The Inhibitory Effects Of SiRNA Expression Vector On The Expression Of Survivin Gene Of Prostate Cancer PC-3 Cell

ObjectiveProstate cancer is one of the most prevalent malignant tumors of the urinary tract in older males,takes the first place for American old men on the list of male tumor incidence rate.The incidence rate has been increasing year by year with the change of average life span and dietary structure in China. Except operation, there is no optimal protocol to treat the prostatic carcinoma and it's very necessary to explore some new strategies for this malignant disease.In order to observe the effects of RNAi-mediated survivin gene silencing in Hormone-Refractory Prostate Cancer cells line PC-3, we construct the plasmids encoding survivin siRNA,and investigate its effects on.MethodsAccording to the computer aided design software,Two siRNAs of survivin and one negative control group and one blank control group were designed and synthesized respectively,then all of them were inserted into plasmids.The recombinant plasmids were confirmed by sequencing. Human prostate cancer cell PC-3 was purchased from medical experimental animal center of peking medical university.The cells were cultured in RPMI 1640 medium supplemented with 100 ml/L fetal bovine serum in a humidified incubator containing 50 ml/L CO₂ at 37℃.Transfection reagent LipofectamineTM2000 was perchased from Invitrogen Corporation. The vectors were transfected into PC-3 cells with liposomes. After transfection of it to PC-3 cells using Lipofectamine^TM2000 for 24 hours, Stable shRNA expressing cell lines were obtained by puromycin selection, the transcription of survivin mRNA was detected with RT-PCR, the Expression of protein was detected with Western blotting, Apoptosis was detected with flow cytometry at different times after transfection. Data were expressed as mean±SD .All statistical analysis were performed with the statistical software SPSS 11.0 for windows, p < 0.05 was considered to be statistically significant.ResultsThe recombinant plasmids were constructed and the aim sequence was confirmed by DNA sequencing.Survivin protein expression is 18.94%±0.63%,16.35±0.23%,46.41%±0.76%,46.20±1.47% respectively by plasmids A,B,negative control group and blank control group with Western blotting. The expressions of survivin protein were inhibited by plasmids A,B significantly. Survivin mRNA expression is 27.94%±1.43%,24.51%±1.37%,49.46%±0.71%,48.49%±1.32% respectively by plasmids A,B,negative control group and blank control group with RT-PCR. The expressions of survivin mRNA were inhibited by plasmids A,B significantly.PC-3 cells apoptosis rate is 12.80%±1.33%,16.48%±1.00%,3.03%±0.62%,2.96%±0.41% respectively by plasmids A,B,negative control group and blank control group with flow cytometry, the apoptosis of PC-3 cells increased by plasmid A,B significantly.ConclusionIn this study, transfection of siRNA-expressing vectors, which targeted against specific sequences of survivin in the PC-3 cells, could result in a significant reduction in the corresponding survivin transcripts and protein,and induce apoptosis of PC-3 cells. The result was significantly lower than that in controls.This inhibition was highly selective,and RNAi activity is sequence-specific.In a ddition,two diferent survivin-targeting siRNAs were proved to be functional with diferent efficacies, plasmid B is better than plasmid A.Thus,there is room for improvement in searching for the ideal sequence or region within a target gene that may yield an optimal gene silencing activity.