GABRD促进肝细胞癌恶性进展及美金刚靶向干预作用研究

GABRD promotes malignant progression of hepatocellular carcinoma and is a therapeutic target of memantine

摘要:

背景与目的 γ-氨基丁酸A型受体δ亚基（GABRD）在多种恶性肿瘤中异常表达并参与肿瘤进展，但其在肝细胞癌（HCC）中的生物学作用及药物干预价值尚不明确。本研究旨在探讨GABRD在HCC中的表达特征、促癌作用及其潜在分子机制，并筛选针对GABRD的候选药物。方法 基于HCCDB和TCGA数据库分析GABRD表达及预后价值，并结合临床组织样本进行验证。采用CRISPR/Cas9敲除和慢病毒过表达技术构建GABRD功能获得与缺失模型，通过CCK-8、EdU、克隆形成、划痕愈合及Transwell实验评价其对HCC细胞增殖、迁移和侵袭能力的影响；利用皮下移植瘤、自发性肝癌及肺转移模型验证其体内作用。通过RNA测序结合GO、KEGG及GSEA分析探索其潜在机制。进一步采用结构基础虚拟筛选、分子对接及分子动力学模拟筛选靶向GABRD的小分子药物，并通过体内外实验验证其抗肿瘤作用。结果 GABRD在HCC组织及细胞中显著高表达，其高表达与患者总体生存期、疾病特异性生存期及无进展生存期缩短密切相关。敲除GABRD显著抑制HCC细胞增殖、迁移及侵袭能力，而过表达GABRD则产生相反效应。动物实验显示，GABRD缺失可明显抑制肿瘤生长和肺转移。转录组分析提示，GABRD可能通过调控细胞因子-细胞因子受体相互作用、JAK-STAT及上皮-间充质转化相关通路促进HCC进展。基于结构的虚拟筛选发现，美金刚与GABRD具有较高结合亲和力，并可下调GABRD表达水平。体内外实验进一步证实，美金刚能够显著逆转GABRD介导的促增殖、促侵袭及促转移作用。结论 GABRD在HCC中高表达并促进肿瘤恶性进展，是潜在的不良预后标志物和治疗靶点。美金刚可通过靶向GABRD抑制HCC生长与转移，为HCC的靶向治疗及老药新用提供新的实验依据。

Abstract:

Background and Aims Gamma-aminobutyric acid type A receptor delta subunit (GABRD) has been implicated in the progression of several malignancies, whereas its biological role and therapeutic relevance in hepatocellular carcinoma (HCC) remain largely unknown. This study aimed to investigate the expression pattern, oncogenic function, and potential molecular mechanisms of GABRD in HCC and to identify candidate small-molecule inhibitors targeting GABRD.Methods Public datasets from HCCDB and TCGA, together with clinical specimens, were used to evaluate GABRD expression and prognostic significance. Gain- and loss-of-function models were established using lentiviral overexpression and CRISPR/Cas9-mediated knockout approaches. Cell proliferation, migration, and invasion were assessed by CCK-8, EdU incorporation, colony formation, wound-healing, and Transwell assays. The in vivo effects were evaluated using subcutaneous xenograft, spontaneous HCC, and pulmonary metastasis models. RNA sequencing combined with GO, KEGG, and GSEA analyses was performed to explore downstream mechanisms. Structure-based virtual screening, molecular docking, and molecular dynamics simulations were applied to identify potential GABRD-targeting compounds, followed by in vitro and in vivo validation.Results GABRD was significantly upregulated in HCC tissues and cell lines, and its elevated expression was associated with unfavorable overall survival, disease-specific survival, and progression-free survival. GABRD knockout markedly inhibited HCC cell proliferation, migration, and invasion, whereas GABRD overexpression exerted opposite effects. In vivo studies further demonstrated that GABRD deficiency suppressed tumor growth and pulmonary metastasis. Transcriptomic analyses suggested that GABRD might promote HCC progression through cytokine-cytokine receptor interaction, JAK-STAT signaling, and epithelial-mesenchymal transition-related pathways. Structure-based virtual screening identified memantine as a potential GABRD-binding compound with high binding affinity. Memantine reduced GABRD expression and significantly reversed the pro-tumorigenic effects mediated by GABRD both in vitro and in vivo.Conclusion GABRD acts as an oncogenic regulator in HCC and may serve as a prognostic biomarker and therapeutic target. Memantine suppresses HCC growth and metastasis by targeting GABRD, providing experimental evidence for drug repurposing and novel targeted therapeutic strategies in HCC.