| Objective:Thin withered cell death of the disorder was closely related to tumor development.PI3K/AKT pathway in a wide range of human tumor spectrum disorders. To promote tumor cell proliferation by regulating downstream effect molecules of apoptosis. inhibitor of apoptosis protein family, was to promote proliferation of tumor cells by inhibited apoptosis. In ovarian cancer, high expression of PI3K/AKT.XIAP.cIAP2and its resistance had a certain relationship study on the regulator} mechanisms of the three ovarian cancer had not been reported.In this study.the use of PI3K specific inhibitor LY294002role in ovarian cancer cell lines NIH:OVCAR-3.to observe cell proliferation.apoptosis changed and the XIAP and cIAP2.and to discuss the relation between expression of XIAP and cIAP2and PI3K/AKT. the theoretical basis for targeted therapy of tumors.Method:NIH:OVCAR-3(human ovarian tumor) was the main object of this study Firstly, we culture tumor cells in CO2gas incubator, which the environment were37℃5%CO2,20%O2and95%moisture. then extract cells of exponential phase of growth which was supposed to be used in following experiments.(1)Make different concentrations of LY294002(0、5、10、20、40ummol/L)acted on the cells.and collected cells after24h、48h72h、96h.The proliferation and apoptosis of NIH:OVCAR-3in each group, which detected by MTT assay, flow cytometry Annexin V-FITC:(2) Used RT-PCR. Western blot to detection the PI3K mRNA. AKT mRNA and PI3K protein.AKT protein, which acted by LY294002.40ummol/L for48hours, and observe the effect of LY294002inhibition of PI3K.simultaneously detected the suppression of XIAP mRNA in the PI3K/AKT.to find out cIAP2mRNA and of XIAP prtein.cIAP2protein whether changed.(3) The experimental data were assessed by SPSS17.0. if P>0.05, the results were statistics significance, if P<0.05.the results were non-statistics significance.Results:(1) LY294002(5、10、20、40ummol/L)acted on cells for24hours, the survival rate was respectively92.27%、91.32%、84.86%、71.84%:LY294002(5、10、20、40ummol/L) acted on cells for48hours, the survival rate was respectively91.4%、87.56%、82.8%、 66.83%:LY294002(5、10、20、40ummol/L) acted on cells for72hours.the survival rate was respectively88.79%、84.87%、79.21%、66.74%:LY294002(5、10、20、40ummol/L) acted on cells for96hours, the survival rate was respectively88.62%、81.83%、74.66%、55.4%.5ummol/L LY294002.when the duration of action, the proliferative activity did not change significantly, the results were non-statistics significant (p>0.05);10.20.40ummol/L.LY294002.when the duration of action, viability of tumor cells decreased in each group.the difference was statistical significant(p<0.05).5,10ummol/L.LY294002.24h.cell proliferative activity (p>0.05):48h.72h.96h when increased LY294002concentration, cell viability was reduced in each group, the difference was statistical significance(p<0.05).(2) LY294002(5,10,20,40ummol/L) acted on cells for24hours, the apoptosis rate was respectively2.2±0.33%、2.48±0.42%、3.37±0.17%、5.13±0.32%:LY294002(5、10、20、40ummol/L) acted for48hours.the apoptosis rate was respectively6.51±0.51%,7.28±0.28%、9.62±0.25%、10.49±0.86%:LY294002(5、10、20、40ummol/L) acted for72hours、 the apoptosis rate was respectively6.78±0.43%、7.73±0.21%、9.22±0.55%、12.14±0.34%:LY294002(5、10、20、40ummol/L) acted for96hours.the apoptosis rate was respectively7.23±0.24%、8.21±0.64%、11.57±0.43%、15.43±0.36%:5.10ummol/L LY294002.the role of apoptosis after24hours did not significant change in the different statistically significant(p>0.05).48h.72h.96h when with the increase of LY294002concentration.the apoptotic rate increased.the difference was statistically significant (p <0.05).(3) The relative content of PI3K mRNA in blank group was197.21±53.31. in experiment group was52.96=5.49. The relative content of AKT2mRNA in blank group is159.86±50.81、65.99±1.14in experimental group. The relative content of XIAP mRNA in blank group was173.89±44.2、61.48±15.63in experiment group. The relative content of cIAP2mRNA in blank group was168.33±23.19、60.71±9.39in experiment group. The experimental groups compared with the blank groups, the expression were decreased, the differences were statistical significant(p<0.05).(4) The expression of protein of PI3K in blank group was25.53±1.21,9.45±0.88in experiment group:The expression of protein of AKT2in blank group is34.75=2.33,13.03=1.47in experimental group. The expression of protein of XIAP in blank group was3.18±0.18,0.84±0.01in experiment group. The expression of protein of cIAP2in blank group is6.90±1.15,2.18±0.84in experiment group. The protein expression in experimental groups were lower than the blank groups, and the difference between all the groups were statistics significant (p<0.05).Conclusion:(1)LY294002could restrain PI3K.then reduce the expression of AKT, that decrease the proliferation and induce the increasing of apoptosis of NIH:OVCAR-3.(2) The increasing of NIH:OVCAR-3apoptosis was caused by the reduced expression of XIAP mRNA、cIAP2mRNA and protein content. So, the expression of XIAP、cIAP2in NIH:OVCAR-3was regulated by the pathway PI3K/AKT. |
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