p21saRNA真核表达质粒的构建及其在泌尿系肿瘤细胞中的功能

Construction of p21saRNA eukaryotic expression vector and identification of its function in urinary cancer cells

目的构建可表达具有RNA激活功能的小分子双链RNA（dsRNA）真核表达质粒，并探讨其调控前列腺癌细胞株PC。3和膀胱癌细胞株T一24中抑癌基因p21 WAFI/CIPI表达的功能。方法构建真核表达质粒dsRNAP21-pGenesil-1，并分别转染至PC-3和T-24细胞株，通过实时定量聚合酶链反应（Real—timeqPCR）和Westernblot检测p21mRNA转录水平和蛋白表达的变化。结果测序结果证实目的表达质粒dsRNAP21-pGenesil-1构建成功，将其转染入PC-3细胞和T-24细胞中，Real—timeqPCR检测p21基因分别被上调4．35倍和2．83倍，Westernblot进一步证明在两种细胞株中p21蛋白表达水平的增加与p21mRNA水平的上调一致，且与对照组比较，差异均有统计学意义（P〈0．05）。结论构建的dsRNAP21-pGenesil-1质粒具备在泌尿系肿瘤中上调抑癌基因p21表达的能力。

Objective To construct small double stand RNA （dsRNA） eukaryotic expression plasmid possessing the ability of RNA activation, and investigate its capability to regulate the tumor suppressor gene p21 WAFI/CIPI expression in human prostate adenocarcinoma cell line PC-3 and human bladder cancer cell line T-24. Methods The dsRNA sequences targeting the p21 promoter at position-322 relative to the transcription start site were synthesized and inserted into dsRNA eukaryotie expression plasmid pGenesil-1, and the positive clone was named dsRNAP21-pGenesil-1. The eukaryotie expression plasmid dsR- NACon-pGenesil-1 was also constructed and used as a negative control. PC-3 and T-24 cells were cultured in vitro and transfected with dsRNAP21-pGenesil-1 or dsRNACon-pGenesil-1 by Lipofectamine 2000. Real- time quantitative polymerase chain reaction （Real-time qPCR） and Western blotting were applied to detect the expression levels of p21 mRNA and protein respectively. Results The eukaryotic expression plasmids dsRNAP21-pGenesil-1 or dsRNACon-pC, enesil-1 were confirmed by DNA sequencing. Seventy-two h after transfection, Real-time qPCR showed that dsRNAP21 -pGenesil-1 caused a significant induction in p21 mRNA expression in two cell lines. Compared with mock transfections, induction of p21 mRNA was 4. 35- and 2. 83-fold in PC-3 and T-24 cells, respectively. Western blotting analysis further verified that the elevated levels of p21 protein were strongly correlated to the increase of p21 mRNA expression in above cell lines. The p21 protein expression level in dsRNAP21-pGenesil-1 transfeetions was significantly higher than in dsCon-pGenesil-1 transfections and mock transfections （P 〈 0. 05 ）. Conclusion The constructed dsR- NAP21-pGenesil-1 could have the ability to increase the expression of tumor suppressor gene p21 in urinary cancer.