Differential Expression And Correlation Analysis Of Global Transcriptome For Hemorrhagic Transformation After Acute Ischemic Stroke

Objective:The purpose of this study is to explore the differentially expressed miRNA,mRNA,lncRNA and circRNA in peripheral blood of patients with hemorrhagic transformation（HT）after acute ischemic stroke and those without hemorrhagic conversion by full transcriptome sequencing technique,and to explore the complex interaction between related transcripts and related signal pathways,so as to obtain the possible molecular mechanism of hemorrhagic transformation after stroke,so as to provide a research basis for the early diagnosis and potential therapeutic targets of HT.Methods:In this study,we selected 6 patients with acute ischemic stroke diagnosed as acute macrovascular occlusion and treated with interventional thrombectomy in the Department of Emergency Stroke,affiliated Hospital of Jining Medical College from October 2020 to June 2021.The patients were divided into two groups according to the results of cerebral CT 24 hours after operation:hemorrhagic group（HT group,cerebral CT indicated hemorrhagic transformation）,non-hemorrhagic group（non-HT group,cerebral CT did not indicate hemorrhagic transformation）.The venous blood 5mL of the above subjects was extracted by EDTA anticoagulant tube,and the human peripheral blood mononuclear cells were isolated by Ficoll-Hypaque gradient method,and then the total RNA was extracted.The differential expression of mRNA,lncRNA,circRNA and miRNA in HT group and non-HT group was analyzed by full transcriptome sequencing.The differentially expressed genes were functionally annotated by GO enrichment and KEGG pathway enrichment analysis.Arraystar data,TargetScan and miRanda were used to predict the interaction among mRNA,lncRNA,circRNA and miRNA.Combined with Cytoscape software to visually display the map of ceRNA regulatory network,the pathogenesis and related signal pathways of HT were discussed from the perspective of lncRNA/circRNA-miRNA-mRNA interactive regulation.Finally,we verified the differentially expressed genes by real-time fluorescence quantitative PCR.Results:（1）In this study,compared with non-HT group,there were 2575 differential expression of mRNA,1051 differential expression of lncRNA,447 differential expression of circRNA and 47 differential expression of miRNA in patients with HT.（2）The results of GO enrichment and KEGG pathway enrichment analysis suggest that ubiquitin-mediated proteolysis,MAPK signal pathway,axon guidance,HIF-1 signal pathway,NOD-like receptor signal pathway,β-alanine metabolism,Wnt signal pathway,sphingolipid signal pathway,neuroactive ligand-receptor interaction and intestinal immune network used in IgA production may play an important role in hemorrhagic transformation.Terms such as iron homeostasis,defense response,immune system process,DNA conformational change,production of transforming growth factor β-2 and oxidoreductase activity are enriched in the gene list,suggesting a potential correlation with hemorrhagic transformation.（3）Through the construction of co-expression network,we have obtained a total of 261 lncRNA-miRNA relationship pairs,of which 29 lncRNA have established a relationship with hsa-miR-365a-5p,and a total of 21 circRNA-miRNA relationship pairs have been obtained,of which 8 circRNA are linked to hsa-miR-708-5p.5 circRNA and 13 lncRNA were selected as ceRNA,which competed with miRNA in the co-expression network,which could be associated with the occurrence of hemorrhagic transformation by regulating the function of mRNA in the network.（4）The results of qRT-PCR showed that hsa_circ₀005954、hsa_circ₀008216、hsa_circ 0004849、hsa_circ₀001680、hsa_circ₀093655、hsa_circ₀000647 had a statistical difference（P<0.05）and was consistent with the sequencing results.Conclusion:There are a large number of differentially expressed mRNA,lncRNA,circRNA and miRNA in peripheral blood of patients with and without hemorrhagic transformation after acute ischemic stroke.The results of GO enrichment and KEGG pathway enrichment showed that MAPK signal pathway,HIF-1 signal pathway,Wnt signal pathway and NOD-like receptor signal pathway may be involved in the occurrence of hemorrhagic transformation.The mutual regulation of lncRNA/circRNA-miRNA-mRNA may play a role in the occurrence of HT.The differentially expressed genes were verified by qRT-PCR,and the results were consistent with the results of sequencing.