The Study On Expression Of XIAP And CIAP2Regulated By AKT2in Ovarian Cancer Cell NIH:OVCAR-3

Objective:In recent years the studies have shown that PI3K/AKT may inhibit apoptosis and promote cell survival by a variety of ways. Inhibitor of apoptosis protein family inhibits apoptosis thereby promoting tumor development. At present, it has not been studied on the regulation among the PI3K/AKT and tumor apoptosis inhibitory protein of XIAP, cIAP2in ovarian cancer at china and abroad. In this study, transfecting PEGFP-N3-AKT2into the ovarian cancer NIH:OVCAR-3irradiated by ultraviolet10minutes.Detectiong the mRNA and protein expression of AKT2, XIAP and cIAP2.As well as to detect the expression of early apoptosis rate and proliferation rate.So as to discuss the relationship among the PI3K/AKT and XIAP, cIAP2.Providing a new strategy for ovarian cancer gene therapy.Methods:The study object of this was human ovarian cancer cell NIH:OVCAR-3.(1) Construction of plasmid for PEGFP-N3-AKT2.(2)The NIH:OVCAR-3cells were cultured by1640medium.(3)Liposome was transfected into the cells to exposed ultraviolet radiation10minutes,and set up the blank control group (Blank),empty plasmid (negative control group),AKT(experimental group).(4)Through AnnexinV-FITC Flow cytometry observe cell apoptosis.(5)MTT method were used to detect the proliferation rate In each group after transfected48hours.(6)The expression of AKT2mRNA,XIAP mRNA and cIAP2mRNA in each group after transfected48hours was detected by RT-PCR.(7)In each group,when transfected48hours the AKT2,XIAP and cIAP2protein were detected by Western Blot.Results:(1)Flow cytometric showed that ovarian cancer cell NIH:OVCAR-3in early apoptosis rate:blank group,the empty plasmid group,AKT group apoptosis rate respectively were15.6%,16.53%,5.27%.The AKT apoptosis rate was lower than the blank group and the empty plasmid group (P<0.05) apoptosis between the blank group and the empty plasmid group were no significant change (P>0.05).(2)MTT observed the proliferation rate of the cell OVCAR-3:cell proliferation rate of blank group,the empty plasmid group and AKT group respectively were100%,91.3%,197%(P<0.05),while the OD values of the AKT group was higher than the other two groups,the value-added rate of the cells significantly increased.The cell proliferation rate between the blank group and the empty plasmid group showed no significant change (P>0.05).(3)RT-PCR detected the mRNA expression AKT2,XIAP and cIAP2gene in each group after transfected48hours:the AKT2mRNA expression,the blank group was28.0±10.0,the empty plasmid group was23.8±5.4,AKT group was482.1±291.3,the mRNA expression of AKT2in AKT group was higher than in the blank group and the empty plasmid group,it was statistically significant (P<0.05),while the blank group and the empty plasmid group,the relative expression was no significant (P>0.05);the XIAP mRNA expression,the blank group was88.1±30.2,the empty plasmid group was84.9±37.3,AKT group was248.6±38.6,the mRNA expression of XIAP in AKT group was higher than the other two groups,the difference was statistics significance (P<0.05),while the blank group and the empty plasmid group,the relative expression was no significant (P>0.05);the cIAP2mRNA relative expression:the blank group was42.4±12.6,the empty plasmid group was39.0±4.5,the AKT group was270.7±65.9,the mRNA expression of cIAP2in AKT group was higher than the other two groups,the difference was statistically significant (P<0.05),while the blank group and the empty plasmid group,the relative expression was no significant (P>0.05).(4)Western blot showed that the expression protein of AKT2,XIAP and cIAP2gene in the cells transfected48hours in each group:The expression protein of AKT2,the blank group was39.85±0.3,the empty plasmid group was50.5±2.2,the AKT group was105.5±2.6;The XIAP protein expression,the blank group was35.05±0.4,the empty plasmid group was40.5±1.2,the AKT group was73.1±1.5;The cIAP2protein,the blank group was10.1±0.1,the empty plasmid group was11.8±0.6,AKT group was29.6±0.2,AKT protein content of the content group were higher than the blank group and the empty plasmid group.It was statistically significant (P<0.05);but the protein content of the blank control group and the empty plasmid group was not statistically significant (P>0.05).Conclusion:In this experiment,transfected PEGFP-N3-AKT2plasmid could inhibit the apoptosis of ovarian cancer cell NIH:OVCAR-3through the ultraviolet ray,while transfected AKT2,the increase of AKT expression could make the XIAP mRNA,cIAP2mRNA expression increasing,also increased the protein expression of XIAP and cIAP2,inhibited apoptosis,that promote cell proliferation rate increased.So that,in ovarian cancer cell NIH:OVCAR-3,XIAP,cIAP2expression levels regulated by the PI3K/AKT pathway, providing a new strategy for ovarian cancer gene therapy.