Inhibition Of CD59 Gene Expression By SiRNA In Tumor Cells

Objective To investigate the effect of retrovirus-mediated RNA interference(RNAi)on the expression of CD59 gene of human cervix cancer cell line HeLa and human ovary cancer cell line A2780,study the effect of cytolysis and apoptosis and the inhibitory effects of xenografts in nude mice.Methods Three pairs of single-stranded oligo encoding CD59 shRNA sequence were designed and synthesized.After annealing ds oligo were inserted into lined pSUPER expression vectors to construct recombinant vectors.Then three recombinant vectors and one negative-control vector(siCD59-C)were transfected into the packaging cell Phoenix A using liposome.HeLa cells were infected by the virus supernants which contained the four recombinant vectors.CD59 mRNA and protein level was detected by RT-PCR and Western blots.Its function was analysed by dye release assay.The ovary cancer cell A2780 was transfected with siCD59 and siCD59-C using liposome2000 and the stable strains were selected by using G418-medium,the cell viability and cell damage was tested by MTT and LDH release assay,flow cytometry and Hoechst stain was performed to assess the cell apoptosis when incubated with 8%NHS.Eighteen 4-5 week old nude mice were divided randomly into two groups,the xenografts of ovary cancer cells A2780 siCD59 and siCD59-C were established.All mice were sacrificed after 6-8 w.The weights of the tumors were measured and the tumor growth rates were calculated.The distribution of CD59 protein was detected by immunohistochemistry.Results Recombinant vectors were successfully constructed and transfected. Two of four recombinant vectors(siCD59-T2 and siCD59-T3)could effectively inhibit the expression of CD59 mRNA and protein.The mRNA expression in siCD59-T2 and siCD59-T3 infected HeLa cells was significantly reduced to 42.60%(P＜0.01)and 49.23 %(P＜0.01),respectively.Western blots demonstrated CD59 protein expression was significantly reduced to 51.61%(P＜0.05)and 52.18%(P＜0.05),respectively.When incubated with fresh normal human serum(8%,v/v)for 1 h at 37℃,the dye release assays between siCD59-T3 group and siCD59-C group suggested that the dye release rate was 6.03%/40.40%,86.83%/30.97%,66.83%/19.51%with 1:5,1:10,1:20 complement dilution.There was difference between them,which suggested that CD59's protection against human complement decreased.The difference among siCD59-T1,siCD59-C,and blank control group had no statistical significance(P＞0.05).The cell viability was decreased and cell damage was increased in siCD59 group compared to siCD59-C transfected cells.Furthermore,this led to the activation of caspase-3 and the hypercondensed nuclei using flow cytometry and Hoechst stain.Meanwhile,the weight of ovary tumor graft in nude mice was significantly decreased in siCD59 group(0.27±0.092) compared to the siCD59-C group(1.57±0.176),the inhibited rate was 82.80%.And the stained cell of CD59 protein of tumor tissue in siCD59 group(296±70)was significantly decreased compared to the siCD59-C group(814±112).There was significant difference between them(P＜0.05).Conclusion Recombinant retroviral vectors and stable virus-producing packaging cell lines were successfully constructed and identified,CD59 silencing by siRNA increased complement-mediated cytolysis and led to cell apoptosis,inhibited growth of xenografts in nude mice,which may pave the way for studying the role of CD59 in the immune escape of tumors cells as well as in tumor therapy.