SH-SY5Y细胞中EZH2调控GFRα1启动子DNA甲基化的机制研究  

Regulatory effect of EZH2 upon GFRα1 promoter DNA methylation in SH-SY5Y cells

作　　者：赵凡[1] 周崇高[1] 彭琨 夏仁鹏[1] 马体栋[1] 许光[1] 邹婵娟[1] 李碧香[1] Zhao Fan;Zhou Chonggao;Peng Kun;Xia Renpeng;Ma Tidong;Xu Guang;Zou Chanjuan;Li Bixiang(Department of Neonatal Surgery,Hunan Children's Hospital,Changsha 410007,China)

机构地区：[1]湖南省儿童医院新生儿外科,长沙410007

出　　处：《中华小儿外科杂志》2024年第4期347-353,共7页Chinese Journal of Pediatric Surgery

基　　金：湖南省自然科学基金(2020JJ5284);湖南省出生缺陷协同防治科技重大专项(2019SK1010)。

摘　　要：目的探讨SH-SY5Y细胞中果蝇zeste基因增强子同源物2(enhancer of zeste homolog 2,EZH2)调控胶质细胞源性神经营养因子受体(glial cell linederived neurotrophic factor receptor,GFR)α1启动子DNA甲基化的分子机制。方法SH-SY5Y细胞中EZH2及DNA甲基转移酶(DNA methyltransferase,DNMT)3B过表达及干扰后,采用实时定量聚合酶链反应(real time fluorescent quantitative polymerase chain reaction,qRT-PCR)及蛋白质印迹法检测EZH2、DNMT3B、GFRα1表达水平,染色质免疫共沉淀qPCR检测转染各组DNMT3B启动子区EZH2结合水平及组蛋白H3第27位赖氨酸(H3K27)甲基化水平,亚硫酸氢盐测序检测转染各组GFRα1启动子DNA甲基化的程度。采用独立样本t检验比较两组间差异;多组数据的组内比较使用one-way ANOVA分析,并使用LSD-t检验进行两两比较。结果qRT-PCR及蛋白质印迹法显示,EZH2过表达组EZH2的mRNA及蛋白相对表达量高于过表达对照组,EZH2干扰组EZH2的mRNA及蛋白相对表达量低于干扰对照组(均P<0.05);DNMT3B过表达组DNMT3B的mRNA及蛋白的相对表达量高于过表达对照组;DNMT3B干扰组DNMT3B的mRNA及蛋白的相对表达量低于干扰对照组(均P<0.05)。染色质免疫共沉淀qPCR显示,EZH2过表达组DNMT3B启动子区EZH2结合水平为2.25±0.15,高于过表达对照组的1.00±0.05(t=-12.82,P=0.006);EZH2过表达组DNMT3B启动子区H3K27甲基化水平为2.47±0.44,高于过表达对照组的1.01±0.09(t=-6.17,P=0.025)。EZH2干扰组DNMT3B启动子区EZH2结合水平为0.57±0.08,低于干扰对照组的1.00±0.06(t=5.80,P=0.029);EZH2干扰组DNMT3B启动子区H3K27甲基化水平为0.31±0.09,低于干扰对照组的1.08±0.17(t=12.24,P=0.007)。qRT-PCR及蛋白质印迹法显示,EZH2过表达组DNMT3B的mRNA相对表达水平为0.58±0.09,低于过表达对照组的1.01±0.13(t=17.75,P=0.003);EZH2过表达组DNMT3B的蛋白质相对表达水平为0.32±0.06,低于过表达对照组的0.54±0.05(t=23.64,P=0.002)。EZH2干扰组DNMT3B的mRNA�Objective To explore the molecular mechanism of EZH2 regulating GFRα1 promoter DNA methylation in SH-SY5Y cells.Methods After EZH2/DNMT3B overexpression and interference in SH-SY5Y cells,the expression levels of EZH2,DNMT3B and GFRα1 were detected by quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot.EZH2 binding level and H3K27me3 in DNMT3B promoter were detected by chromatin immunoprecipitation assay-PCR(ChIP-qPCR).DNA methylation of GFRα1 promoter in all transfected groups was detected by BSP.Independent sample t-test was utilized for comparing the mean of two groups.Multiple groups of data were examined by one-way ANOVA and LSD test.Results qRT-PCR and Western blot results showed that mRNA and protein relative expression levels of EZH2 in EZH2 overexpression group were higher than those in overexpression control group,and those in EZH2 interference group were lower than those in interference control group(all P<0.05).The relative expression levels of mRNA and protein were higher in DNMT3B overexpression group than those in overexpression control group.The relative expression levels of DNMT3B mRNA and protein were lower in DNMT3B interference group than those in interference control group(all P<0.05).The results of ChIP-qPCR showed that EZH2 binding level was higher in DNMT3B promoter region of EZH2 overexpression group than that of overexpression control group[(2.25±0.15)vs(1.00±0.05),t=-12.82,P=0.006].Methylation level of H3K27 was higher in DNMT3B promoter region of EZH2 overexpression group than that of overexpression control group[(2.47±0.44)vs(1.01±0.09),t=-6.17,P=0.025].EZH2 binding level of DNMT3B promoter in EZH2 interference group was lower than that in control group[(0.57±0.08)vs(1.00±0.06),t=5.80,P=0.029].The methylation level of H3K27 was lower in DNMT3B promoter of EZH2 interference group than that of control group[(0.31±0.09)vs(1.08±0.17),t=12.24,P=0.007].qRT-PCR and Western blot results showed that the relative expression level of DNMT3B mRNA in EZH2 overexpres

关 键 词：先天性巨结肠 DNA甲基化 胶质细胞源性神经营养因子 

分 类 号：R726.5[医药卫生—儿科]