Effect Of DsRNA Upregulation Of Numb On Human Prostate Carcinoma PC-3 Cells

Objective Screen the high efficiency small activating RNA(sa RNA) molecule which can active the target gene Numb, basing on the tumor treatment strategies of sa RNA. Then to investigate the influence of androgen and antiandrogen drug on human prostate carcinoma PC-3 cells into which were transfected Numb-sa RNA. Methods 1. Three pairs of candidate small double-stranded RNA(ds RNA) molecules were designed and synthesized for target gene Numb, and the control ds RNA(ds Control) was designed to be non-homologous sequence with the human genome. The ds RNA molecules were transfected into human prostate carcinoma PC-3 cells. 2. Real-time quantitative PCR(RT-q PCR) was employed to detect the m RNA expression levels of the target gene Numb in human prostate carcinoma PC-3 cells after transfection. 3. Western Blot was used to verify the protein expression levels of the target gene Numb in human prostate carcinoma PC-3 cells after transfecting the sa RNA into the cells. 4. The Numb-sa RNAs selected by us were transfected into the PC-3 cells. Then the experiment groups were dispart into two groups, one group was the transfection group, while the second group was the non-transfection. The negative control group was without transfectting and using drugs. After a 48-hour treatment with dihydrotestosterone(DHT), flutamide and dihydrotestosterone-flutamide mixture of different concertrations, changes in cell growth and proliferation were reflected by MTT colorimetric analysis. 5. According to the MTT colorimetric analysis results, the effective drug dose were selected. After a 48-hour treatment with different drugs, the apoptotic cells was detected by flow cytometry to detect labbled Annexin V-FITC/PI. Results 1. ds Numb-870 and ds Numb-948 failed to up-regulation the m RNA expression level of the target gene Numb in human prostate carcinoma PC-3 cells, while ds Numb-298 was succeed to elevate the m RNA and the protein significantly. 2. Adopting MTT colorimetric analysis, we found the differences of cell viability in transfection groups treated with 10-3mol/L、10-4mol/L、10-5mol/L DHT were all significantly lower than that in the negative control group(P<0.05). And the cell treated with 10-3mol/L and 10-4mol/L DHT in transfection groups demonstrated restrain growth with significantly decreased cell viability compare with that of the negative control group(P<0.05). Similarly, We also found the differences of cell viability in transfection groups treated with 10-3mol/L、10-4mol/L、10-5mol/L flutamide were all dramatically lower than that in the negative control group(P<0.05). And the cell treated with 10-4mol/L flutamide in transfection groups demonstrated much slow growth with remarkablely reduced cell viability compare with that of the negative control group(P<0.05).Finally, when we using all different concertration of the drugs, the different survival rates in transfection groups treated with dihydrotestosterone-flutamide mixture were all significantly lower than that in the negative control group(P<0.05). The same effect had been obtained that the cell viability of the different concertration of transfection groups were significantly highr than that of the negative control group(P<0.05). 1. The apoptotic cells was detected by flow cytometry to detect labbled Annexin V-FITC/PI. We demonstrated that the apoptosis rate of transfection goup which treated with DHT was decreased obviously than that of non-transfection group and negative control group(P<0.05). When all experiment groups were treated with flutamide, the result was the percentage of cell apoptosis of transfection group was significantly higher than that of non-transfection group and negative control group(P<0.05). As a result, the apoptosis rate in transfection goup was much more higher than that of non-transfection group and negative control group after using dihydrotestosterone-flutamide mixture(P<0.05). Conclusion 1. The ds Numb-298 molecule has the function to specially activate the gene Numb expression in human prostate carcinoma PC-3 cells. 2. When the Numb-sa RNAs are transfected into the PC-3 cells, the result is that the sensitivity of PC-3 cells to hormone might be restored to a certain extent and the cells can be induced apoptosis.