saRNA在转录水平激活人膀胱癌细胞p21^(WAF1/CIP1)的表达研究  

Study of saRNA-directed transcriptional activation of p21^(WAF1/CIP1) expression in human bladder cancer cells

作　　者：胡嘏[1] 陈忠[1] 吴嘉[1] 张勇[1] 王骥[2] 郭小林[1] 徐华[1] 李龙承[2] 杨为民[1] 叶章群[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院泌尿外科,武汉430030 [2]美国加州大学旧金山分校

出　　处：《现代泌尿生殖肿瘤杂志》2012年第3期165-168,173,共5页Journal of Contemporary Urologic and Reproductive Oncology

基　　金：国家自然科学基金资助项目(30873018)

摘　　要：目的探讨小激活RNA-dsP21-322上调人膀胱癌细胞系T24中抑癌基因p21WAF1/CIP1(p21)表达的效应。进一步分析dsP21-322是否还可上调p21前体mRNA的表达,以及增强RNA聚合酶Ⅱ蛋白与dsP21-322靶向的p21启动子区的联系。方法合成靶向p21启动子区的小激活RNA序列dsP21-322及阴性对照(dsControl),并分别转染至T24细胞,RT-qPCR和WesternBlot分别检测p21mRNA、前体mRNA和蛋白表达的变化。ChIP检测RNA聚合酶Ⅱ蛋白与dsP21-322靶向的p21启动子富集的改变。结果 RT-qPCR和WesternBlot结果显示,dsP21-322可以明显上调T24细胞系中p21成熟mRNA、前体mRNA及p21蛋白的表达,且与dsControl相比,差异均有统计学意义(P<0.001)。此外,dsP21-322的转染可以明显增强RNA聚合酶Ⅱ蛋白与dsP21-322靶向的p21启动子区的联系。结论 dsP21-322上调抑癌基因p21表达是发生在转录水平的基因调控过程,这一特征的发现为后续RNA激活机制的研究提供了部分理论依据。Objective To study the effect of a small activating RNA （dsP21-322）-mediated tumor suppressor gene p21 activation in T24 human bladder cancer cell line. Further analysis wheth- er activated dsP21-322 could also increase the expression of p21 pre-mRNA, and enhance association of RNA pol Ⅱ with the dsP21-322-targeted promoters. Methods A dsRNA, dsP21-322, targeting the p21 promoter at position -322 relative to the transcription start site was synthesized. A dsControl was also synthesized and used as a negative control. T24 cell line was cultured in vitro and transfected with dsP21-322 or dsControl, then RT-qPCR and Western Blot were applied to detect the expression level of p21 mRNA, p21 pre-mRNA and p21 protein, respectively. Enrichment of RNA pol Ⅱ at dsP21-322-targeted promoters was detected by CHIP. Results The results of RT-qPCR and Western Blot showed that dsP21-322 caused an obvious induction of p21 mRNA, p21 pre-mRNA and p21 protein in T24 cell line, which was more than in dsControl transfections （P〈0. 001）. In ad- dition, treatment with dsP21-322 enhanced association of RNA pol Ⅱ with the dsP21-322-targeted promoters. Conclusions These findings reveal the feature that up-regulation of tumor suppressor gene p21 mediated by activated dsP21-322 occurs at transcriptional level, which offer certain theory basis for exploring RNAa mechanism.

关 键 词：RNA激活 小激活RNA p21前体mRNA RNA聚合酶Ⅱ p21启动子 

分 类 号：R737.14[医药卫生—肿瘤]