shRNA下调人端粒酶逆转录酶基因抑制膀胱癌细胞生长作用的实验研究

Experimental study on shRNA targeted hTERT gene to suppress bladder cancer cell growth

目的 探讨靶向人端粒酶逆转录酶(hTERT)基因shRNA载体,下调c myc和TGF -β1表达抑制膀胱癌细胞生长的作用机理。 方法 采用RNAi DNA载体技术,构建靶向hTERT基因不同片段的shRNA -hTERT pTZU6+1真核表达载体,转染入膀胱癌T24细胞内, RT -PCR法检测hTERT基因表达筛选最有效的shRNA hTERT pTZU6+1载体,并转染至T24细胞内,流式细胞术检测对细胞生长周期的影响, RT PCR和免疫组织化学检测转染前后hTERT、c- myc和TGF -β1的表达。 结果 成功构建3个shRNA- hTERT pTZU6+1真核表达质粒,以1. 0μg的ph2 shRNA为最佳浓度的最有效力载体,该载体转染至T24细胞后,引起细胞生长减慢,细胞周期中S期细胞数由65. 2%减少至38. 6%,G1 /G0细胞由32. 0%增至57. 9%,细胞内hTERT、c -myc和TGF β1表达减弱。 结论 RNAi可通过下调hTERT基因表达抑制膀胱癌细胞生长,其过程是通过下调癌细胞内c -myc和TGF-β1表达途径来进行的。

Objective To explore the mechanism of siRNA targeted hTERT gene to inhibit bladder cancer T24 cell growth by decreasing c-myc and TGF-β1 expression. Methods shRNA-hTERT-pTZU6+1 vectors were constructed by RNAi-DNA vector technique, then the vectors were transfected into bladder cancer T24 cells,and the most effective vector and its optimal concentration were screened using RT-PCR to detect hTERT expression in T24 cells.The T24 cell growth, the alternative of cell phase,the expression of hTERT,c-myc and TGF-β1 were detected by flow cytometry,RT-PCR and immunohistochemistry. Results Three shRNA-hTERT-pTZU6+1 vectors were successfully constructed.The most effective vector was ph2-shRNA vector,and its optimal concentration was 1.0 μg.This vector decreased the cell growth and the cell number of S phase from 65.2% to 38.6%,increased the cell number of G0/G1 phase from 32.0% to 57.9%,and attenuated both mRNA and protein expressions of hTERT,c-myc and TGF-β1 in T24 cells. Conclusions Targeted hTERT gene with siRNA may inhibit the cell proliferation of bladder cancer;down-regulating hTERT expression by attenuating the expression of c-myc and TGF-β1 is probably involved in the mechanism.