| Background and Objectives: Prostate cancer (Pca) is the most common malignancymale genitourinary system. In our country, the morbidity of prostate cancer was1.71per100,000cases in1993, and increased to2.0per100,000cases in1997, In2000it was up to4.55per100,000cases with increasing tendency year by year. The development and theprogression of prostate cancer have a variety of mechanisms which involved in a greatnumber of gene change and related cell signaling cross-talk, which provide challenge fordeveloping a safe and desirable therapeutic strategy for prostate cancer. Hence, thecriticaltask of recent research is to find an effective and safe therapeutic method forprostate cancer, and gene therapy is perhaps the most effective way for the treatment ofprostate cancer. Current studies showed that the relationship between siRNA (microRNA/siRNA) and oncogene, anti-oncogene might be closely correlated with the developmentand the progression of prostate cancer. This study is to compare the efficacy ofoverexpression of microRNA-494to knockdown survivin gene with RNA interferencemediated survivin gene inhibition in prostate cancer PC-3cell line.Methods:Pairing result of mature form of has-microRNA-494and survivin mRNA waspredicted by biological software TargetScan5.2. The expression status of microRNA-494in normal prostate epithelial cell RWPE-1cell line and prostate cancer PC-3cell line wasanalyzed by quantitative reverse transcriptive polymerase chain reaction(qPCR).Adenovirus vector that can overexpressed microRNA-494(Ad-miR494) was constructedaccordingly, and another adenovirus vector that can express survivin shRNA(Ad-sur) wasprovided by our Lab. These two virus amplification and titration were carried out inHEK-293A cells, and were used to infect PC-3cells both respectively and simultaneously in vitro. The growth inhibition in blank control,negative control (Ad-1、Ad-2、Ad-1+Ad-2),Ad-494, Ad-sur and Ad-494＋Ad-sur group were measured by MTT method. Reversetranscriptase polymerase chain reaction (RT-PCR) and western blot were used to detectsurvivin mRNA and protein expression status. Flow cytometry was used to measure cellcycle and apoptosis condition.Results: Software prediction showed that microRNA-494mature form canincomplete complementary match with terminal3’ UTR of survivin mRNA.mirSVR andphast Cons score is satisfactory. Q-PCR results indicated that microRNA-494expression inRWPE-1cells is much lower than that in human prostate cancer PC-3cells. EffectiveAd-494was successfully constructed. The results indicated that survivin expressionreduced,cell proliferation inhibited, cell apoptosis increased and cell number in G1stage inexperiment groups as compared with blank and negative groups. More importantly, theeffectiveness was more apparently in group Adsur+Ad494than that in other groups.,thisindicated that these two methods can effectively inhibite survivin gene expression andhave synergetic anticancer effect.Conclusion:1. Ad-pre-microRNA-494as an overexpessed vector is first successfullyconstructed.2. Biologic software result showed that microRNA-494can target survivin mRNA.compared with, the expression status of microRNA-494is obviously down-regulated inprostate cancer PC-3cells than that in human prostate glandular epithelium RWPE-1cells.3. Ad-pre-microRNA-494and Ad-sur can significantly inhibit survivin geneexpression, cell proliferation and induce apoptosis in PC-3cells in vitro. these twomethods can effectively inhibite survivin gene expression and have synergetic anticancereffect. |
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