancer cells.Method：The transcriptional activity of hTERT promoter in ovarian cancer cell line A2780 were verified by the report plasmid carried Luc gene.The hTERT promoter mediated siRNA expression vector phTERT-siMDR1B which targeted mdr1 gene were constructed.Co-transfected inhibition experiment of phTERT-siMDR1B and report plasmid which carried mdr1 gene target sequence was carried out.A2780 cells were furtherly co-transfected by phTERT-siMDR1B expression vector and mdr1 gene expression vector，and the expression level of mRNA and P-gp protein of mdr1 gene in cells were detected.The drug resistance were evaluated by using A2780 cells which has a resistance to Taxol.Result：hTERT promoter had good transcriptional activity in ovarian cancer cell line A2780，whereas without transcriptional activity in normal human diploid cell lines MRC-5.hTERT promoter mediated siMDR1B had good inhibitory effect and specificity.hTERT promoter mediated siMDR1B could si