失巢凋亡相关基因在脑梗死中的表达及电针治疗的可能机制研究

The potential therapeutic mechanism of electroacupuncture treatment in ischemic stroke by improving anoikis

摘要:

目的 探讨失巢凋亡相关基因和microRNA（miRNA）在脑梗死中的作用，并探索电针治疗的潜在机制。方法 从GSE16561和GSE22255数据集中收集脑梗死患者和对照组的mRNA表达数据，通过基因集变异分析（GSVA）和基因集富集分析（GSEA）评估失巢凋亡的活化状态，并利用差异表达分析鉴定差异表达mRNAs（DEmRs）。结合Lasso回归、随机森林和XGBoost模型筛选与脑梗死诊断相关的关键基因，并绘制受试者工作特征（ROC）曲线评估其诊断效能。同时，从GSE110993数据集中获取microRNAs表达数据，鉴定DEmiRs，并通过ENCORI和miRtarbase数据库预测调控关键基因的候选miRNAs，与DEmiRs进行交集分析。此外，复制大鼠大脑中动脉栓塞模型并收集脑组织样本，分为对照组、模型组和模型+电针治疗组，通过TTC染色、酶联免疫吸附试验、实时荧光定量聚合酶链反应和Western blotting检测脑损伤程度、炎症因子水平、失巢凋亡相关基因及蛋白的表达变化。结果 GSVA和GSEA表明失巢凋亡在脑梗死中活化。共鉴定出27个失巢凋亡相关DEmRs，并通过多模型分析筛选出踝蛋白1（TLN1）、丙酮酸脱氢酶激酶4（PDK4）、p21活化激酶2（PAK2）和肉碱棕榈酰转移酶1A（CPT1A）对脑梗死具有诊断价值，其曲线下面积均较高。在miRNA层面，预测了hsa-miR-369-3p调控TLN1、PDK4和PAK2，hsa-miR-26b-5p调控PAK2，hsa-miR-20a-5p和hsa-miR-17-5p调控PDK4。与对照组比较，模型组大鼠脑损伤和炎症因子水平升高，hsa-miR-369-3p、hsa-miR-26b-5p、hsa-miR-20a-5p和hsa-miR-17-5p的表达降低，而TLN1、PDK4、PAK2及失巢凋亡相关蛋白水平升高（P <0.05）。与模型组比较，模型+电针治疗组显著缓解了脑缺血损伤，上述miRNAs表达升高，而TLN1、PDK4、PAK2及相关蛋白的表达降低（P <0.05）。结论 失巢凋亡在脑梗死病理过程中发挥重要作用，电针治疗可能通过调控失巢凋亡相关miRNAs及靶基因表达改善脑缺血损伤，为脑梗死的治疗提供了新思路。

Abstract:

Objective To investigate the role of anoikis-related genes and microRNAs (miRNAs) in cerebral infarction and explore the potential mechanism of electroacupuncture therapy.Methods mRNA expression data from patients with cerebral infarction and control subjects were collected from the GSE16561 and GSE22255 datasets. The activation status of anoikis was evaluated using Gene Set Variation Analysis (GSVA) and Gene Set Enrichment Analysis (GSEA), and differentially expressed mRNAs (DEmRs) were identified through differential expression analysis. Key genes related to the diagnosis of cerebral infarction were screened by combining Lasso regression, random forest, and XGBoost models, and receiver operating characteristic (ROC) curves were plotted to assess their diagnostic efficacy. Meanwhile, miRNA expression data were obtained from the GSE110993 dataset to identify differentially expressed miRNAs (DEmiRs). Candidate miRNAs regulating the key genes were predicted using the ENCORI and miRtarbase databases, and intersection analysis was performed with DEmiRs. In addition, a rat middle cerebral artery occlusion (MCAO) model was established, and brain tissue samples were collected. The rats were divided into a control group, a model group, and a model + electroacupuncture treatment group. The degree of brain injury, levels of inflammatory factors, and expression changes of anoikis-related genes and proteins were detected using 2,3,5-triphenyltetrazolium chloride (TTC) staining, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.Results GSVA and GSEA analyses showed that anoikis was activated in cerebral infarction. A total of 27 anoikis-related DEmRs were identified, and through multi-model analysis, TLN1, PDK4, PAK2, and CPT1A were screened out as having diagnostic value for cerebral infarction, with high area under the curve values. At the miRNA level, hsa-miR-369-3p was predicted to regulate TLN1, PDK4, and PAK2; hsa-miR-26b-5p to regulate PAK2; and hsa-miR-20a-5p and hsa-miR-17-5p to regulate PDK4. Compared with the control group, the MCAO model group showed increased brain injury and levels of inflammatory factors, decreased expression of hsa-miR-369-3p, hsa-miR-26b-5p, hsa-miR-20a-5p, and hsa-miR-17-5p, and increased levels of TLN1, PDK4, PAK2, and anoikis-related proteins (P < 0.05). Compared with the model group, the model + electroacupuncture treatment group showed alleviated cerebral ischemic injury, increased expression of the aforementioned miRNAs, and decreased expression of TLN1, PDK4, PAK2, and related proteins (P < 0.05).Conclusion Anoikis plays an important role in the pathological process of cerebral infarction. Electroacupuncture therapy may improve cerebral ischemic injury by regulating the expression of anoikis-related miRNAs and their target genes, providing a new idea for the treatment of cerebral infarction.