AGPAT5在肝癌中的功能与机制研究  

Functional and mechanistic of AGPAT5 in liver cancer

作　　者：陈奕君 刘雨航 段海波 王雄军 CHEN Yijun;LIU Yuhang;DUAN Haibo;WANG Xiongjun(Precision Editing and Health Research Center,Guangzhou University,Guangzhou 510405,Guangdong Province,China)

机构地区：[1]广州大学精准编辑与健康研究中心,广东广州510405

出　　处：《中国癌症杂志》2024年第9期838-847,共10页China Oncology

基　　金：国家自然科学基金面上项目(82272714)。

摘　　要：背景与目的:肿瘤的发生、发展过程中会发生代谢重编程,1-酰基甘油-3-磷酸O-酰基转移酶(1-acylglycerol-3-phosphate O-acyltransferase,AGPAT)作为三酰甘油(triacylglycerol,TAG)从头合成的关键酶,与肿瘤的进展密切相关。但目前作为亚型之一的AGPAT5在癌症中的研究还十分有限,本研究深入剖析AGPAT5在肝癌发生、发展中发挥的作用及潜在的分子机制,旨在为肝癌诊断和治疗策略提供新思路。方法:利用慢病毒感染将多种肝癌细胞系中的AGPAT5敲减,并通过锥虫蓝计数、划痕、transwell及平板克隆等实验在体外检测AGPAT5对肝癌细胞增殖、迁移及抗失巢凋亡能力的影响。通过回复野生型或酶活性缺失型的AGPAT5,探究其作为代谢酶是否发挥经典代谢作用调控肝癌细胞迁移。构建BALB/c裸鼠尾静脉注射移植瘤模型,从体内层面验证体外的细胞表型。采用免疫沉淀质谱联用(immunoprecipitation mass spectrum,IP-MS)鉴定出与AGPAT5相互作用的蛋白,并进行免疫共沉淀(co-immunoprecipitation,coIP)验证。蛋白质翻译后通过修饰鉴定分析AGPAT5潜在的修饰位点,通过体外实验探究点突变前后对肝癌细胞迁移的影响。通过coIP探究该位点突变前后AGPAT5与相互作用蛋白结合的情况。通过敲低相互作用蛋白确定其在细胞表型中的作用。通过回复实验验证AGPAT5是否通过相互作用蛋白发挥作用。检测野生型肝癌细胞系中的AGPAT5和相互作用蛋白的表达水平,检验两者之间是否具有相关性。结果:肝癌细胞敲减AGPAT5后会更加耐受无血清饥饿,并促进细胞迁移,但不会影响细胞增殖和失巢凋亡。而酶活性缺失并不影响AGPAT5对肝癌细胞迁移的抑制。敲减AGPAT5可促进肝癌细胞在裸鼠体内的肺转移和肝转移。AGPAT5可以与原纤维蛋白(fibrillarin,FBL)相互作用,并在无血清饥饿刺激下加强两者的结合。遏制FBL的表达会抑制肝癌细胞迁移,且效果与Background and purpose:Metabolic reprogramming occurs during tumor progression,and 1-acylglycerol-3-phosphate O-acyltransferase(AGPAT),as a key enzyme in the de novo synthesis of triacylglycerol(TAG),is closely associated with tumor progression.However,one of the isoforms,AGPAT5,has been studied in cancer in a very limited way,and this study aimed to provide a new perspective on the role of AGPAT5 in hepatocellular carcinoma and its potential molecular mechanisms,providing novel ideas for the diagnosis and treatment strategies of liver cancer.Methods:AGPAT5 was knocked down in a variety of hepatocellular carcinoma cell lines using lentiviral infection,and the effects of AGPAT5 on the functions of hepatocellular carcinoma cell proliferation,migration and resistance to anoikis were detected in vitro by experiments such as Taipan blue counting,scratching,transwell and plate cloning.The wild-type or enzyme activity-deficient form of AGPAT5 was rescued to investigate whether AGPAT5,as a metabolic enzyme,plays a classical role in regulating the migration of hepatocellular carcinoma cells.We constructed a tail vein metastasis model in nude mice to validate the cellular phenotype in vitro from the in vivo level.Immunoprecipitation mass spectrum(IP-MS)identified proteins interacting with AGPAT5 and verified by co-immunoprecipitation(coIP).Protein post-translational modification identification was performed to analyze the potential modification sites of AGPAT5,and in vitro experiments were performed to explore the effects of the point mutation before and after the point mutation on the migration of hepatocellular carcinoma cells.CoIP was performed to explore the binding of AGPAT5 to the interacting protein before and after the mutation of the site.We determined its role in cell phenotype by knocking down interacting proteins.Rescue experiments were used to verify whether AGPAT5 exerts its effects through the interacting protein.We detected the expression levels of AGPAT5 and the interacting protein in wild-type hepatocellu

关 键 词：1-酰基甘油-3-磷酸O-酰基转移酶5 肝癌转移 原纤维蛋白 K201位点乙酰化 非代谢作用 

分 类 号：R735.7[医药卫生—肿瘤]