雄黄主要成分As2S2通过“ANP32A-INHAT-H3乙酰化”轴抑制三阴性乳腺癌细胞增殖、迁移的表观遗传调控机制

目的 探究雄黄主要成分二硫化二砷（As2S2）对三阴性乳腺癌（triple negative breast cancer，TNBC）的作用及表观遗传调控机制。方法 〖JP2〗采用CCK-8、平板克隆形成和细胞划痕实验探究As2S2对人正常乳腺上皮细胞MCF-10A及TNBC细胞增殖和迁移的影响；4D-label free定量蛋白质组学分析挖掘As2S2抗TNBC的潜在干预靶点酸性核磷蛋白家族成员32A（acidic nuclear phosphoprotein family member 32A，ANP32A）；慢病毒感染法构建ANP32A过表达敲低细胞株,探究潜在靶点ANP32A对As2S2抗TNBC作用的影响；蛋白质免疫共沉淀和Western blot实验探究As2S2是否通过ANP32A调控TNBC细胞H3乙酰化。结果 As2S2对人正常乳腺上皮细胞MCF-10A影响甚微，但显著抑制TNBC细胞增殖和迁移，且呈剂量依赖性；4D-lable free定量蛋白质组学分析结果显示，促癌因子ANP32A被As2S2显著下调，且ANP32A表达影响As2S2在TNBC中的抗增殖和迁移效果。As2S2能下调ANP32A的蛋白水平，抑制乙酰转移酶抑制剂复合物亚基的招募，增加H3乙酰化水平。结论 As2S2通过下调ANP32A蛋白调控TNBC细胞中H3乙酰化，抑制TNBC细胞增殖和迁移。

The Epigenetic Regulatory Mechanism of As2S2 from Realgar in Inhibiting the Proliferation and Migration of Triple-negative Breast Cancer Cells Through the ANP32A-INHAT-H3 Acetylation Axis

Objective To investigate the effect of As2S2, the main component of realgar, on triple-negative breast cancer (TNBC) cells and its epigenetic regulatory mechanism.Methods CCK-8 assay, plate colony formation assay, and wound healing assay were used to explore the effect of As2S2 on the proliferation and migration of the normal breast epithelial cell line MCF-10A and TNBC cells; 4D-label free quantitative proteomic analysis was used to investigate the potential intervention target acidic nuclear phosphoprotein family member 32A(ANP32A) of As2S2 in anti-TNBC therapy; lentivirus transfection was used for the overexpression and knockdown of ANP32A to investigate the role of ANP32A in modulating the anti-TNBC activity of As2S2; protein co-immunoprecipitation and Western blot were used to investigate whether As2S2 regulated H3 acetylation of TNBC cells through ANP32A.Results As2S2 had little effect on the normal breast epithelial cell line MCF-10A, but it significantly inhibitied the proliferation and migration of TNBC cells in a dose-dependent manner. 4D-label free quantitative proteomic analysis showed that As2S2 significantly downregulated the oncogenic factor ANP32A, and the expression of ANP32A significantly affected the anti-proliferation and ant-migration effects of As2S2 in TNBC. As2S2 downregulated the protein expression level of ANP32A, inhibited the assembly of complex subunits of acetyltransferase inhibitor, and increased the level of H3 acetylation.Conclusion As2S2 inhibits the proliferation and migration of TNBC cells by downregulating the level of ANP32A protein and regulating H3 acetylation in TNBC cells.