Activation Of Endogenous P53 By Small Activating Rna Combines With Cp-31398 To Induce Du-145 Cells Apoptosis

Section One P53-saRNA could activate the expression of p53 in DU-145 cells while not induce them apoptosis in vitroObjective:To explored the effect of P53-Sa-RNAs in the androgen-independent prostate cancer in vitro.Methods:A previously identified P53 promoter-targeted small activating RNAs (saRNA, double-stranded ds-P53-285) and a negative control dsRNAs were chemically synthesized and transfected into DU-145 cells. Seventy-two hours later after transfection, the cells were collected. Among un-treated cells group, control-RNA transfected group and P53-Sa-RNA transfected group, the expression of P53 messenger RNA (mRNA) was evaluated by reverse transcription polymerase chain reaction (RT-PCR) and the expression of p53 protein was evaluated by Western blotting assay. Meanwhile, flow cytometery (FCM) were performed to analysis the cell cycle by PI assay and the induction of apotosis by AnnexinV-PI assay.Results:Compared to ds-control RNA, P53-Sa RNA could increases the expression of P53 gene at the messenger RNA levels in DU-145 cells, but doesn't induce p21WAF1 and DU-145 cleavage indicative of caspase-dependent apoptosis and Cell cycle arrest.Conclusion:P53-Sa RNA could up-regulate the expression of P53 gene in DU-145 cells while not induce them apoptosis. This suggested that the effect of Sa RNA is related to whether the targeted gene is mutant and implied that the upregulated expression of mutant p53 is the result that the expression file of micro-RNAs changed. Section Two Construction and identification of Eukaryotic expression plasmid encoding Sa-RNA targeting P53objective:To constructed eukaryotic expressing saRNA sections targeting P53 and to observe their effects on P53 gene expression in Du-145 cell.Methods:the shRNA sequence targeting P53-285 mRNA were designed according to the methods published in PLOS by LI, then double strands oligonucleotide (ds oligo) was synthesized and cloned into the pENTRTM/U6 plasmid. Connection product was inverted competent cells and multiplied. Plasmid was extracted and sequenced. After sequencing analysis, the recombinant pENTR/U6-P53-saRNA plasmid were transfecfed into DU-145 cells. The expressions of P53 were detected by RT-PCR.Results:Sequencing results indicated that pENTR/U6-P53- sa-RNA was positive clone. RT-PCR and western blotting test showed that the recombinant plasmid can activated P53 gene expression.Conclusion:the vector expressing P53-sa-RNA was successfully constructed. It was sugguested that the mothed of vector-expressing to get si-RNA is applied to get sa-RNA. Section ThreeP53-saRNAs combine with CP-31398 to induce DU-145 cells ApoptosisObjective:to explored the effect of P53-Sa-RNAs combined with CP-31398 in the androgen-independent prostate cancer in vitro.Methods:Seventy-two hours later after transfection with the plasmid expressing P53-Sa-RNA, the cells were treated with CP-31398 for another 24 hours. Among un-treated cells group, P53-Sa-RNA transfected group, CP-31398 treated group and the combination group, the expressions of P53 and P21 messenger RNA (mRNA) were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and the expressions of p53 and p21 protein were evaluated by Western blotting assay. Meanwhile, flow cytometery (FCM) were performed to analysis the cell cycle by PI assay and the induction of apotosis by AnnexinV-PI assay. MTT assay were performed to evaluate the viability of them.Results:when the P53-Sa RNA-transfected cells further treated with CP-31398, the growth and viability of DU-145 cells were inhibited and the expression level of p21WAF1 was increased.Conclusion:These findings suggest that RNA activation targeting mutant p53 combined with CP-31398 may serve as a novel therapeutic strategy for the treatment of androgen-independent prostate cancer.