摘要目的 探索联合RNA干扰TR及TERT[小型干扰RNA(siRNA)、人端粒酶RNA(hTR)及人端粒酶逆转录酶(hTERT)]基因对膀胱移行细胞癌细胞株BIU-87端粒酶活性及其增殖的影响.方法 根据hTRmRNA和hTERTmRNA的序列,分别设计合成3对不同的且能干扰TR和TERT基因表达的siRNA并筛选出最高效抑制序列(pRNAT-TR-Ⅲ、pRNAT-TERT-Ⅲ),构建以hTR、hTERT基因为靶点的联合RNA干扰装置pRNAT-TR-Ⅲ、pRNAT-TERT-Ⅲ(脂质体法)并将其转染BIU-87细胞株,采用荧光定量PCR分析hTR及hTERT mRNA表达,TRAP-ELISA检测端粒酶活性,MTT法检测细胞增殖.结果 分别及联合RNA干扰hTR及hTERT基因后,pRNAT-TERT-Ⅲ,pRNAT-TR-Ⅲ及pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ的联合干扰可以特异性抑制BIU-87细胞株中TERT和TR表达(分别减少pRNAT-TERT-Ⅲ TERTmRNA:67%、pRNAT-TR-Ⅲ TRmRNA:41%、pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ TRmRNA:57%、pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ TERTmRNA:70%.P＜0.05).且膀胱移行细胞癌细胞株BIU-87的生长和端粒酶活性均明显受到抑制,以联合RNA干扰尤为明显(pRNAT-TERT-Ⅲ:1.80±0.014,pRNAT-TR-Ⅲ:1.95±0.016,pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ:1.25±0.012,P＜0.05).结论 联合RNA干扰hTERT及hTR基因能更明显抑制膀胱移行细胞癌BIU-87细胞株的生长,为其临床应用奠定了基础.

abstractsObjective To investigate the influence of combining RNAi-hTR plus hTERT genes upon the telomerase activity of bladder cancer BIU-87 cell line and provide new methods and evidence for RNAi in gene therapy of bladder transitional cell cancer.Methods Three hTR-specific double-stranded siRNAs and 3 hTERT-speeific double-stranded siRNAs were designed targeting different regions of hTR and hTERT mRNA.siRNAs (systems-PhTR-siRNA,PhTERT-siRNA and combining systems-PhTR plus PhTERT-siRNA) were transfeeted into bladder transitional cancer BIU-87 cell line.And hTR and hTERT mRNA expression were determined by fluorescence quantitative RT-PCR while telomerie repeat amplification protocol (TRAP) was applied to detect the telomerase activity and the growth inhibition of BIU-87 cells detected by MTT assay.Results RNAi-pRNAT-hTERT-Ⅲ,RNAi-pRNAT-hTR-Ⅲ and combining RNAi-hTR plus hTERT could inhibit the expression of hTERT and hTR mRNA in bladder cancer BIU-87 cell lines by RNAi-pRNAT-hTERT-Ⅲ hTERTmRNA 67%,RNAi-pRNAT-hTR-Ⅲ hTRmRNA 41% and pRNAT-hTR-Ⅲ hTRmRNA:57%,pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ hTERTmRNA:70% (P＜0.05).The growth of bladder cancer BIU-87 ceil was inhibited and telomerase activity considerably decreased,especially in combining RNAi-hTR and hTERT.Conclusion hTR-siRNA,hTERT-siRNA and combing siRNA hTR plus hTERT have been successfully designed and constructed.They can suppress specifically and effectively both hTR and hTERT mRNA expression and telomerase activity,especially in combining siRNA-hTR+hTERT.Combining siRNA-hTR plus hTERT are needed to explore its clinical applications.