Cheng Wen, Su Yun, Ma Hong-qing, Wei Zhi-feng, Gao Jian-ping, Zhang Zheng-yu, Ge Jing-ping, Jing Kang-zhen, Xu Feng
Department of Urological Surgery, Jinling Hospital, Nanjing, China.
Zhonghua Yi Xue Za Zhi. 2009 Nov 3;89(40):2847-52.
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.
Three hTR-specific double-stranded siRNAs and 3 hTERT-specific 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 transfected into bladder transitional cancer BIU-87 cell line. And hTR and hTERT mRNA expression were determined by fluorescence quantitative RT-PCR while telomeric repeat amplification protocol (TRAP) was applied to detect the telomerase activity and the growth inhibition of BIU-87 cells detected by MTT assay.
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 cell was inhibited and telomerase activity considerably decreased, especially in combining RNAi-hTR and hTERT.
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.
探讨RNA干扰（RNAi）-hTR基因联合hTERT基因对膀胱癌细胞株BIU-87端粒酶活性的影响，为RNAi技术应用于膀胱移行细胞癌的基因治疗提供新方法和依据。
针对hTR和hTERT mRNA的不同区域设计3条hTR特异性双链小干扰RNA（siRNA）和3条hTERT特异性双链siRNA，将其转染至膀胱移行癌细胞株BIU-87。采用荧光定量逆转录聚合酶链反应（RT-PCR）检测hTR和hTERT mRNA的表达；应用端粒重复序列扩增法（TRAP）检测端粒酶活性；采用噻唑蓝（MTT）比色法检测BIU-87细胞的生长抑制情况。
RNA干扰质粒RNAi-pRNAT-hTERT-III、RNAi-pRNAT-hTR-III及联合干扰质粒RNAi-hTR联合hTERT可通过RNA干扰抑制膀胱癌细胞株BIU-87中hTERT和hTR mRNA的表达，RNAi-pRNAT-hTERT-III使hTERT mRNA表达降低67%，RNAi-pRNAT-hTR-III使hTR mRNA表达降低41%，联合干扰使hTR mRNA表达降低57%、hTERT mRNA表达降低70%（P<0.05）。膀胱癌细胞株BIU-87的生长受到抑制，端粒酶活性显著降低，尤其是联合干扰RNAi-hTR联合hTERT组。
成功设计并构建了hTR-siRNA、hTERT-siRNA及联合干扰siRNA-hTR联合hTERT质粒，三者均可特异性、有效地抑制hTR和hTERT mRNA表达及端粒酶活性，尤其是联合干扰siRNA-hTR联合hTERT组。联合干扰siRNA-hTR联合hTERT有待进一步探讨其临床应用价值。
