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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 investigate the effect of small activation RNA-dsP21-322 on the expression of p21WAF1/CIP1 (p21), a tumor suppressor gene in human bladder cancer cell line T24. Further analysis of dsP21-322 could also upregulate the expression of p21 precursor mRNA and enhance the association of RNA polymerase II protein with the p21 promoter region targeted by dsP21-322. Methods The small activation RNA sequence dsP21-322 and dsControl targeting p21 promoter region were synthesized and transfected into T24 cells respectively. The expression of p21 mRNA, precursor mRNA and protein were detected by RT-qPCR and Western Blot, respectively. ChIP detects changes in RNA polymerase II protein and dsP21-322-targeted p21 promoter enrichment. Results The results of RT-qPCR and Western Blot showed that dsP21-322 could significantly up-regulate the expression of p21 mature mRNA, precursor mRNA and p21 protein in T24 cell line. Compared with dsControl, the difference was statistically significant (P<0.001). In addition, transfection of dsP21-322 significantly enhanced the association of the RNA polymerase II protein with the p21 promoter region targeted by dsP21-322. Conclusion The up-regulation of p21 expression by dsP21-322 is a gene regulation process at the transcriptional level. The discovery of this feature provides some theoretical basis for the study of subsequent RNA activation mechanisms.