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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- 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-3 western 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 treat 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 SW620 cells or downregulation in HCT8, B7-H3 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 In addition, the results of cell cycle and active caspase-3 Western blotting demonstrated that B7-H3 overexpression inhibited apoptosis in colorectal cancer cell lines (P <0.05) .B7-H3 overexpression improved Jak2 and STAT3 phosphorylation 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 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 suggests 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.