CD59 Silencing via Retrovirus-Mediated RNA Interference Enhanced Complement-Mediated Cell Damage in Ovary Cancer

【摘要】 CD59,belonging to membrane complement regulatory proteins(mCRPs) ,inhibits the cytolytic activity of complement and is over-expressed in solid cancers,including ovary cancer. The aim of the present study was to construct recombinant retrovirus encoding shRNA targeted human CD59 and infect A2780 cells in order to investigate the relationship between decreased CD59 expression and tumorigenesis of ovary cancer. siCD59 and siCD59-C were successfully constructed and identified by PCR,restriction endonuclease analyses and DNA sequencing,respectively. The siCD59 was able to efficiently infect A2780 cells,which was confirmed by Western blotting. When incubated with fresh normal human serum(8%,v/v) for 1 h at 37°C,the cell viability was decreased and cell damage was increased in siCD59 infected A2780 cells compared to siCD59-C infected cells. This led to the activation of caspase-3. The apoptosis in siCD59 infected cells was shown with hypercondensed nuclei using Hoechst staining. Meanwhile,the weight of ovary tumor graft in nude mice was significantly decreased in siCD59 group compared to that of siCD59-C group. And the expression of CD59 protein in tumor tissue in siCD59 group was significantly decreased. These results suggested that CD59 silencing in ovary cancer cells via retrovirusmediated RNAi can enhance complement-mediated cell damage,inhibiting growth of ovary cancer. CD59 might be a potential target for gene therapy in ovary cancer.更多还原

【Abstract】 CD59,belonging to membrane complement regulatory proteins(mCRPs) ,inhibits the cytolytic activity of complement and is over-expressed in solid cancers,including ovary cancer. The aim of the present study was to construct recombinant retrovirus encoding shRNA targeted human CD59 and infect A2780 cells in order to investigate the relationship between decreased CD59 expression and tumorigenesis of ovary cancer. siCD59 and siCD59-C were successfully constructed and identified by PCR,restriction endonuclease analyses and DNA sequencing,respectively. The siCD59 was able to efficiently infect A2780 cells,which was confirmed by Western blotting. When incubated with fresh normal human serum(8%,v/v) for 1 h at 37°C,the cell viability was decreased and cell damage was increased in siCD59 infected A2780 cells compared to siCD59-C infected cells. This led to the activation of caspase-3. The apoptosis in siCD59 infected cells was shown with hypercondensed nuclei using Hoechst staining. Meanwhile,the weight of ovary tumor graft in nude mice was significantly decreased in siCD59 group compared to that of siCD59-C group. And the expression of CD59 protein in tumor tissue in siCD59 group was significantly decreased. These results suggested that CD59 silencing in ovary cancer cells via retrovirusmediated RNAi can enhance complement-mediated cell damage,inhibiting growth of ovary cancer. CD59 might be a potential target for gene therapy in ovary cancer.更多还原