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Screening of differential miRNAs and target genes associated with ischemic strokeObjective: This study used bioinformatics methods to integrate published strokes related mRNA and miRNA high-throughput data,analyze and predict miRNA-mRNA relationships using a variety of tools and algorithms,and then construct them with Cytoscape software.A bioinformatics regulatory network composed of miRNAs and mRNAs predicts that miRNAs are involved in the regulation of key molecular pathways in stroke.Methods: First,mRNA and miRNA expression data derived from ischemic stroke patients using second-generation sequencing technology were retrieved and downloaded from the GEO database(https://www.ncbi.nlm.nih.gov/geo/)using R software.Analysis,miRNA expression data were normalized to analyze the differentially expressed miRNAs,Gene ontology(GO)analysis of molecular functions,biological processes and cell groups through KEGG database,analysis of genomic sequences,functions and pathways information.Target genes for differentially expressed miRNAs were screened using a miRNA target gene prediction tool database.Results: Differentially expressed miRNAs and mRNA were screened by analyzing the miRNA and mRNA raw data associated with the included stroke patients.A total of 15 differentially expressed miRNAs were obtained by co-screening,of which 10 were significantly down-regulated and 5 were significantly up-regulated.A total of 1178 differentially expressed genes were screened,of which 550 were significantly down-regulated and 628 were significantly up-regulated.Using Cytoscape software,we have a network map of target genes composed of 971 relationships.Conclusion: This part of the preliminary screening of the above miRNA and key target genes is associated with the metastasis of stroke patients,providing some candidate targets for the mechanism of ischemic stroke onset,followed by further experimental confirmation of the results of the analysis,and in vitro culture of ischemic stroke animals.The model studies the mechanism of its regulation.To study the function of differentially expressed target genes,we separately performed GO function annotation and KEGG pathway enrichment analysis on 1178 differentially expressed genes,respectively.A total of 1026 genes were significantly enriched in GO function and KEGG pathway,of which the first 15 top significantly enriched GO function and KEGG pathway.Part ? Verification of stroke candidate miRNAsObjective: We used fluorescence real-time quantitative PCR to detect and analyze collected stroke samples to verify the expression and targeting relationship between miRNA and target genes,exploring the pathogenesis of stroke and find new molecular markers and potentials.Diagnostic or therapeutic targets.Methods: We recruited 60 patients(age 65.1 ± 8.4 years)from the neurology clinic and were diagnosed with ischemic stroke patients.General enrollment was performed on all enrolled subjects,including the date of birth,gender,and medical history,including history of hypertension,diabetes,and so on.Determination of biochemical indicators.The patient was extracted for RNA extraction and the expression of the gene was detected by qRT-PCR.Results: The ischemic stroke patients and the control group included in the analysis were 80 patients.Compared with the normal group,the ischemic stroke patients with hypertension and diabetes were significantly higher;the proportion of smoking and alcohol was also higher than the control group;biochemical index SBP,DBP,blood glucose,TG,HDL,LDL indicators were significantly worse than the control group(P<0.05).The differentially expressed miRNAs and the five key target genes with the highest connectivity were screened by qRT-PCR to detect the expression of some miRNAs and key target genes in the collected stroke blood.The results showed that the expression of NOTCH1,PIP4K2 B and DIP2 B was up-regulated in stroke patients compared with the normal group,while the expression of IGFBP5 and hsa-miR-424 was down-regulated.Conclusion: This part of the study initially indicated that the above miRNAs and key target genes are related to stroke rice,and provide some candidate targets for the mechanism of stroke onset.Further experiments will confirm the results of the analysis,and in vitro culture of cells and in-depth study Its regulation mechanism.Part three MIR-424 was involved in the molecular mechanism of ischemia stroke development.Objective: It's of great significance to investigate the novel targets of drugs for the treatment of ischemia stroke.In this study,we explored the neuroprotective role of miR-424 in oxygen glucose deprivation(OGD)-induced injuries in PC-12 cells.Methods: PC-12 cells were subjected to OGD stimulation to mimic ischemic injury.The expressions of miR-424 and mitogen activated protein kinase phosphatase-1(MKP-1)were altered by transient transfection with miR-424 mimic,miR-424 inhibitor,pEX-MKP-1,or sh-MKP-1.Cell counting kit-8(CCK-8)assay,flow cytometry,quantitative reverse transcription polymerase chain reaction(qRT-PCR)was conducted to respectively detect cell viability,apoptotic cells,and the expression of miR-424 and MKP-1.The protein expressions of several factors were determined by western blot.Meanwhile,relative luciferase activity assay was done to verify the predicted targets association.Results: OGD induced injury in PC-12 cells by suppressing cell viability and inducing apoptosis.OGD also induced the expression of miR-424 in PC-12 cells.Overexpression of miR-424 protected PC-12 cells from OGD-induced injury by increasing cell viability and decreasing apoptosis.MKP-1 was a direct target of miR-424 and its expression was negatively regulated by miR-424.Up regulation of expression of MKP-1 aggravated OGD-induced cell injury by inhibiting the expression of hypoxia inducible factor 1?(HIF-1?)and thus inhibiting the PI3K/AKT/mTOR pathways.Conclusions:miR-424 protected PC-12 cells from OGD-induced injury through direct suppression of MKP-1 expression,as MKP-1 promoted OGD-induced cell injury by inhibiting the expression of HIF-1? and PI3K/AKT/mTOR pathways. |
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