【作者】 苏鑫；

【导师】 王丽娜； 王允山；

【作者基本信息】 山东大学 ， 临床检验诊断学（专业学位）， 2025， 硕士

【摘要】 目的:胃癌是全球范围内发病率和死亡率均位居前列的恶性肿瘤,对人类健康构成严重威胁。由于其早期症状隐匿,大多数患者在确诊时已进入中晚期,导致临床治疗效果不佳,患者预后较差。因此,深入解析胃癌发生发展的分子机制,开发新型早期诊断标志物和有效治疗靶点,对于改善患者预后具有重要意义。近年来,乳酸化修饰作为一种新型翻译后修饰类型,在肿瘤代谢重编程和表观遗传调控过程中的重要作用逐渐被揭示,但其在胃癌中的具体功能和分子机制仍不明确。羟酰基辅酶A脱氢酶α亚基(Hydroxyacyl-CoA dehydrogenase alpha subunit,HADHA)作为线粒体三功能蛋白的关键组分,不仅是长链脂肪酸β-氧化的核心酶,还在多种癌症进展中发挥重要调控作用。本研究旨在揭示乳酸化修饰及其调控酶HADHA在胃癌中的生物学功能及分子机制,为胃癌的精准诊疗提供新的理论依据和潜在干预靶点。方法:1.采用非标记蛋白质定量技术,对胃癌组织及对应癌旁组织进行乳酸化修饰组学分析,探寻潜在的蛋白质乳酸化修饰位点。利用免疫组织化学染色评估胃癌及癌旁组织中的泛乳酸化修饰水平,并用Kaplan-Meier方法评估泛乳酸化修饰水平高低与胃癌预后的关系。2.通过对差异表达蛋白进行通路富集分析寻找合适的乳酸化修饰蛋白,通过免疫印迹实验(Western blot)、质谱分析及免疫组化方法验证胃癌组织和细胞中HADHA的K350和K406位点的乳酸化修饰情况。3.通过蛋白质结构预测与分析明确乳酸化修饰对HADHA结构的影响,通过酶活性测定法检测胃癌细胞中HADHA酶活性,通过免疫共沉淀实验检测胃癌细胞中HADHA与HDHAB的结合情况。4.通过试剂盒检测胃癌细胞代谢产物浓度的变化。借助液相色谱-串联质谱技术,进行基于13C标记的碳代谢流分析。通过MTT和Transwell实验检测HADHA-K406乳酸化对胃癌细胞增殖和迁移能力的影响。通过构建裸鼠皮下移植瘤模型明确HADHA-K406乳酸化对胃癌发生发展的影响。5.通过RNA测序及染色质免疫沉淀测序技术探寻HADHA-K406乳酸化可能的作用靶点,即 3-羟基-3-甲基戊二酰辅酶 A 还原酶(3-Hydroxy-3-methylglutaryl-CoA reductase,HMGCR)。通过逆转录定量聚合酶链式反应(Reverse transcription-quantitative polymerase chain reaction,RT-qPCR)或Western blot检测胃癌细胞中HMCCR的表达水平。通过构建小鼠皮下移植瘤模型,探究胆固醇水平对胃癌进展的影响。结果:1.与癌旁组织相比,胃癌组织中乳酸化修饰水平显著升高,且泛乳酸化修饰水平与胃癌患者的不良预后呈正相关。2.在临床胃癌组织样本中,HADHA的K350和K406位点均表现出明显的乳酸化修饰。在体外培养的胃癌细胞中,仅检测到HADHA-K406位点的乳酸化修饰,而K350位点没有发生明显的乳酸化修饰。3.HADHA-K406位点的乳酸化会对其酶结构产生影响,乳酸处理或K406位点发生乳酸化修饰可使HADHA酶活性升高,与HADHB的结合能力增强。4.HADHA-K406乳酸化影响胃癌细胞的脂肪酸β-氧化及糖酵解,乳酸化修饰与乳酸生成之间存在反馈回路。HADHA-K406乳酸化能增强胃癌细胞增殖和迁移的能力,并促进裸鼠皮下移植瘤的生长。5.HADHA-K406乳酸化能够增强胃癌细胞HMGCR启动子区域的组蛋白乙酰化水平,促进其转录及蛋白表达,进而增加胃癌细胞中胆固醇的合成。胆固醇水平的升高促进了小鼠皮下移植瘤的生长。结论:1.与癌旁组织相比,胃癌组织中的乳酸化修饰水平升高,且与患者不良预后密切相关。其中HADHA-K406位点的乳酸化修饰水平明显上调。2.HADHA-K406位点发生乳酸化修饰,能够改变其酶结构与活性,重塑胃癌细胞代谢,并通过表观遗传机制上调HMGCR介导的胆固醇合成途径,促进胃癌的进展。更多还原

