联合RNA干扰TR及TERT对膀胱移行细胞癌BIU-87细胞株端粒酶活性及其增殖的影响

Effects of combining siRNA-TR and TERT upon teiomerase activity and in vitro growth inhibition in transitional cancer of bladder BIU-87 cell line

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hTERTmRNA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-ⅢTERTmRNA：67％、pRNAT—TR-ⅢTRmRNA：41％、pRNAT—TERT—Ⅲ、pRNAT—TR—ⅢTRmRNA：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。

Objective 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-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. Results RNAi-pRNAT-hTERT-m, 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. 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.