HDAC6通过介导FLOT2去乙酰化维持其在鼻咽癌中的稳定及促瘤作用  

HDAC6-mediated deacetylation of FLOT2 maintains stability and tumorigenic function of FLOT2 in nasopharyngeal carcinoma

作　　者：罗辰骅 温彬斌 刘洁[3] 杨文龙[4] LUO Chenhua;WEN Binbin;LIU Jie;YANG Wenlong(Xiangya School of Medicine,Central South University,Changsha 410013;Xiangya School of Nursing,Central South University,Changsha 410013;Department of Pathology,Affiliated Changsha Central Hospital,University of South China,Changsha 410004;Department of Gastrointestinal Surgery II,Third Xiangya Hospital,Central South University,Changsha 410013,China)

机构地区：[1]中南大学湘雅医学院,长沙410013 [2]中南大学湘雅护理学院,长沙410013 [3]南华大学附属长沙中心医院病理科,长沙410004 [4]中南大学湘雅三医院胃肠外科II科,长沙410013

出　　处：《中南大学学报（医学版）》2024年第5期687-697,共11页Journal of Central South University ：Medical Science

基　　金：湖南省自然科学基金(2021JJ40623)。

摘　　要：目的:浮舰蛋白(flotillin-2,FLOT2)是典型的致瘤蛋白和潜在的肿瘤治疗靶点,但靶向FLOT2的干预策略仍未确定。翻译后修饰作为表观调控的重要方式,对蛋白质的活性、定位和稳定性等特性具有重要的调控作用,揭示蛋白质翻译后修饰的调控机制和功能是靶向治疗开发的一种有效手段。本研究旨在研究鼻咽癌中FLOT2赖氨酸乙酰化修饰的调控机制及其功能,为靶向FLOT2的肿瘤干预手段提供新思路。方法:利用PhosphoSitePlus数据库分析FLOT2的赖氨酸乙酰化位点,并构建赖氨酸乙酰化位点突变的FLOT2突变体[FLOT2(K211R)];用组蛋白去乙酰化酶(histonedeacetylases, HDAC)抑制剂曲古菌素A(trichostatin A, TSA)、 Sirt家族去乙酰化酶抑制剂烟酰胺(nicotinamide,NAM)处理鼻咽癌细胞,TSA处理转染FLOT2突变体质粒的人胚肾细胞(human embryonic kidney,HEK)-293T细胞;利用免疫共沉淀(co-immunoprecipitation,Co-IP)检测FLOT2的总赖氨酸乙酰化水平以及特定赖氨酸(K)位点突变对FLOT2总赖氨酸乙酰化水平的影响。用蛋白质印迹法检测TSA处理未转染/转染FLOT2突变体质粒后的鼻咽癌细胞中FLOT2/FLAG-FLOT2的蛋白质表达,实时反转录聚合酶链反应(real-time reverse transcription PCR,real-time RT-PCR)检测TSA处理后鼻咽癌细胞中FLOT2 mRNA的表达。用TSA分别联合MG132或氯喹(chloroquine,CQ)处理鼻咽癌细胞后,检测FLOT2的蛋白质表达。用放线菌酮(cycloheximide,CHX)分别处理已转染FLAG-FLOT2(WT)或FLAG-FLOT2(K211R)质粒的HEK-293T细胞,检测FLAG-FLOT2、FLOT2(K211R)的蛋白质表达水平以反映蛋白质的降解速率。通过BioGrid数据库查询FLOT2与HDAC6之间是否可能存在相互作用,并采用Co-IP验证。用FLAG-FLOT2(WT)/FLAG-FLOT2(K211R)质粒分别联合空白对照(Vector)/HDAC6质粒转染HEK-293T细胞,分为FLAG-FLOT2(WT)+Vector、 FLAG-FLOT2(WT)+HDAC6、 FLAG-FLOT2(K211R)+Vector、 FLAG-FLOT2(K211R)+HDAC6共4组,分析K211R突变对FLOT2总赖氨�Objective:Flotillin-2(FLOT2)is a prototypical oncogenic and a potential target for cancer therapy.However,strategies for targeting FLOT2 remain undefined.Post-translational modifications are crucial for regulating protein stability,function,and localization.Understanding the mechanisms and roles of post-translational modifications is key to developing targeted therapies.This study aims to investigate the regulation and function of lysine acetylation of FLOT2 in nasopharyngeal carcinoma,providing new insights for targeting FLOT2 in cancer intervention.Methods:The PhosphoSitePlus database was used to analyze the lysine acetylation sites of FLOT2,and a lysine acetylation site mutation of FLOT2[FLOT2(K211R)]was constructed.Nasopharyngeal carcinoma cells were treated with histone deacetylase(HDAC)inhibitor trichostatin A(TSA)and Sirt family deacetylase inhibitor nicotinamide(NAM).TSA-treated human embryonic kidney(HEK)-293T were transfected with FLOT2 mutant plasmids.Co-immunoprecipitation(Co-IP)was used to detect total acetylation levels of FLOT2 and the effects of specific lysine(K)site mutations on FLOT2 acetylation.Western blotting was used to detect FLOT2/FLAG-FLOT2 protein expression in TSAtreated nasopharyngeal carcinoma cells transfected with FLOT mutant plasmids,and realtime reverse transcription PCR(real-time RT-PCR)was used to detect FLOT2 mRNA expression.Nasopharyngeal carcinoma cells were treated with TSA combined with MG132 or chloroquine(CQ)to analyze FLOT2 protein expression.Cycloheximide(CHX)was used to treat HEK-293T cells transfected with FLAG-FLOT2(WT)or FLAG-FLOT2(K211R)plasmids to assess protein degradation rates.The BioGrid database was used to identify potential interactions between FLOT2 and HDAC6,which were validated by CoIP.HEK-293T cells were co-transfected with FLAG-FLOT2(WT)/FLAG-FLOT2(K211R)and Vector/HDAC6 plasmids,and grouped into FLAG-FLOT2(WT)+Vector,FLAGFLOT2(WT)+HDAC6,FLAG-FLOT2(K211R)+Vector,and FLAG-FLOT2(K211R)+HDAC6 to analyze the impact of K211R mutation on total lysine acetyl

关 键 词：浮舰蛋白2 赖氨酸乙酰化 鼻咽癌 组蛋白去乙酰化酶6 

分 类 号：R739.63[医药卫生—肿瘤]