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

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 WAF1/CIP1 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 eukaryotic expression plasmid pGenesil-1,and the positive clone was named dsRNAP21-pGenesil-1.The eukaryotic 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.Realtime 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-pGenesil-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 mR-NA expression in two cell lines.Compared with mock transfections,induction of p21mRNA 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 transfections was significantly higher than in dsCon-pGenesil-1 transfections and mock transfections ( P ＜ 0.05 ).Conclusion The constructed dsRNAP21-pGenesil-1 could have the ability to increase the expression of tumor suppressor gene p21 in urinary cancer. Key words: RNA;  Eukaryotic expression plasmid;  p21 ;  Urinary cancer