Experimental Study On Biological Diagnostic Markers Of Large Artery Atherosclerosis Subtype Of Acute Ischemic Stroke

Acute ischemic stroke(AIS)is a common neurological disease characterized by high morbidity and mortality.The diagnosis of this disease was mainly based on clinical manifestations combined with imaging examination.However,the diagnosis of patients with uncertain onset time,atypical symptoms,delayed or negative imaging examination was still difficult.The search for biomarkers in the early stages of large artery atherosclerosis subtypeacute ischemic stroke was helpful in shortening the following recanalization time and improving the outcome of patients.Atherosclerosis was considered to be one of the major causes of acute ischemic stroke,and the mortality and recurrence rate of large-artery atherosclerosis(LAA)acute ischemic stroke were high.Studies had shown that miRNA in patient plasma could be used as biomarkers for many diseases.However,there were few studies on the early expression of miRNA in the hyperacute cerebral infarction of large-artery atherosclerosis.And the molecular mechanisms of miRNA involved in the regulation of stroke pathological process were not fully understood.In this study,high-through put miRNA microarrays were used to screen miRNA for the expression of early large-artery atherosclerotic AIS,target gene prediction and bioinformatics analysis were performed on different expressed miRNA.And then to explore the biological processes these miRNA may be involved in,and to lay the experimental and theoretical basis for the diagnosis and treatment of large-artery atherosclerosis AIS.Objective:To search for biomarkers of hypersensitivity and specificity in the early stage of large-artery atherosclerosis AIS,and to further explore the biological effects that may be involved in the pathophysiological process of large-artery atherosclerosis AIS.Methods:1.The patients were selected from the 981 th Hospital of the Chinese People’s Liberation Army.12 cases of large-artery atherosclerosis AIS were classified by TOAST,13 cases of atherosclerosis,and 15 cases of healthy physical examination were used as control group.The microarray technique was used to detect the expression profile of miRNA in plasma of each group,and the miRNA specifically expressed by large-artery atherosclerosis AIS were screened.2.Target genes for specifically expressed miRNA were predicted by three target gene prediction databases,TargetScan,miRwalk and miRDB.3.The STRING database was used to obtain the protein interaction between the target genes of the different expressed miRNA.And the interaction network between the target genes was constructed using Cytoscape software.4.GO(Gene Ontology)functional analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis were performed by the target genes of the differently expressed miRNA through the DAVID database.5.The cortical neuron cells of Wistar rats were cultured,and miR-129-1-3p mimics transfection group,miR-129-1-3p inhibitor transfection group and negative control transfection group were established to verify miR-129-1-3p.Effects on neuronal axon growth and the expression level of the target gene Runt-related transcription factor 2(Runx2).6.The value of miRNA for the diagnosis of Large-artery atherosclerosis AIS was analyzed by ROC(Receiver operating characteristic curve)curve.Results:1.Compared with the healthy control group,27 miRNA were differentially expressed in the large-artery atherosclerosis AIS group,of which 20 miRNA were up-regulated and 7 miRNA were down-regulated.Likewise,compared with the atherosclerosis group,9 miRNA were differentially expressed in the large-artery atherosclerosis AIS group,including 5 up-regulated miRNA and 4 down-regulated miRNA.2.miR-129-1-3p,miR-4312,miR-5196-3p were specifically expressed in large-artery atherosclerosis AIS.The expression of miR-129-1-3p and miR-4312 were gradually increased in the atherosclerosis group and the Large-artery atherosclerosis AIS group.3.Target gene prediction results indicated that miR-129-1-3p targets 172 common genes,of which Runx2 was predicted to be the target gene of miR-129-1-3p.4.The protein interaction network showed that there was a wide relationship between miR-129-1-3p target genes.And Runx2 interacted with various target genes,suggesting that Runx2 may play a key role in molecular network of various biological processes.5.DAVID analysis revealed that the target genes of miR-129-1-3p were enriched into a variety of biological processes,cellular components,molecular functions and signaling pathways involved in axons and synapses.It was suggested that miR-129-1-3p may be involved in the regulation of neuronal axons and synaptic functions.6.Transfection experiments showed that after the overexpression of miR-129-1-3p in cortical neuronal cells of Wistar rats,neuronal axon extension was significantly inhibited,while Runx2 protein expression level was also reduced.7.The ROC curve showed that miR-129-1-3p and miR-4312 are sensitive and Specific of large-artery atherosclerosis AIS.When inhibiting the expression of miR-129-1-3p,the length of axon elongation was significantly increased,and the protein expression level of Runx2 was increased.Conclusion:1.miR-129-1-3p and miR-4312 may be biomarkers for the diagnosis of acute atherosclerotic acute ischemic stroke with good sensitivity and specificity.2.miR-129-1-3p may affect cortical neuron axon growth by targeting Runx2.3.Promoting axon growth of cortical neurons by inhibiting miR-129-1-3p overexpression may provide new ideas for the treatment of aortic atherosclerotic acute ischemic stroke.