Biological Effects Of B7-H4 Gene In Ovarian Cancer Cells

Aim:To investigate the changes of biological behavior of SKOV3 after B7-H4 transfection.To analyze the effect of overexpression of B7-H4 gene in ovarian cancer cells on CTL cytotoxicity.Methods:RT-PCR was applied to amplify human B7-H4 cDNA from human ovarian serous cystadenocarcinoma.B7-H4 cDNA was inserted into PEGFP-N1 vector to construct B7-H4 eukaryotic expression vector.The recombinant vector (PEGFP-N1/B7-H4) was transfected into SKOV3 cells.Positive cell clones were selected and amplified.The mRNA and EGFP/B7-H4 fusion protein expressions of the cells were detected by RT-PCR and fluorescence microscopy.Cells with(negative control group) and without(blank control group) transfected empty plasmid were served as control groups.In vitro,the biology effects of B7-H4 on SKOV3 cells were analyzed through the methyl thiazolyl tetrazolium test,cell cycle and apoptosis through flow cytometry,wound healing assay,well adhesion assay,transwell chamber cell invasion assay.After isolated and purified from umbilical cord blood by a higher gradient magnetic cell sorting system(MASC),CD34~+ cells were induced to differentiate into DC by cocktail cytokines.Antigens were extracted from SKOV3 cells by freeze-thaw method,and then loaded on DC.The cytotoxicity of CTL activated by DC loading with freeze-thaw ovarian cancer antigen to SKOV3/B7-H4, SKOV3/neo and SKOV3 was evaluated by MTT assay.Results:(1) B7-H4 eukaryotic expression vector was constructed correctly. SKOV3/B7-H4 cells could steadily express B7-H4 protein and mRNA,while in negative control group and blank control group cells,the expression of B7-H4 protein or mRNA was not detected.(2) SKOV3/B7-H4 cells displayed an enhanced rate of proliferation compared with the negative control group or the blank control group (P＜0.05),and there was no significant difference between the negative control group and the blank control group(P＞0.05).(3) There was no significant difference among SKOV3/B7-H4 cell group(15.25±7.27),negative control group(13.50±7.55) and blank group(13.75±8.73) in colony-forming experiment(P＞0.05).(4) Flow cytometry showed that(20.3±2.1)%of cells were arrested at G2/M stage in SKOV3/B7-H4 group,which was significantly higher than that of the blank control group(14.5±0.6)%or the negative group(13.2±1.4)%(P＜0.05).While apoptosis rate in SKOV3/B7-H4 group was(6.33±1.21)%,which was less than the negative control group(10.50±2.88) or the blank control group(10.17±3.13)%(P＜0.05).(5) SKOV3/B7-H4 cell group had a significantly lower percentage than the other cell lines in the dimensions of the wound areas(P＜0.05).(6)The invasion number of SKOV3/B7-H4 cell group and the negative control group were 47.00±15.90 and 26.88±12.99 respectively,with a significant difference between the two groups (P＜0.05).(7) The cytotoxicity rate of CTL to SKOV3/B7-H4 was significantly lower than that to the negative control cells(20.12%±3.14%VS 36.34%±4.53%) (P＜0.05).Conclusion:B7-H4 could directly promote proliferation rate and increase activities of cell migration and invasion.B7-H4 inhibited the cytotoxicity rate of CTL in vitro. B7-H4 played a more pivotal role in tumor formation and metastasis and it can be a new promising target for future antitumoral therapy.