共刺激分子B7H3通过调控DNA拓扑异构酶Ⅱα的表达对乳腺癌多柔比星化疗的影响

Effect of costimulatory molecule B7H3 on doxorubicin chemotherapy in breast cancer by regulating the expression of DNA topoisomeraseⅡalpha

目的探讨共刺激分子B7H3(costimulatory molecule B7H3,B7H3)通过调控DNA拓扑异构酶Ⅱα(DNA topoisom eraseⅡalpha,TOP2A)的表达对乳腺癌多柔比星化疗敏感性的影响。方法收集临床标本,采用免疫组织化学染色验证B7H3表达与多柔比星化疗敏感性的相关性。采用成簇规律间隔短回文重复/Cas9基因编辑技术敲除B7H3,通过蛋白质印迹法和流式细胞术验证敲除结果。采用半抑制浓度值分析、集落形成、蛋白质印迹法检测凋亡蛋白、荧光双染色检测凋亡细胞以及细胞周期测定等方法验证B7H3表达与多柔比星作用的敏感性。小干扰TOP2A及其过表达TOP2A回复实验检测细胞凋亡。构建小鼠皮下成瘤模型验证体内B7H3表达与多柔比星化疗的作用。结果高表达B7H3的肿瘤组织对多柔比星治疗更加敏感。成功构建B7H3敲除的乳腺癌细胞。B7H3高表达的乳腺癌细胞对多柔比星作用更敏感。B7H3通过激活核因子κB(nuclear factor-κB,NF-κB)途径促进TOP2A的表达,进而增加乳腺癌细胞对多柔比星作用的敏感性。小鼠体内实验证明,B7H3高表达可增加乳腺癌对多柔比星化疗的敏感性。结论B7H3通过激活NF-κB通路调控TOP2A的表达使乳腺癌对多柔比星化疗更敏感。

Objective To investigate the effect of costimulatory molecule B7H3(B7H3)on breast cancer sensitivity to doxorubicin chemotherapy by regulating the expression of DNA topoisomeraseⅡalpha(TOP2A).Method The clinical specimens were collected,and the correlation between B7H3 expression and the sensitivity of doxorubicin treatment was verified by immunohistochemical staining.B7H3 was knocked out by clustered regularly interspaced short palindromic repeat/Cas9 gene editing technology,and the results were verified by Western blotting and flow cytometry.The sensitivity of B7H3 expression to the action of doxorubicin was verified by using experimental methods such as half maximal inhibitory concentration analysis,colony formation assay,Western blotting to detect apoptosis-related proteins,fluorescence dual staining to detect apoptotic cells,and cell cycle analysis.Experimental detection of cell apoptosis by small interference with TOP2A and its overexpression reversal assay.The relationship between B7H3 expression and doxorubicin therapy was verified by establishing a mouse subcutaneous tumor formation model.Result Tumor tissues with high expression of B7H3 were more sensitive to doxorubicin therapy.Breast cancer cells with B7H3 expression knocked out were successfully constructed.Breast cancer cells with high expression of B7H3 were more sensitive to doxorubicin.B7H3 promoted the expression of TOP2A by activating the nuclear factor-κB(NF-κB)pathway,thereby increasing the sensitivity of breast cancer cells to doxorubicin.In vivo experiments in mice demonstrated that high expression of B7H3 increased the sensitivity of breast cancer to doxorubicin therapy.Conclusion B7H3 regulates the expression of TOP2A by activating NF-κB pathway,making breast cancer sensitive to doxorubicin chemotherapy.