甜菜碱对猪胎儿成纤维细胞m6A甲基化修饰的影响  

Effect of betaine on m6A methylation modification in porcine fetal fibroblasts

作　　者：陈梦佳 李思佳 闫茜 王燕新 陈莫斯楠 石德顺[1] 陆凤花[1] CHEN Mengjia;LI Sijia;YAN Xi;WANG Yanxin;CHEN Mosinan;SHI Deshun;LU Fenghua(State Key Laboratory of Subtropical Conservation and Utilization of Agricultural Biological Resources,College of Animal Science and Technology,Guangxi University,Nanning 530004,China)

机构地区：[1]广西大学动物科学技术学院,亚热带农业生物资源保护与利用国家重点实验室,广西南宁530004

出　　处：《中国兽医学报》2023年第12期2554-2560,共7页Chinese Journal of Veterinary Science

基　　金：广西研究生教育创新计划资助项目(YCSW2022116);国家自然科学基金资助项目(32160788)。

摘　　要：旨在探究甜菜碱对猪胎儿成纤维细胞(PFFs)m6A甲基化修饰的影响,以期揭示甜菜碱与PFFs RNA表观遗传修饰的关系。取适宜体长的胎猪分离PFFs,添加不同浓度甜菜碱培养PFFs不同时间,利用CCK-8检测细胞增殖活性;并通过免疫荧光染色法检测细胞整体m6A甲基化修饰水平;采用qRT-PCR检测细胞凋亡相关基因(Bcl,Bax)、m6A甲基转移酶基因(METTL3,METTL14,WYAP)、去甲基酶基因(ALKBH5,FTO)和阅读蛋白(YTHDC1,YTHDF1,YTHDF2)的表达情况。结果表明,添加20,50 mmol/L甜菜碱对细胞凋亡和增殖活性影响最小,高于100 mmol/L甜菜碱可抑制细胞增殖。免疫荧光染色结果显示,甜菜碱可显著提高PFFs中m6A修饰整体水平(P<0.05),且以剂量依赖模式增加m6A水平。qRT-PCR结果显示,20 mmol/L甜菜碱可抑制细胞凋亡,且显著改变m6A修饰酶基因的表达水平(P<0.05)。综上所述,甜菜碱通过改变m6A相关酶的表达,从而有效提升PFFs m6A甲基化修饰水平,为进一步挖掘RNA表观遗传修饰与体细胞克隆供体细胞重编程的关系提供理论依据。To explore the effect of betaine on m6A methylation modification of porcine fetal fibroblasts(PFFs)to reveal the intrinsic relationship between betaine and PFFs RNA epigenetic modification.In this experiment,PFFs were isolated from fetal pigs,different concentrations of betaine were added to treat PFFs at different times,and cell proliferation activity was detected by CCK-8.The overall level of m6A methylation modification of cells was detected by cellular immunofluorescence staining.qRT-PCR was used to detect the expression of apoptosis-associated gene(Bcl,Bax),m6A methyltransferase gene(METTL3,METTL14,WYAP),demethylase gene(ALKBH5,FTO)and reading protein(YTHDC1,YTHDF1,YTHDF2).The results showed that the addition of 20 and 50 mmol/L betaine had the least effect on the proliferative activity of apoptotic cells,and more than 100 mmol/L betaine inhibited cell proliferation.Cytoimmunofluorescence staining showed that betaine significantly increased the overall m6A modification level(P<0.05)in PFFs and increased m6A levels in a dose-dependent mode.qRT-PCR results showed that 20 mmol/L betaine inhibited apoptosis and significantly changed the expression level of m6A modifying enzyme gene(P<0.05).In summary,betaine effectively increases the level of m6A methylation modification of PFFs by changing the expression of m6A-related enzymes.This study discusses the epigenetic modification of PFFs RNA,which provides a new theoretical basis and practical basis for further improving the cloning efficiency of pig somatic cells.

关 键 词：甜菜碱 猪胎儿成纤维细胞 m6A甲基化 

分 类 号：S852.21[农业科学—基础兽医学]