Inhibition of Bcl-2 expression by a novel tumor-specific RNA interference system increases chemosensitivity to 5-fluorouracil in Hela cells
Inhibition of Bcl-2 expression by a novel tumor-specific RNA interference system increases chemosensitivity to 5-fluorouracil in Hela cells
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DOI：
10.1111/j.1745-7254.2006.00247.x
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发表时间：
2006-02-01
影响因子：
8.2
通讯作者：
Zhao, HF
中科院分区：
文献类型：
--
作者：
Huang, SL;Wu, Y;Zhao, HF
目的：RNA干扰（RNAi）已被提出作为一种潜在的癌症治疗方法，但缺乏细胞靶点限制了其在癌症基因治疗中的应用。目前还没有技术利用RNAi实现直接的肿瘤特异性基因沉默。在目前的研究中，我们试图利用人类端粒酶逆转录酶（hTERT）启动子开发肿瘤特异性RNAi系统；进一步分析其对Bcl-2表达的抑制作用。方法：构建含有外源报告基因[萤火虫荧光素酶和增强型绿色荧光蛋白（EGFP）]的小发夹RNA （shRNA）和内源基因（Bcl-2）的载体。双荧光素酶法检测荧光素酶的表达。采用逆转录聚合酶链反应（RT-PCR）、荧光显微镜和荧光活化细胞分选（FACS）检测EGFP的表达。RT-PCR和Western blotting检测Bcl-2的抑制作用。采用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基- 2h -溴化四氮唑（MTT）法测定细胞增殖和活力。采用流式细胞仪分析细胞周期分布。结果：我们发现hTERT启动子直接驱动shRNA转录，在体外特异性抑制肿瘤细胞中外源报告基因（LUC和EGFP）的表达，而非正常细胞。hTERT启动子驱动的shRNA也抑制了Bcl-2的表达。抑制Bcl-2不影响细胞增殖，但增加了HeLa细胞对5-氟尿嘧啶的化学敏感性。结论：本研究描述了一种利用hTERT启动子对肿瘤细胞特异性基因沉默的高效RNAi系统。利用该系统抑制Bcl-2使HeLa细胞对5-氟尿嘧啶敏感。该系统可能对RNAi治疗有用。
Aim: RNA interference (RNAi) has been proposed as a potential treatment for cancer, but the lack of cellular targets limits its use in cancer gene therapy. No current technology has achieved direct tumor-specific gene silencing using RNAi. In the present study we attempt to develop a tumor-specific RNAi system using the human telomerase reverse transcriptase (hTERT) promoter; furthermore, we analyzed its inhibitive effect on Bcl-2 expression. Methods: The vectors containing a small hairpin RNA (shRNA) to target exogenous reporters [firefly luciferase and enhanced green fluorescent protein (EGFP)] and endogenous gene (Bcl-2) were constructed. Luciferase expression was determined by dual luciferase assay. Reverse transcription-polymerase chain reaction (RT-PCR), fluorescence microscopy and fluorescence-activated cell sorting (FACS) were used to measure EGFP expression. Inhibition of Bcl-2 was evaluated by RT-PCR and Western blotting. Cell proliferation and viability were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. FACS was used to analyze the cell cycle distribution profile. Results: We showed that with the hTERT promoter directly driving shRNA transcription, expression of the exogenous reporters (LUC and EGFP) in tumor cells, but not normal cells, was specifically inhibited in vitro. The hTERT promoter-driven shRNA also depressed the expression of Bcl-2. Inhibition of Bcl-2 did not affect cell proliferation, but increased the chemosensitivity of HeLa cells to 5-fluorouracil. Conclusion: The present study describes an efficient RNAi system for gene silencing that is specific to tumor cells using the hTERT promoter. Suppression of Bcl-2 by using this system sensitized HeLa cells to 5-fluorouracil. This system may be useful for RNAi therapy.