【Abstract】 Objective:Gastric cancer is a malignant tumor with high incidence and mortality rates worldwide,posing a serious threat to human health.Due to its insidious early symptoms,most patients have already reached the middle or advanced stage at the time of diagnosis,resulting in poor clinical treatment outcomes and a poor prognosis for patients.Therefore,in-depth analysis of the molecular mechanisms underlying the development and progression of gastric cancer,as well as the development of novel early diagnostic biomarkers and effective therapeutic targets,is of great significance for improving patient prognosis.In recent years,lactylation modification,as a novel type of post-translational modification,has gradually revealed its important role in the processes of tumor metabolic reprogramming and epigenetic regulation.However,its specific functions and molecular mechanisms in gastric cancer remain unclear.The hydroxyacyl-CoA dehydrogenase alpha subunit（HADHA）,as a key component of the mitochondrial trifunctional protein,is not only a core enzyme in the β-oxidation of long-chain fatty acids but also plays an important regulatory role in the progression of various cancers.This study aims to reveal the biological functions and molecular mechanisms of lactylation modification and its regulatory enzyme HADHA in gastric cancer,providing new theoretical basis and potential intervention targets for the precise diagnosis and treatment of gastric cancer.Methods:1.Label-free quantitative proteomics technology was employed to conduct a lactylation modification omics analysis of gastric cancer tissues and their corresponding adjacent tissues,aiming to explore potential protein lactylation modification sites.Immunohistochemical staining was used to evaluate the pan-lactylation modification levels in gastric cancer tissues and adjacent tissues,and the Kaplan-Meier method was applied to assess the relationship between the high or low levels of pan-lactylation modification and the prognosis of gastric cancer.2.Pathway enrichment analysis of differentially expressed proteins was performed to screen for candidate lactylated proteins.Validation of HADHA lactylation at K350 and K406 was conducted using Western blotting,mass spectrometry,and IHC in gastric cancer tissues and cell lines.3.Protein structure prediction was used to elucidate the impact of lactylation on HADHA conformation.Enzymatic activity assays were performed to measure HADHA activity in gastric cancer cells,and co-immunoprecipitation（Co-IP）was used to investigate HADHA-HDHAB interactions.4.Metabolite concentration changes were detected using commercial kits.¹³C-labeled metabolic flux analysis via liquid chromatography-tandem mass spectrometry（LC-MS/MS）was performed to track carbon flow.The functional effects of HADHA-K406 lactylation on cell proliferation and migration were assessed using MTT assays and Transwell assays.A nude mouse xenograft model was established to evaluate the role of HADHA-K406 lactylation in tumorigenesis.5.RNA sequencing and chromatin immunoprecipitation sequencing were used to explore the possible targets of HADHA-K406 lactylation,namely 3-Hydroxy-3-methylglutaryl-CoA reductase（HMGCR）.The expression level of HMGCR in gastric cancer cells was detected by reverse transcription-quantitative polymerase chain reaction（RT-qPCR）or Western blot.A subcutaneous xenograft tumor model in mice was established to investigate the effect of cholesterol levels on the progression of gastric cancer.Results:1.Compared with adjacent normal tissues,the level of lactylation modification in gastric cancer tissues is significantly increased,and the level of pan-lactylation modification is positively correlated with the poor prognosis of gastric cancer patients.2.In clinical gastric cancer tissue samples,both the K350 and K406 sites of HADHA show obvious lactylation modification.In in-vitro cultured gastric cancer cells,lactylation modification is only detected at the HADHA-K406 site,while no obvious lactylation modification occurs at the K350 site.3.Lactylation at the HADHA-K406 site affects the enzyme structure of HADH A.Treatment with lactate or lactylation modification at the K406 site can increase the enzymatic activity of HADH A and enhance its binding ability to HADHB.4.HADHA-K406 lactylation affects fatty acid β-oxidation and glycolysis in gastric cancer cells.There is a feedback loop between lactylation modification and lactate production.HADHA-K406 lactylation can enhance the proliferation and migration abilities of gastric cancer cells and promote the progression of subcutaneous xenograft tumors in nude mice.5.HADHA-K406 lactylation can enhance the histone acetylation level in the promoter region of HMGCR in gastric cancer cells,promote its transcription and protein expression,and further increase cholesterol synthesis in gastric cancer cells.The increase in cholesterol level promotes the growth of subcutaneous xenograft tumors in mice.Conclusions:1.Compared with adjacent non-cancerous tissues,the level of lactylation modification in gastric cancer tissues is elevated and is closely associated with poor prognosis in patients.In particular,the level of lactylation modification at the HADHA-K406 site is significantly upregulated.2.Lactylation modification at the HADHA-K406 site can alter its enzyme structure and activity,reshape the metabolism of gastric cancer cells,and upregulate the HMGCR-mediated cholesterol synthesis pathway through epigenetic mechanisms,thereby promoting the progression of gastric cancer.更多还原