ABSTRACT
Objective To investigate the effect of dsP21-555 transfection on the expression of tumor suppressor gene p21 in renal clear cell carcinoma cell lines ACHN and 786-O.Methods Renal clear cell carcinoma cells were transfected with dsControl and dsP21-555 with Lipofectamine 3000 respectively.Real-time quantitative PCR (RT-qPCR) and Western blotting were used to detect the expression of p21 mRNA and protein.Cell cycle distribution was detected by flow cytometry (FCM).Cell viability and proliferation were analyzed by cell viability assay (MTS method) and colony culture assay.Results In ACHN and 786-O cells, the expressions of p21 mRNA in dsP21-555 group (2.86±0.33, 1.96±0.35) were significantly higher than those in dsControl group (1.05±0.34, 1.01±0.14), which were increased to 2.72 times (t=7.640, P<0.001) and 1.95 times (t=5.058, P=0.002).Western blotting showed that the expressions of P21 protein were up-regulated in both renal cell lines, which was consistent with p21 mRNA up-regulation.The result of FCM showed that the cell cycle was blocked in G0-G1 phase (57.08%±5.66% vs.46.06%±4.60%, t=3.023, P=0.023;61.58%±6.23% vs.42.25%±6.08%, t=4.444, P=0.004) after transfection of dsP21-555 in renal clear cell carcinoma cells.MTS result showed that the vitality of both cell lines after transfection of dsP21-555 decreased compared with dsControl group, their absorbance values were 0.85±0.20 vs.1.27±0.13, t=3.410, P=0.014;1.04±0.25 vs.1.55±0.10, t=3.758, P=0.009.Colony culture experiments showed that the numbers of colonies formed by ACHN and 786-O in the dsControl group were 110.91±26.21 and 129.99±22.87 respectively, and the numbers of colonies formed in the dsP21-555 group were 59.37±14.23 (t=3.456, P=0.014) and 71.26±21.38 (t=3.745, P=0.010), indicating that the proliferation of cells in the dsP21-555 group was significantly reduced.Conclusion dsP21-555 can up-regulate the expression of p21 gene in renal clear cell carcinoma cells and inhibit the growth of carcinoma cells, suggesting that dsP21-555 may become a new gene therapy tool.