MeCP2诱导的视网膜色素上皮细胞转录组和m6A的改变  

MeCP2-induced alterations of transcriptome and m6A modification in retinal pigment epithelial cell

作　　者：张咏雅 李晓华 赵雪茹 李雪 Zhang Yongya;Li Xiaohua;Zhao Xueru;Li Xue(Department of Ophthalmology,Zhengzhou University People's Hospital,Henan Provincial People's Hospital,Henan Eye Hospital,Henan Key Laboratory of Ophthalmology and Visual Science,Henan Academy of Innovations in Medical Science,Zhengzhou 450003,China)

机构地区：[1]郑州大学人民医院眼科、河南省人民医院眼科、河南省立眼科医院、河南省眼科与视觉科学重点实验室、河南省医学科学院眼科研究所,郑州450003

出　　处：《中华实验眼科杂志》2024年第5期408-416,共9页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金面上项目(81770952)。

摘　　要：目的探讨重组人甲基CpG结合蛋白2(MeCP2)处理的视网膜色素上皮(RPE)细胞中mRNA和N6-甲基腺嘌呤(m6A)改变及其机制。方法将传代ARPE-19细胞贴壁培养后分为正常对照组和MeCP2组,正常对照组细胞采用正常培养液培养,MeCP2组细胞于含终质量浓度20 ng/ml重组人MeCP2蛋白培养液中,连续培养72 h。提取细胞内总RNA进行转录组测序(RNA-seq)和甲基化免疫共沉淀测序(MeRIP-seq)分析。采用edgeR软件包根据P<0.05筛选差异表达基因(DEGs)和差异甲基化基因(DMGs)。采用基因本体论(GO)富集分析对差异基因进行生物学功能描述,采用京都基因和基因组百科全书(KEGG)进行通路富集分析。筛选出DEGs与DMGs交集的基因,采用实时荧光定量PCR技术检测各组差异基因mRNA表达水平。结果共筛选出DEGs 100个,DMGs 7441个,富集分析发现DEGs与细胞外基质(ECM)-受体相互作用、细胞分裂、细胞周期和磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路等相关,DMGs与微管细胞骨架、血管生成、表皮生长因子受体(ErbB)信号通路、晚期糖基化终末产物(AGEs)-糖基化终末产物受体(RAGE)信号通路、哺乳动物雷帕霉素靶蛋白(mTOR)信号通路、Notch信号通路和转化生长因子β(TGF-β)信号通路等相关。DEGs中24个基因表达增加,76个基因表达减少;DMGs中5个基因含有高甲基化峰,7439个基因含有低甲基化峰,注释峰后,正常对照组有7626个基因发生m6A甲基化,MeCP2组有8006个基因发生m6A甲基化,2个组间有7360个交集基因。正常对照组和MeCP2组的m6A甲基化富集于转录本的CDS、内含子和3'-非翻译区(3'UTR)区域,其甲基化比例分别为23.62%/22.27%、48.53%/48.35%和23.66%/25.28%。联合分析发现2个上皮-间充质转化(EMT)相关基因CSPG 5和RBP1的mRNA和m6A水平均降低。荧光定量PCR结果显示,MeCP2组GSPG5、RBP1、ZNF484 mRNA相对表达量均明显低于正常对照组,差异均有统计学意义(t=7.885、7.613、Objective To investigate mRNA and N6-methyladenosine(m6A)changes in retinal pigment epithelium(RPE)cells treated with recombinant human methyl-CpG binding protein 2(MeCP2)and the mechanisms.Methods The passaged ARPE-19 cells were divided into normal control and MeCP2 groups after adhesion culture.Cells in the normal control group were continuously cultured in normal culture medium,and the cells in the MeCP2 group were cultured in culture medium containing a final concentration of 20 ng/ml of recombinant human MeCP2 protein for 72 hours.Transcriptomic sequencing(RNA-seq)and methylated RNA immunoprecipitation sequencing(MeRIP-seq)were used to extract and analyze total RNA.Differentially methylated genes(DMGs)and differentially expressed genes(DEGs)were screened using the edgeR software package based on P<0.05.The biological function of differential genes was determined by gene ontology(GO)enrichment analysis,and the pathway enrichment analysis was performed by Kyoto Encyclopedia of Genes and Genomes(KEGG).Intersection of genes between DEGs and DMGs were screened,and real-time fluorescence quantitative PCR was used to determine the mRNA expression levels of differential genes.Results A total of 100 DEGs and 7441 DMGs genes were screened.According to enrichment analysis,the DEGs were enriched to extracellular matrix(ECM)-receptor interaction,cell division,phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT)signaling pathway and so on.The DMGs were associated with microtubule cytoskeleton,angiogenesis,epidermal growth factor receptor(ErbB)signaling pathway,advanced glycation end-products(AGEs)-glycation end-products receptor(RAGE)signaling pathway,mammalian target of rapamycin(mTOR)signaling pathway,Notch signaling pathway and transforming growth factors-β(TGF-β)signaling pathway and so on.There were 24 up-regulated and 76 down-regulated DEGs.Five DMGs had hypermethylation peaks,and 7439 DMGs had hypomethylation peaks.After annotation of peaks,7626 genes in the normal control group and 8006 genes in the MeCP2 gr

关 键 词：增生性玻璃体视网膜病变 视网膜色素上皮细胞 MECP2 m6A MeRIP-seq 上皮-间充质转化 

分 类 号：R774.1[医药卫生—眼科]