摘要
AIM:To investigate the role of the overexpression of B7-H3 in apoptosis in colorectal cancer cell lines and the underlying molecular mechanisms.METHODS:SW620 cells that highly overexpressed B7-H3(SW620-B7-H3-EGFP)and HCT8 cells stably transfected with B7-H3 sh RNA(HCT8-sh B7-H3)were previously constructed in our laboratory.Cells transfected with p IRES2-EGFP were used as negative controls(SW620-NC and HCT8-NC).Real-time PCR and western blotting analysis were used to detect the m RNA and protein expressions of the apoptosis regulator proteins Bcl-2,Bcl-xl and Bax.A cell proliferation assay was used to evaluate the survival rate and drug sensitivity of the cells.The effect of drug resistance was detected by a cell cycle assay.Active caspase-3western blotting was used to reflect the anti-apoptotic ability of cells.Western blotting was also performed to determine the expression of proteins associated with the Jak2-STAT3 signaling pathway and the apoptosis regulator proteins after the treatment with AG490,a Jak2 specific inhibitor,in B7-H3 overexpressing cells.The data were analyzed by Graph Pad Prism 6 using a non-paired t-test.RESULTS:Whether by overexpression in SW620cells or downregulation in HCT8,B7-H3 significantly affected the expression of anti-and pro-apoptotic proteins,at both the transcriptional and translational levels,compared with the negative control(P<0.05).A cell proliferation assay revealed that B7-H3overexpression increased the drug resistance of cells and resulted in a higher survival rate(P<0.05).In addition,the results of cell cycle and active caspase-3western blotting proved that B7-H3 overexpression inhibited apoptosis in colorectal cancer cell lines(P<0.05).B7-H3 overexpression improved Jak2 and STAT3phosphorylation and,in turn,increased the expression of the downstream anti-apoptotic proteins B-cell CLL/lymphoma 2(Bcl-2)and Bcl-xl,based on western blotting(P<0.05).After treating B7-H3 overexpressing cells with the Jak2-specific inhibitor AG490,the phosphorylation of Jak2 and STAT3,and the expression of Bcl-2 and Bcl-xl,decreased accordingly(P<0.05).This finding suggested that the Jak2-STAT3 pathway is involved in the mechanism mediating the anti-apoptotic ability of B7-H3.CONCLUSION:The overexpression of B7-H3 induces resistance to apoptosis in colorectal cancer cell lines by upregulating the Jak2-STAT3 signaling pathway,potentially providing new approaches to the treatment of colorectal cancer.
AIM:To investigate the role of the overexpression of B7-H3 in apoptosis in colorectal cancer cell lines and the underlying molecular mechanisms.METHODS:SW620 cells that highly overexpressed B7-H3(SW620-B7-H3-EGFP)and HCT8 cells stably transfected with B7-H3 sh RNA(HCT8-sh B7-H3)were previously constructed in our laboratory.Cells transfected with p IRES2-EGFP were used as negative controls(SW620-NC and HCT8-NC).Real-time PCR and western blotting analysis were used to detect the m RNA and protein expressions of the apoptosis regulator proteins Bcl-2,Bcl-xl and Bax.A cell proliferation assay was used to evaluate the survival rate and drug sensitivity of the cells.The effect of drug resistance was detected by a cell cycle assay.Active caspase-3western blotting was used to reflect the anti-apoptotic ability of cells.Western blotting was also performed to determine the expression of proteins associated with the Jak2-STAT3 signaling pathway and the apoptosis regulator proteins after the treatment with AG490,a Jak2 specific inhibitor,in B7-H3 overexpressing cells.The data were analyzed by Graph Pad Prism 6 using a non-paired t-test.RESULTS:Whether by overexpression in SW620cells or downregulation in HCT8,B7-H3 significantly affected the expression of anti-and pro-apoptotic proteins,at both the transcriptional and translational levels,compared with the negative control(P<0.05).A cell proliferation assay revealed that B7-H3overexpression increased the drug resistance of cells and resulted in a higher survival rate(P<0.05).In addition,the results of cell cycle and active caspase-3western blotting proved that B7-H3 overexpression inhibited apoptosis in colorectal cancer cell lines(P<0.05).B7-H3 overexpression improved Jak2 and STAT3phosphorylation and,in turn,increased the expression of the downstream anti-apoptotic proteins B-cell CLL/lymphoma 2(Bcl-2)and Bcl-xl,based on western blotting(P<0.05).After treating B7-H3 overexpressing cells with the Jak2-specific inhibitor AG490,the phosphorylation of Jak2 and STAT3,and the expression of Bcl-2 and Bcl-xl,decreased accordingly(P<0.05).This finding suggested that the Jak2-STAT3 pathway is involved in the mechanism mediating the anti-apoptotic ability of B7-H3.CONCLUSION:The overexpression of B7-H3 induces resistance to apoptosis in colorectal cancer cell lines by upregulating the Jak2-STAT3 signaling pathway,potentially providing new approaches to the treatment of colorectal cancer.
基金
Supported by Project of Natural Science Foundation of Jiangsu Province,No.BK2012542
the Project of Hospital Management Center of Wuxi City,No.YGZ1108
