基于转录组数据探究MEF2D在癫痫中的作用机制  

Role and mechanism of MEF2D in epilepsy based on transcriptomic data

作　　者：韩登峰[1] 焦腾飞 热娜·阿不都萨拉木 HAN Dengfeng;JIAO Tengfei;Rena Abudusalamu(Department of Neurology,First Affiliated Hospital of Xinjiang Medical Univer-sity,Urumqi 830054,China)

机构地区：[1]新疆医科大学第一附属医院神经内科,乌鲁木齐830054

出　　处：《山西医科大学学报》2024年第10期1314-1323,共10页Journal of Shanxi Medical University

基　　金：新疆维吾尔自治区自然科学基金项目(2022D01C759)。

摘　　要：目的 探究肌细胞增强因子2D(myocyte enhancer factor 2D,MEF2D)在癫痫(epilepsy, EP)致病中的作用机制。方法选取小鼠海马神经元细胞(HT22)作为研究对象,对MEF2D选取3个基因位点(686、912、1283)进行慢病毒敲低转染,实验分为sh686组、sh912组、sh1283组以及空白载体对照(shNC)组,通过qRT-PCR和Western blot筛选出敲低成功的细胞,并对筛选出的细胞进行凋亡检测和RNA测序(RNA-seq),通过转录组数据筛选差异表达基因(DEG),检测发生的可调控选择性剪接事件(RASE)并得到可调控选择性剪接基因(RASG)。对DEG和RASG进行基因本论(GO)富集分析、京都基因与基因组百科全书(KEGG)通路富集分析。结果 qRT-PCR和Western blot结果显示,sh912组MEF2D敲低成功。流式细胞术检测发现,sh912组早期凋亡率明显高于shNC组(P<0.05)。对转录组数据分析共得到638个DEG;GO富集分析显示,DEG的生物学过程主要富集在糖酵解、糖代谢、氧化还原反应的调节等方面,KEGG通路主要富集在Nod样受体信号传导通路、肿瘤坏死因子(TNF)信号传导途径、缺氧诱导因子(HIF)-1信号传导途径、IL-17信号传导途径等通路。在之后的AS分析中,共检出1 265个差异性且受调控的选择性剪接事件(RASE),其中最主要的RASE为内含子保留(IR),其次为可变的5′剪切位点(A5SS)、可变的3′剪切位点(A3SS);GO富集分析显示,RASG的生物学过程主要富集在DNA修复、RNA剪切、蛋白质转运,KEGG功能通路富集在Rap1信号传导、PI3K/Akt信号传导、磷脂酰肌醇信号传导等通路;DEG和RASG的重叠基因共28个。结论 敲低MEF2D可以促进海马神经元细胞的凋亡;内含子保留是敲低MEF2D后发生的主要选择性剪切事件;MEF2D可能通过影响细胞的糖酵解、氧化还原反应等功能参与细胞凋亡反应。Objective To investigate the role of myocyte enhancer factor 2D(MEF2D)in the pathogenesis of epilepsy(EP).Methods Mouse hippocampal neuronal cell line HT22 was used as the research model.Three distinct gene loci(686,912,and 1283)within MEF2D were targeted for knockdown using lentiviral vectors.The study was divided into four groups:sh686 group,sh912 group,sh1283 group,and shNC group.RT‐qPCR and Western blot were performed to verify the successful knockdown of MEF2D.Cells with successful MEF2D knockdown were subjected to apoptosis detection.RNA‐seq was performed on the selected cells to identify differen‐tially expressed genes(DEGs),regulated alternative splicing events(RASE),and regulated alternative splicing genes(RASGs).Gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were per‐formed on both DEGs and RASGs.Results RT‐qPCR and Western blot results confirmed successful MEF2D knockdown in sh912 group.Flow cytometry analysis revealed a significantly higher rate of early apoptosis in sh912 group than in shNC group(P<0.05).RNA sequencing analysis identified 638 differentially expressed genes(DEGs).GO analysis of DEGs primarily enriched biological pro‐cesses related to glycolysis,carbohydrate metabolism,and regulation of redox reactions.KEGG pathway analysis highlighted signifi‐cant enrichment in pathways such as Nod‐like receptor signaling,tumor necrosis factor(TNF)signaling,hypoxia‐inducible factor(HIF)‐1 signaling,and IL‐17 signaling.A total of 1265 differentially RASEs were identified in the AS analysis,and intron retention(IR)was the most prevalent type,followed by alternative 5′splice site(A5SS)and alternative 3′splice site(A3SS).GO analysis of RASGs predominantly enriched biological processes related to DNA repair,RNA splicing,and protein transport.KEGG pathway analysis of RASGs highlighted significant enrichment in pathways such as Rap1 signaling,PI3K/Akt signaling,and phosphatidylinosi‐tol signaling.A total of 28 gene

关 键 词：癫痫 MEF2D 差异表达基因 选择性剪切 内含子保留 细胞凋亡 RNA测序 

分 类 号：R742.1[医药卫生—神经病学与精神病学]