The Study Of UBE2C Gene Silencing On Human Prostate Cancer Cell Lines By Lentivirus Mediated RNA Interference

ObjectiveTo investigate the effects the silencing of ubiquitin-conjugating enzyme E2C(UBE2C)gene by the recombinant lentiviral particles mediated RNA interference( RNAi) and detect its silencing effect on the proliferation,migration and invasion,Colony-forming of human prostate cancer PC3 and DU145 cell lines.Methods1. The expression of UBE2 C m RNA and protein levels were discovered by real-time reverse transcription and polymerase Chain reaction(RT-PCR) and Western blot in LNCap, PC3, DU145 and C4-2 these four kinds of human prostate cancer cells,according to the results to screen experiments using cells.2. According to the design principles of RNAi,the recombinant lentiviral vector carrying short hairpin RNA(sh RNA) targeting the UBE2 C gene(LV-sh RNAUBE2C) and recombinant lentiviral vector carrying sh RNA targeting the negative control gene(LV-sh RNA-NC)were designed and compounded by American Gene Copoeia company.3. Human prostate cancer PC3 and DU145 cells were randomly divided into three groups,the LV-sh RNA- UBE2 C experimental group, the LV-sh RNA-NC negative control group and the mock control group. The LV-sh RNA- UBE2 C experimental group, prostate cancer PC3 and DU145 cells were infected with the lentiviral particles(LV-sh RNA- UBE2C), while the LV-sh RNA-NC negative control group were infected with lentiviral particles( LV-sh RNA-NC), the mock control group with no treatment. The m RNA and protein level of UBE2 C were analyzed by RT-PCR and Western blot, and then determined the interference efficiency to got the effective target.4. Cell proliferation was detected by methylthiazol tetrazoliu(MTT)assay,cell invasion and migration were detected by transwell assays,cell colony formation was detected by Colony-forming assay, the cell cycle were observed by flow cytometry(FCM).Results1. The RT-PCR results showed that the expression of UBE2 Cm RNA in prostate cancer cell lines PC3 and DU145 were increase in four kinds of cells. And the expression of UBE2 Cm RNA in human prostate cancer cells PC3 were highest in four cells.2. The Western blot results showed that the expression of UBE2 C protein levels in human prostate cancer cells PC3 and DU145 were higher than in four cells.And the expression of UBE2 Cm RNA levels in PC3 cells were highest in four cells. And then choose PC3 and DU145 cells as the experimental group.3. After transfecting cells, The UBE2 Cm RNA levels of the UBE2 C in LV-sh RNAUBE2 C experimental group was downregulated as compared with the LV-sh RNA-NC negative control group and the mock control group(p<0.05)was shown by RT-PCR results.4. MTT assays results,as compared with the mock control group untransfected and LV-sh RNA-NC negative control group the proliferation activity of PC3 and DU145 cells transfected with LV-sh RNA- UBE2 C was significantly inhibited(p< 0.05).5. Colony-forming assays results showed that the clone formation of LV-sh RNA-UBE2 C group significantly downregulate the blank control group,and the LV-sh RNA-NC negative control group and significantly suppressed experimental cell growth(p< 0.05).6. Transwell assays results showed that the imgration and invasion ablity of LV-sh RNA-UBE2 C group significantly lower than the blank control group,and the negative control group and significantly suppressed experimental imgration and invasion ablity(p< 0.05).7. Flow cytometry analysis showed that the percentage of cells in G1 phase increased in cells transfected with the LV-sh RNA-UBE2C(p<0.05).Conclusion1. The recombinant lentiviral particles for sh RNA of UBE2 C gene can effectively suppress the m RNA and protein expression of UBE2 C gene.2. The recombinant lentiviral particles for shRNA of UBE2 C gene can effectively suppress the expression of UBE2 C gene. UBE2 C down-regulation could better inhibit the cell growth of human prostate caner cells, suppress the formation of cell cloning, could better inhibit the cell imgration and invasion of human prostate caner PC3 and DU145 cells. It suggests that UBE2 C may be a new useful target for gene therapy of prostate cancer in future.