肝细胞癌抗原587基因甲基化调节HCA587-TAF9互作介导肝细胞癌的侵袭和迁移

目的：研究肝细胞癌抗原587（HCA587）在肝细胞癌（HCC）组织中的表达和启动子区甲基化状态，探讨HCA587对肝细胞癌细胞迁移和侵袭的调节机制。方法：纳入肝细胞癌肿瘤组织114 例，分为HCC组和癌旁非癌组织（NT）组。荧光定量PCR 检测HCA587的mRNA表达量；免疫组织化学和免疫印迹检测HCC组和NT组组织中HCA587的蛋白表达。基于甲基化敏感酶和甲基化依赖酶酶切，并结合荧光定量PCR 方法分析肝细胞癌组织中HCA587基因启动子区甲基化状态。构建重组HCA587过表达质粒（pcDNA3.1-HCA587），培养人肝细胞癌细胞系BEL-7404，转染pcDNA3.1-HCA587 或者使用甲基化抑制剂5-Azacytidin 处理细胞。细胞分为对照组、5-Azacytidin 组、pcDNA3.1-HCA587 组和pcDNA3.1-HCA587 空载体（EV） 组。免疫沉淀法分析HCA587与TATA 盒结合蛋白相关因子9（TAF9）的结合，Transwell 小室检测细胞的迁移和侵袭能力。结果： 与癌旁非癌组织组比，HCC组的HCA587在mRNA和蛋白水平均上调。免疫组织化学证实HCC组组织中HCA587阳性表达。HCC组中HCA587的启动子区CpG 岛甲基化水平降低。体外实验结果显示，5-Azacytidin 促进BEL-7404 中HCA587的表达、HCA587与TAF9的结合、BEL-7404 细胞的迁移和侵袭能力，但抑制HCA587甲基化水平；另外，HCA587过表达增强BEL-7404 细胞的迁移和侵袭能力。结论：HCA587高表达或抑制其启动子区甲基化可以促进HCA587与TAF9的结合及导致肝细胞癌细胞的迁移和侵袭。

HCA587 gene methylation regulates HCA587-TAF9 interaction to mediate invasion and migration of hepatocellular carcinoma

Objective To study the expression of hepatocellular carcinoma antigen 587（ HCA587） in hepatocellularcarcinoma（ HCC） tissues and the methylation status of promoter region， and to explore the mechanism of HCA587involvement in the regulation of HCC cell migration and invasion. Methods 114 HCC tumor tissues were includedand divided into HCC group and non-cancerous tissue（ NT） group adjacent to cancer. Both the HCC and NTgroups were used to observe the expression and methylation changes of HCA587. Fluorescence quantitative PCRwas used to detect the mRNA expression of HCA587. Immunohistochemistry and Western blotting were used todetect the expression of HCA587 in HCC tissues and NT. The methylation status of HCA587 gene promoter regionin HCC tissues was analyzed based on methylation-sensitive enzyme and methylation-dependent enzyme digestioncombined with fluorescent quantitative PCR. Recombinant HCA587 overexpression plasmid （pcDNA3.1-HCA587）was constructed. The human HCC cell line BEL-7404 was cultured， and the cells were transfected with pcDNA3.1-HCA587 or treated with methylation inhibitor 5-Azacytidin. The cells were divided into control group， 5-Azacytidingroup， pcDNA3.1-HCA587 group， and pcDNA3.1-HCA587 empty vector（ EV） group. Immunoprecipitation analysisof the binding effect of HCA587 and TATA box binding protein-related factor 9（ TAF9） was performed. The migrationand invasion ability of cells were detected by transwell assay. Results Compared with non-cancerous tissues adjacentto cancer， HCA587 in HCC group was up-regulated atboth mRNA and protein levels. Immunohistochemistryconfirmed the positive expression of HCA587 in HCC— 406 — tissues. In the HCC group ， the methylation level of CpG island in the promoter region of HCA587 decreased. In vitro，5-Azacytidin promoted the expression of HCA587 in BEL-7404， the binding of HCA587 to TAF9， the migrationand invasion ability of BEL-7404 cells， but inhibited the methylation level of HCA587. In addition， HCA587 overexpressionenhanced the migration and invasion ability of BEL-7404 cells. Conclusion High expression of HCA587or inhibition of its promotor methylation can promote the binding of HCA587 to TAF9 and guide the migration andinvasion of HCC cells.