文章摘要
王慧杰，刘晓鸣，陈娇.难治性癫痫患儿外周血microRNAs的表达研究[J].神经损伤功能重建,2025,(5):260-265
难治性癫痫患儿外周血microRNAs的表达研究
Study on the Expression of MicroRNAs in the Peripheral Blood of Children with RefractoryEpilepsy
  
DOI：
中文关键词: 难治性癫痫  儿童  外周血  miRNAs  发病机制
英文关键词: refractory epilepsy  children  peripheral blood  miRNAs  pathogenesis
基金项目:徐 州 市 科 技 项 目 （基于高通量测序 的RNA-seq技术探 讨 miRNAs 在难治 性癫痫儿童中的表 达，No. KC20149）
作者单位
王慧杰，刘晓鸣，陈娇 徐州医科大学附属 徐州儿童医院神经 内科 
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中文摘要:
      目的：研究难治性癫痫患儿外周血中microRNAs（miRNAs）的差异表达。方法：收集癫痫患儿46例， 分为难治性癫痫组（R组）20例、癫痫控制良好组（C组）26例，另选取同期儿童保健科健康体检儿童为健康 对照组（N组）26例，运用高通量测序技术检测3组儿童外周全血中miRNAs的表达，DEseq2软件筛选差异 表达的miRNAs；同样条件R组、C组、N组重新各纳入30例儿童，采集外周血标本，运用荧光定量聚合酶链 反应（qRT-PCR）扩增技术验证目标miRNA在儿童外周血中的表达水平，miRanda、RNAhybrid在线数据库 预测差异表达miRNAs的靶基因并对候选靶基因进行基因功能（GO）分析和基因组百科全书（KEGG）通路 分析。结果：与N组相比，R组共有124个差异表达miRNAs，其中74个miRNAs上调，50个miRNAs下调，C 组共有66个差异表达miRNAs，其中21个miRNAs上调，45个miRNAs下调；与C组相比，R组共有337个差 异表达的miRNAs，其中197个miRNAs上调，140个miRNAs下调，其中差异性最明显的是miR-15a-5p（P= 9.00×10－8 , |log2(foldchange)|=2.93），对miR-15a-5p进行qRT-PCR扩增，发现其在3组儿童外周血中的表达差 异有统计学意义（P＜0.001），且与高通量测序结果一致，受试者工作特征曲线（ROC）显示miR-15a-5p在诊 断儿童难治性癫痫中具有良好的曲线下面积（AUC）值（0.884）、敏感度（90%）及特异度（83.3%）。对差异 miRNAs进行靶基因预测，并对候选靶基因进行GO分析和KEGG分析，显示miR-15a-5p可能参与调控儿童 RE的信号通路包括MAPK信号通路、cAMP信号通路、铂类耐药信号通路、p53信号通路、Ras信号通路、 Fc-γ-R介导的吞噬作用信号通路、神经变性疾病信号通路。结论：难治性癫痫患儿外周血中miRNAs的表 达谱与癫痫控制良好患儿及健康儿童存在明显差异，其中miR-15a-5p差异性表达最显著，并可能通过调控 多条通路参与癫痫的病理生理过程。
英文摘要:
      To investigate the differential expression of microRNAs (miRNAs) in the peripheral blood of children with refractory epilepsy. Methods: A total of 46 pediatric patients with epilepsy were enrolled and divided into a refractory epilepsy group (R group, n=20) and a well-controlled epilepsy group (C group, n= 26). Additionally, 26 healthy children undergoing routine health examinations in the pediatric healthcare department during the same period were included as the healthy control group (N group). High-throughput sequencing technology was used to detect the expression of miRNAs in the peripheral whole blood of children in the three groups, and DEseq2 software was employed to screen for differentially expressed miRNAs. Under the same conditions, an additional 30 children were enrolled in each of the R, C, and N groups, and peripheral blood samples were collected. Fluorescent quantitative polymerase chain reaction (qRT-PCR) amplification technology was used to validate the expression levels of target miRNAs in the peripheral blood of children. The miRanda and RNAhybrid online databases were utilized to predict the target genes of the differentially expressed miRNAs, and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted for the candidate target genes. Results: Compared with the N group, a total of 124 differentially expressed miRNAs were identified in the R group, including 74 upregulated and 50 downregulated miRNAs, while 66 differentially expressed miRNAs were found in the C group, with 21 upregulated and 45 downregulated. When compared with the C group, the R group exhibited 337 differentially expressed miRNAs, of which 197 were upregulated and 140 were downregulated. Among these, miR-15a-5p showed the most significant difference (P=9.00×10－8 , |log2(fold change)|=2.93). qRT-PCR amplification of miR-15a-5p revealed a statistically significant difference in its expression among the three groups of children (P<0.001), consistent with the high-throughput sequencing results. The receiver operating characteristic (ROC) curve demonstrated that miR-15a-5p had a good area under the curve (AUC) value (0.884), sensitivity (90% ), and specificity (83.3% ) in diagnosing refractory epilepsy in children. Target gene prediction for the differentially expressed miRNAs, along with GO and KEGG analysis of the candidate target genes, suggested that miR-15a-5p may be involved in regulating signaling pathways related to refractory epilepsy (RE) in children, including the MAPK signaling pathway, cAMP signaling pathway, platinum drug resistance signaling pathway, p53 signaling pathway, Ras signaling pathway, Fc-γ-R-mediated phagocytosis signaling pathway, and neurodegenerative disease signaling pathway. Conclusion: The expression profile of miRNAs in the peripheral blood of children with refractory epilepsy differs significantly from that of children with well-controlled epilepsy and healthy children. Among these, miR-15a-5p exhibits the most prominent differential expression and may participate in the pathophysiological process of epilepsy by regulating multiple pathways.
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