The Study Of CD59 Gene Active Site As A New Gene Target Of Cervical Cancer Immune Escape

Objective To study the effect of GPI anchored protein CD59 on the proliferation and apoptosis activity of Hela cells and explore the mechanisms of malignant proliferation of cervical cancer cells.Methods The recombinant plasmids were transfected into Hela cells via Lipofectamine 2000.CD59 interfering plasmid,pSUPER-siCD59,was transfected separately owe to it could express green fluorescent protein for the observation of transfection efficiency,while pIRES-WTCD59 and pIRES-MCD59 plasmids were cotransfected with pLeGFP plasmid.Inverted fluorescence Microscope was adopted for the detection of transfection efficiency respectively at 24h,48h and 72h after tranfection.We used G418 to screen the stable transfection cell lines.The CD59 gene or protein expression of every group Hela cells was confirmed by immunofluorescence,RT-PCR and Western-blot.Another Hela cell group we studied was affected by peptide seal to CD59,1ug or 10ug for 6h or 8h.MTT colorimetry was adopted for the cell proliferation activity,and TUNEL and flow cytometry were used to detect the apoptosis activity.Cervical cancer xenograft model in nude mice was established.Results The results under the Inverted fluorescence Microscope showed that the strongest green fluorescene was appeared at 48h after transfection.The transfection efficiency of pSUPER-siCD59 plasmid was approximately 70%,while the cotransfection efficiency of pIRES-WTCD59 and pIRES-MCD59 plasmids with pLeGFP plasmid was reaching 50%.The concentration of G418 for screening is 500ug/ml.The level of CD59 in Hela cells transfected with pSUPER-siCD59 were significantly lower than the other groups.Compared with untransfected group,the results showed that the expression of CD59 was decreased in the pSUPER-siCD59 group and increased in the pIRES-WTCD59 and pIRES-MCD59 transfected group(p<0.05).After antibody cross-linking of CD59 receptors,compared with untransfected group,pSUPER-siCD59 and peptide seal to CD59 slowered proliferation,and large doses of peptide seal worked better,while pIRES-WTCD59 and pIRES-MCD59 accelerated proliferation.The opposite results were appeared in both TUNEL and flow cytometry measurement of cell apoptosis.We successfully created the subcutaneous tumor model in nude mice.Tumor of pIRES-WTCD59 Hela cell group was bigger than normal Hela cell group,while CD59 peptide seal Hela cell group was smaller.Conclusion CD59 is the inhibitory regulatory protein of terminal complement regulation.CD59 could affect the proliferation and apoptosis activity of cervical cancer Hela cells which may be due to it could inhibit the apoptosis signal transduction in Hela cells through some ways.Our study might provide New way for the clinical treatment of cervical cancer created because of our study.