不同启动子相关双链小分子RNA介导激活p21 Waf1/Cip1 基因研究

The study to active gene expression by different small double -stranded RNA which related promot. ers of p21waf1/Cip1

目的探讨针对p21基因启动子序列不同转录起始位点设计的小双链RNA（dsRNA）对p21基因表达的正向调控作用。方法针对p21基因启动子DNA序列设计合成4对dsRNA分子dsP21．382、dsP21-436、dsP21-484、dsP21—625，转染人体外培养的人膀胱肿瘤细胞株5637细胞及T24细胞中，并以具有激活功能的dsRNA分子dsP21—322作为阳性对照，以未转染的5637和T24细胞为空白对照，以dsControl为阴性对照。采用实时定量聚合酶链反应（Real-timePCR）和Westernblot等观察各dsRNA转染后，细胞内p21基因mRNA及蛋白表达量的差异，以及dsRNA转染对肿瘤细胞株生长的影响。结果PCR结果显示，与空白对照组比较，dsP21—382、dsP21-436、dsP21-484、dsP21-625分别上调5637细胞中p21mRNA的表达1．56、1．88、2．41和1．74倍（P〈0．05），dsP21-382、dsP21-436、dsP21-484、dsP21-625分别上调T24细胞中021mRNA的表达1．71、1．69、2．62和1．85倍（P〈0．05）。Westernblot结果显示，与空白对照组比较，p21蛋白表达的升高与021mRNA水平的升高一致。4对dsRNA均能显著抑制膀胱肿瘤细胞株的生长。结论dsRNA对基因表达正向调控作用可能是一种普遍现象。

Objective Research the regulation of the small activating RNA （saRNA） designed for the sites of p21 gene promoter, which increase the expression of the p21 gene. Methods We designed new dsRNAs, dsP21 -382, dsP21 -436, dsP21 -484 and dsP2l -625, targeting the p21 promoter at some sequence position relative to the transcription start site, and transfect them into human bladder cancer cell lines 5637 and T - 24. We also transfect dsP21 - 322, which could activate expression of the p21 gene, into 5637 and 3＂24 as a positive control. Take 5637 and T24 cells without transfeetion as the blank control, take dsControl as negetive control. Take 5637 and T24 cells without any treatments as the blank control groups. Additionally, real - time quantitative polymerase chain reaction （ Real - time PCR） and Western blotting were performed to examine the expression level of p21 gene in each group. We also ob- served the inhibition effect of tumor cell growth by dsRNA transfection. Results PCR analysis showed that dsP21-382, dsP21 -436, dsP21 -484, dsP21 -625 can significantly promote the expression of p21 mRNA in 5637 and T24 cells. Compared with blank control, the overexpression of p21 mRNA was 1.56 - fold, 1.88 - fold, 2. 41 - fold, 1.74 - fold in 5637 cells （P 〈 0.05） and 1.71 - fold, 1.69 - fold, 2.62 -fold, 1.85 -fold in T24 cells （P 〈0. 05）. Western blotting showed that the p21 protein expression levels were consistent with the up - regulation of p21 mRNA expression in 5637 and T24 cells. Four pairs of dsRNAs could significantly inhibite the growth of bladder tumor cell lines. Conclusion These findings indicate that universality of the activation of p21 gene by promoter - associated dsRNA. The mechanism still remains further study.