Synthetic Small Double-stranded RNA Inhibits The Development Of Bladder Cancer By Activating P21 Expression

Part I The comparison of exogenous and endogenous small ds RNAs' activating effects on the expression of p21 in bladder cancer cellsOBJECTIVE: Eight pairs of synthetic candidate small ds RNAs with complete complementarity to mi R-1236-3p or mi R-370-5p target site of p21 promoter: ds P21-242, ds P21-243, ds P21-244, ds P21-245 and ds P21-552, ds P21-553, ds P21-554, ds P21-555. To investigate the differences of ds RNAs' and corresponding mi RNA' activating effects on p21^WAF1/CIP1 expression in bladder cancer cell lines T24 and EJ.METHODS: Eight pairs of ds RNAs were designed and synthesized referring to sa RNA design principles. ds Control?negative control?, mi R-1236-3p or mi R-370-5p?positive control?, ds RNAs were respectively transfected into T24 and EJ cells. q PCR and RT-PCR were conducted to analyze the p21 mRNA expression level. The p21 protein expression level was accessed by Western blot.RESULTS: q PCR showed that both ds P21-245 and ds P21-555 can significantly promote the expression of p21 mRNA in T24 and EJ. Compared with ds Control group, the overexpression of p21 mRNA was 2.32–fold in T24 cells?P<0.01? and 2.84–fold in EJ cells?P<0.001? after ds P21-245 was transfected. And compared with mi R-1236-3p group, the differences was not statistically significant in T24 and EJ cells?P>0.05?. Compared with ds Control group, the expressions of p21 mRNA were both up-regulated in T24 cells?P<0.001? and EJ cells?P<0.001? after ds P21-555 was transfected. And compared with mi R-370-5p group, the differences was not statistically significant in both cell lines?P>0.05?. The corresponding expression changes of p21 mRNA in both cell lines were confirmed by RT-PCR. Western blot showed that the overexpression levels of p21 protein were consistent with that of p21 mRNA in T24 and EJ. Compared with ds Control, the remaining six pairs of ds RNAs failed to up-regulated p21^WAF1/CIP1 gene expression in both cell lines?P>0.05?.CONCLUSIONS: Exogenous ds P21-245 and ds P21-555 can significantly promote the up-regulation of p21^WAF1/CIP1 in bladder cancer cells. Besides, there are no significantly differences about the p21-activated abilities of synthetic ds RNA and corresponding endogenous mi RNA.Part II Exogenous ds RNA inhibits the development of bladder cancer by activating p21 expressionOBJECTIVE: To explore the inhibitory effects of a ds RNA?ds P21-555? on the development of bladder cancer cell lines T24 and EJ after transfected candidate ds RNA.METHODS: ds Control?negative control?, mi R-370-5p?positive control?, ds P21-555?experimental group? were respectively transfected into T24 and EJ cells. q PCR was conducted to evaluate the expression of p21 mRNA and CDK4/6 mRNA. Western blot was applied to analyze the expression of p21 and CDK4/6 proteins. Cell cycle distribution of each group was measured by FCM. Cell proliferation assay was conducted to access the proliferative capacity of cells in each group. Colony formation assay was performed to evaluate the proliferative ability of single cancer cell in each group.RESULTS: q PCR showed that ds P21-555 activated the expressions of p21 mRNA to 2.46-fold?P <0.01? in T24 cells and 2.60-fold?P <0.001? in EJ cells, compared with ds Control group; whereas the expressions of p21 mRNA were no significantly different?P> 0.05? compared with mi R-370-5p group. ds P21-555 inhibited the expressions of CDK4 mRNA to 0.57 –fold?P <0.001? in T24 and 0.46 –fold?P <0.01? in EJ cells compared with ds Control group; besides, CDK6 mRNA expressions were down-regulated to 0.61 –fold?P <0.01? in T24 and 0.64 –fold?P <0.01? in EJ cells; however, the differences were not statistically significant?P> 0.05? compared with mi R-370-5p group. Western blot showed that the overexpression levels of p21 and CDK4/6 protein were consistent with that of p21 and CDK4/6 mRNA in T24 and EJ.Compared with ds Control group, FCM showed that an obvious cell cycle arrest at the G0/1 phase while S, G2 / M phase cells decreased and the G0 / G1 phase cells significantly increased after mi R-370-5p or ds P21-555 was transfected. Cell proliferation assay revealed that the proliferative abilities of transfected mi R-370-5p or ds P21-555 cells decreased obviously?P<0.05? compared with ds Control group. However, the differences of proliferative abilities between mi R-370-5p and ds P21-555 were no statistically significant?P> 0.05?. Colony formation assay showed that the numbers of colonies formed both in mi R-370-5p and ds P21-555 group were much fewer than that in ds Control group.CONCLUSION: Exogenous ds P21-555 can activate the expression of p21^WAF1 / CIPI and obviously inhibited the growth of bladder cancer cells.