Chemoresistance of negative costimulatory molecule B7-H4 to 786-O cells

Human B7-H4 wild type gene was amplified by RT-PCR, digested with restriction endonuclease EcoR I and BamH I, and inserted into eukaryotic expression vector pIRES2-EGFP to construct recombinant eukaryotic expression vector pIRES2-EGFP-B7-H4-WT. The nuclear localization sequence(NLS)mutation type of B7-H4 was obtained by site-directed mutagenesis. The mutation type was then ligated with the vector pIRES2-EGFP and the recombinant eukaryotic expression vector pIRES2-EGFP-B7-H4-NLS-MT was constructed. The recombinant plasmids were transfected into 786-O cell line using Lipofectamine 2000 and three transgenic cells were constructed: Mock/786-O, B7-H4 WT/786-O and B7-H4 NLS MT/786-O. The cells proliferation and the cytotoxicity of chemotherapy drugs were assayed by CCK8 kit. The apoptosis of cells treated by 5-fluorouracil was assayed by Annexin V-PI/7ADD staining which detected by flowcytometry. Results showed that B7-H4 WT could effectively promote cell proliferation. When cells treated by various concentrations of 5-fluorouracil, doxorubicin and cisplatin B7-H4 WT/786-O cells were most resistant to drugs when compared with Mock/786-O and B7-H4 NLS MT/786-O, which suggested that B7-H4 wild type could confer chemoresistance to 786-O cells while B7-H4 NLS MT/786-O could not. The resistance index of B7-H4 WT/786-O cells to 5-5-fluorouracil was 2. 06, to doxorubicin was 1. 81 and to cisplatin was 1. 72. Flowcytometric analysis showed that when cells were treated with 1. 25 μg/mL 5-fluorouracil, the apoptosis of B7-H4 MT/786-O cells was lowest(20. 2%)when compared with Mock/786-O(41. 1%)and B7-H4 NLS MT/786-O(35. 1%). It showed that B7-H4 WT could confer multidrug resistance to 786-O cells through regulating cell apoptosis. However B7-H4 NLS MT could not act as an anti-apoptotic molecule. These results demonstrate that the NLS play an important role in the proliferation and chemoresistance promotion effect of B7-H4.