甜菜碱同型半胱氨酸甲基转移酶在肝细胞癌中的作用

作者：刘文丽1  罗达亚2  刘卓琦2  杨晓红2  朱伟锋2  胡楷恒3  肖影群1

单位：1. 南昌市第九医院（南昌大学附属感染病医院） 病理科 江西 南昌 330006 2. 南昌大学基础医学院 生物化学与分子生物学教研室 江西 南昌 330006 3. 南昌大学临床医学院（中外合作办学） 玛丽女王学院 江西 南昌 330006]

关键词：甜菜碱同型半胱氨酸甲基转移酶 肝细胞癌 预后 免疫浸润

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出版年,卷(期):页码：2025,17(4):31-42

摘要：

摘要：目的 采用生物信息学方法探讨甜菜碱同型半胱氨酸甲基转移酶（betaine homocysteine methyltransferase，BHMT）与肝细胞癌（hepatocellular carcinoma， HCC）的作用及临床联系，为HCC的临床诊疗提供新思路。方法 使用Sanger Box在线 软件分析BHMT在泛癌中的表达水平。通过UCSC Xena数据库分析BHMT mRNA的表 达水平以及HCC中BHMT的甲基化数据和患者的临床特征信息，分析BHMT表达与这 些数据的关系。通过TIMER在线软件分析HCC中BHMT的表达与免疫细胞浸润的相关 性。通过LinkedOmics在线软件对HCC中BHMT进行GO功能与KEGG信号通路富集分 析。收集2008年至2018年南昌市第九医院符合入选标准的28例HCC患者的石蜡标本， 采用免疫组织化学法检测BHMT蛋白表达水平。结果 癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库和基因型组织表达（Genotype-Tissue Expression， GTEx）数据库显示，BHMT在包括HCC在内的多种肿瘤中低表达。UCSC XENA数 据库分析表明，BHMT mRNA在HCC中呈低表达。BHMT mRNA低表达患者的生存 时间显著低于高表达患者（Log rank χ 2 = 5.812，P = 0.0159）；BHMT mRNA低表达 患者中男性比例高于BHMT mRNA高表达者（χ 2 = 46.600，P ＜ 0.001），两组间HCC Edmondson-Steiner分级、AJCC分级、TNM分期及生存状态差异无统计学意义（P均＞ 0.05）。BHMT mRNA表达与其DNA甲基化水平呈负相关（r = －0659，P ＜ 0.001）， 且BHMT表达与cg07418518、cg18483750等9个甲基化位点存在相关性（P均＜ 0.05）， 这9个位点的甲基化水平在HCC组织与正常组织中存在差异。临床样本分析也表明 BHMT蛋白在HCC组织的表达水平显著低于癌旁组织（中位数：3.000比1.000；U = 258，P = 0.0401），BHMT蛋白高表达患者和BHMT蛋白低表达患者微血管浸润程度 差异有统计学意义（χ 2 = 7.133，P = 0.008）。HCC组织CD4蛋白相对表达量显著低于 癌旁组织（中位数：3.000比1.000，U = 215，P ＜ 0.001）。GO分析结果表明，HCC 中BHMT在生物过程（biological process，BP）的富集主要与生物调节、代谢过程、刺 激应答等有关；在细胞成分（cell component，CC）的富集主要与细胞膜、细胞核和膜 腔等结构有关；在分子功能（molecular function，MF）的富集主要与蛋白质结合、离 子结合和核酸结合等过程有关。KEGG信号通路分析表明，与BHMT正相关的通路主 要富集于物质代谢（脂质、氨基酸、固醇等）和应答过程（急性炎症刺激、异生物刺 激等）；而与BHMT负相关的通路主要富集于细胞周期、信号通路、机体生物化学反 应、免疫细胞活化等过程。结论 BHMT在HCC的发生发展中发挥关键作用。

Abstract: Objective To investigate the role and clinical association between betaine homocysteine methyltransferase (BHMT) and hepatocellular carcinoma (HCC) by bioinformatics methods, in order to provide new insights for the clinical diagnosis and treatment of HCC. Methods The expression levels of BHMT in pan-cancer were analyzed by the online software Sanger Box. The expression levels of BHMT mRNA, the methylation data of BHMT in HCC and the clinical characteristics of patients, as well as the relationship between BHMT expression and these data were analyzed by UCSC Xena database. The correlation between BHMT expression and immune cell infiltration in HCC was analyzed by Timer online software. GO functional enrichment and KEGG pathway enrichment analysis of BHMT in HCC were performed with LinkedOmics online software. Paraffin embedded specimens from 28 eligible HCC patients in The Ninth Hospital of Nanchang from 2008 to 2018 were collected, and the expression levels of BHMT protein were detected by immunohistochemical methods. Results Data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project indicated that BHMT was downregulated in multiple tumor types, including HCC. Consistently, UCSC Xena analysis showed significantly reduced BHMT mRNA levels in HCC. Patients with low BHMT mRNA expression had significantly shorter survival time than those with high BHMT mRNA expression (Log rank χ 2 = 5.812, P = 0.0159); the proportion of males was higher in low BHMT mRNA expression group than in high expression group (χ 2 = 46.600，P ＜ 0.001). No statistically significant differences were observed in HCC Edmondson-Steiner grade, AJCC stage, TNM stage, or survival state between two groups (all P ＞ 0.05). A negative correlation was observed between BHMT mRNA expression and its DNA methylation level (r = －0659, P ＜ 0.001). Furthermore, BHMT expression was correlated with 9 methylation sites including cg07418518 and cg18483750 (all P ＜ 0.05), and the methylation levels of these 9 sites differed significantly between HCC tissues and normal tissues. Clinical samples analysis also demonstrated that the expression level of BHMT protein in HCC tissues was significantly lower than that in adjacent non-tumor tissues (median: 3.000 vs. 1.000; U = 258, P = 0.0401). A statistically significant difference was observed in microvascular invasion status between patients with high BHMT protein expression and those with low expression (χ2 = 7.133, P = 0.008). The relative expression level of CD4 protein in HCC tissues was significantly lower than that in adjacent non-tumor tissues (median: 3.000 vs. 1.000, U = 215, P ＜ 0.001). GO analysis revealed that BHMT in HCC was primarily enriched in biological processes (BP) related to biological regulation, metabolic processes, and response to stimuli; cellular components (CC) enrichment was associated with structures including cell membrane, nucleus, and membrane lumen; and molecular functions (MF) enrichment was involved in protein binding, ion binding, and nucleic acid binding. KEGG pathway analysis indicated that pathways positively correlated with BHMT were mainly enriched in substance metabolism (including lipids, amino acids, sterols, etc.) and response processes (such as acute inflammatory stimulus response, xenobiotic stimulus response, etc.); whereas pathways negatively correlated with BHMT were primarily enriched in processes including cell cycle, signaling pathways, organismal biochemical reactions, and immune cell activation. Conclusion BHMT played a crucial role on the development and progression of HCC.

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