安徽医科大学学报

目的 探究黑色素瘤相关抗原D4(MAGED4)对肝细胞癌（HCC）恶性生物学行为的影响，为评估其作为HCC潜在治疗靶点提供实验依据。方法 利用LinkedOmics数据库筛选HCC中与MAGED4表达呈正相关的基因，并进行GO功能与KEGG通路富集分析。选取内源性MAGED4低表达的SMMC-7721、Huh7细胞，并以正常肝细胞LO2作为对照，通过慢病毒感染构建稳定过表达细胞株（MAGED4过表达组），并采用逆转录-聚合酶链式反应（RT-PCR）与Western blot验证其过表达效率。通过CCK-8实验、流式细胞术、划痕愈合实验、Transwell迁移实验及细胞黏附实验，系统分析MAGED4过表达对细胞增殖、周期分布、迁移及黏附能力的影响。结果 生物信息学分析提示，MAGED4共表达基因显著富集于细胞间黏附、细胞外基质组织及PI3K-Akt信号通路等与肿瘤增殖和转移密切相关的生物学过程。成功构建MAGED4稳定过表达细胞模型。体外功能实验结果显示，MAGED4过表达组SMMC-7721和Huh7细胞呈间质样形态，而LO2细胞仍维持上皮样形态；与空病毒对照组相比，MAGED4过表达组三株细胞的增殖活性（48 h:t=3.887、22.070、8.866，均P<0.05;72 h:t=3.231、23.840、9.848，均P<0.05;96 h:t=5.123、49.800、5.909,P<0.05、P<0.01、P<0.05）、划痕愈合率（12 h:F=40.90、44.00、200.50，均P<0.001;24 h:F=112.00、34.89、249.20，均P<0.001）及穿膜细胞数（F=239.20、 59.79、132.80，均P<0.001）均升高，同时黏附细胞数降低（F=73.65、85.91、138.00，均P<0.001），且细胞周期各时相分布无显著差异。结论 MAGED4可增强HCC和正常肝细胞增殖与迁移、抑制黏附，但仅诱导HCC细胞发生间质样形态改变，提示MAGED4能驱动HCC进展，有望成为HCC潜在的治疗靶点。

Objective To investigate the impact of melanoma-associated antigen D4(MAGED4) on the malignant biological behavior of hepatocellular carcinoma(HCC), providing experimental evidence for its evaluation as a po‑tential therapeutic target in HCC.Methods Genes positively correlated with MAGED4 expression in HCC were screened using the LinkedOmics database, followed by GO functional and KEGG pathway enrichment analyses. Endogenously low-expressing SMMC-7721 and Huh7 cells were selected, with normal hepatic LO2 cells serving as controls. Stable overexpression cell lines were established via lentiviral infection(MAGED4 overexpression group), with RT-PCR and Western blot validating overexpression efficiency. The effects of MAGED4 overexpres‑sion on cell proliferation, cell cycle distribution, migration, and adhesion were systematically analyzed using CCK-8 assays, flow cytometry, scratch healing assays, Transwell migration assays, and cell adhesion assays.Results Bioinformatics analysis indicated that MAGED4 co-expressed genes were significantly enriched in biological pro‑cesses closely associated with tumor proliferation and metastasis, including cell-cell adhesion, extracellular matrix organization, and the PI3K-Akt signaling pathway. A stable MAGED4 overexpression cell model was successfully established. In vitro functional assays demonstrated that MAGED4-overexpressing SMMC-7721 and Huh7 cells ex‑hibited a mesenchymal-like morphology, whereas LO2 cells maintained an epithelial-like morphology. Compared with the empty-virus control groups, the MAGED4-overexpressing groups showed significantly increased prolifera‑tive activity(48 h: t=3. 887, 22. 070, 8. 866, all P<0. 05; 72 h: t=3. 231, 23. 840, 9. 848, all P<0. 05; 96 h: t=5. 123, 49. 800, 5. 909, P<0. 05, P<0. 01, P<0. 05), wound healing rates(12 h: F=40. 90, 44. 00, 200. 50, all P<0. 001; 24 h: F=112. 00, 34. 89, 249. 20, all P<0. 001), and numbers of migrated cells(F=239. 20, 59. 79, 132. 80, all P<0. 001), along with significantly decreased numbers of adherent cells(F=73. 65, 85. 91, 138. 00, all P<0. 001), while no significant differences were observed in cell cycle phase distribution in all three cell lines.Conclusion MAGED4 enhances proliferation and migration while suppressing adhesion in both HCC and normal hepatocytes, however, induces mesenchymal-like morphological changes exclusively in HCC cells, suggesting that MAGED4 is capable of driving HCC progression and may serve as a potential therapeutic target for HCC.