| Background:Ischemic stroke,as a serious life quatity-threating illness,is one of the most common causes of death and disability in a worldwide,the disease is not only bringing a heavy burden to the family,social medical resources is facing the severe test epidemiological investigation and study found that morbidity is not only getting higher,and also is becoming younger.This disease currently has no efficient therapeutic strategy in clinic except for thrombolysis treatment in time window(3-4.5 hours).therefore,the stroke prevention and control have become the important subject to need to solve urgent due to irreversible functional neurons loss and neural tissue injury.Further study on the molecular mechanism of stroke development is the key to solve the problems of stroke treatment.With the further studies on this disease,the correlation between mi RNA and stroke disease has been gradually recognized in recent years.one-third of the human gene expressions are regulated by highly conserved miRNAs affiliated to different species and are involved in important biological regulatory processes such as proliferation and differentiation,autophagy and apoptosis.In addition,half of miRNAs in vertebrate homology with other species.These characteristics provide the basis for the exploration of the correlation between miRNA and disease,and also lay a good theoretical foundation for the smooth development of clinical work with transformation.In view of this,we inferred that(1)owe to the extensive biological functions of miRNAs in cells,the coaction of multiple miRNAs modeled must be play an important role in the cascade of stroke injuries;(2)the rapid development of high-throughput microarray technology has provided us with the possibility to study the molecular mechanism related to diseases at the genomic level.By studying the combined functions of multiple miRNAs in ischemic stroke,it has opened up a new way to study the mechanism of stroke injury;(3)it is feasible to screen the miRNA expression profiles in the ischemic penumbra between the mouse model group and the control group via data modeling in the bioinformatics technology and data mining the specific expression miRNAs related to cerebral ischemia;(4)the mechanism of differentially expressed genes in ischemic areas of brain tissue in stroke was explored through the construction of miRNAs/mRNA co-expression network,GO analysis,KEGG pathway analysis and dual-lucifer enzyme reporting experiment,it will lay a theoretical foundation for the discovery of new therapeutic targets.Objectives:In this study,based on the microarray data of mice suffering from cerebral ischemia reper-fusion injury in GEO database and advanced data processing and analysis methods,we mined expressed miRNAs significantlly,and constructed a data model related to the coordinated regulation of miRNAs on stroke progression used Weka software;and then the effect and partial mechanism of target miRNAs(miR-216,miR-124)and CADM2 synergistically regulating target genes on cerebral ischemiareper-fusion injury mice were preliminarily discussed from the cellular level and the animal level in order to further improve the molecular mechanisms of stroke occurrence development,to provide a new therapeutic target for clinical prevention and control of cerebral apoplexy.Methods:1.Download mice miRNAs chip data involved in cerebral ischemia-reperfusion injury in the GEO database and analysis data;build significant mode of miRNAs coordinated stroke progress related Using the Weka software;mine target genes of miRNAs using bioinformatics software and verify the relationship between miRNAs and target genes.2.The OGD model in vitro will be constructed;and then miRNAs mimic/ inhibitor were transfected into N2 a cells treated with OGD model in all experimental groups;Parallel detection of biological effects and functional analysis were conducted.3.MCAO model of mice to obtain the best modeling time;miRNAs into brain tissure in all experimental groups;At the same time the biological effects were detected and analyzed.Results:1.30 differentially expressed miRNAs related to ischemia reperfusion injury in mice,including miR-124 and miR-216,were obtained through microarray data and processing.the target miRNAs(miR-124/miR-216)associated with stroke disease and their potential common target gene CADM2 were mined through Weka software,and the potential pathways and functions were analyzed.2.N2 a cells were treated with oxygen and sugar deprivation(OGD),and then transfected and co-transfected with mimic/inhibitor of mir-216 and mir-124 to test the cell survival rate,cell apoptosis rate,ducting ability and expression of gene,protein and apoptosis pathway protein in each groups.We found that the high expression of miR-216 and/or miR-124 can increase the survival rate of cells and inhibit the occurrence of apoptosis.,the apoptosis of the stroke model cells was significantly reduced.Especially,survival rate of cells were statistical significance in co-transfection with mimic of miR-216/miR-124 groups.3.MCAO model was adopted and transfected with lentivirus vectors with overexpression of mirna-216/124.We found that mice with overexpression of mirna-216/124 had better neural function scores and less apoptosis,however CADM2 of target gene with overexpression of mirna-216/124 was statistical significance reduced.Conclusions:Through data mining and analysis of public databases,it was found that mirna-216/124 was involved in the development of stroke diseases.Based on the analysis of cell experiment,it was believed that miR-216 and miR-124 could activate the signaling pathway PI3K/AKT and negatively regulate the expression of common target gene CADM2 to improve the apoptosis of nerve cells.Animal experiment further validatied that miR-216/124 involved in nerve cells regulation of ischemia-reperfusion mice,and we speculated that miR-216/124 may have be negative regulated CADM2 involved in ischemia-reperfusion mice and give play to the role of the protection of the nerve cells,these results indicate that the miR-216/124 can be used as potential markers for stroke treatment of candidate drugs provides a new direction. |
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