利用生物信息学对缺血性脑卒中相关microRNA和mRNA的分析筛选与qRT-PCR验证

Bioinformatics analysis and qRT-PCR validation of ischemic stroke-related microRNA and mRNA

目的利用生物信息学分析筛选与缺血性脑卒中(IS)相关的microRNA(miRNA)和mRNA,通过实验鉴定关键基因、利用Cytoscape构建miRNA-mRNA相互作用网络,探讨其在IS中的作用及其主要调控的相关信号通路。方法从基因表达数据库(GEO)中下载微阵列数据集GSE58294(mRNA谱)、GSE16561(mRNA谱)和GSE43618(miRNA谱),对GEO2R鉴定差异基因和mRNA、DAVID数据库筛选得到的差异表达基因(DEGs)进行功能及通路富集分析,通过STRING构建差异基因的蛋白质-蛋白质相互作用(PPI)网络、MCODE插件从PPI中提取相互作用最密切的网络和关键基因,通过TargetScan预测差异表达miRNA(DEMs)的靶基因;利用氧糖剥夺/再灌注(OGD/R)细胞模型、实时荧光定量PCR(qRT-PCR)验证筛选得到的关键基因,利用蛋白免疫印迹(WB)检测铁死亡标志蛋白SLC2A3及GPX4的蛋白表达水平。结果共筛选到198个DEGs,基因本体(GO)及京都基因与基因组百科全书(KEGG)结果显示,198个基因主要参与Toll样受体信号通路、肌动蛋白细胞骨架调控及细胞凋亡;从PPI网络中鉴定出12个关键基因(CEACAM1、CKAP4、ITGAM、STOM、SLC2A3、ADAM8、DEGS3、MOSPD2、FPR2、MCEMP1、CLEC4D及ADGRG3)和26个DEMs;根据miRNA和mRNA的靶向关系构建miRNA-mRNA作用网络对12个关键基因进行了验证,除MCEMP1外,其余11个关键基因都随再灌注时间延长而上调;利用生物信息学分析人外周血样本发现,SLC2A3与GPX4呈负相关;WB结果显示,当SLC2A3表达上调时、GPX4表达下调(P<0.05)。结论IS发生后DGEs和DEMs表达上调,本研究共筛选到198个DEGs,确定了12个关键DEGs和26个DEMs,构建了12个miRNA-mRNA调控网络,这些基因可能是IS的发病的潜在调控机制,在炎症过程的发生中起着重要作用。

Objective To identify ischemic stroke(IS)-related microRNAs(miRNAs)and mRNAs using bioinformatics,validate key genes experimentally,construct miRNA-mRNA interaction networks,and elucidate their roles in IS and associated signaling pathways.Methods Microarray datasets GSE58294,GSE16561(mRNA profiles),and GSE43618(miRNA profile)were obtained from the Gene Expression Omnibus(GEO)database.Differentially expressed genes(DEGs)and mRNAs were identified using GEO2R.The DAVID database was employed for functional and pathway enrichment analyses of DEGs.A protein-protein interaction(PPI)network was constructed using STRING,and key genes were extracted using the MCODE plugin.TargetScan predicted target genes of differentially expressed miRNAs(DEMs).An oxygen-glucose deprivation/reoxygenation(OGD/R)cell model and quantitative real-time PCR(qRT-PCR)were used to validate key genes.Western blot analysis assessed protein expression of ferroptosis markers SLC2A3 and GPX4.Results The study identified 198 DEGs primarily involved in Toll-like receptor signaling,actin cytoskeleton regulation,and apoptosis.Twelve key genes(CEACAM1,CKAP4,ITGAM,STOM,SLC2A3,ADAM8,DEGS3,MOSPD2,FPR2,MCEMP1,CLEC4D,and ADGRG3)and 26 DEMs were identified from the PPI network.Eleven of the 12 key genes were upregulated with prolonged reoxygenation,except MCEMP1.Bioinformatics analysis of human peripheral blood samples revealed a negative correlation between SLC2A3 and GPX4.Western blot analysis confirmed that SLC2A3 upregulation corresponded with GPX4 downregulation(P<0.05).Conclusion This study identified 198 DEGs,12 key DEGs,and 26 DEMs upregulated after IS,constructing 12 miRNA-mRNA regulatory networks.These genes may represent potential regulatory mechanisms in IS pathogenesis,particularly in inflammatory processes.