| Part I Screening and Bioinformatics Analysis of Serum Exosomal miRNAs in Acute Myocardial InfarctionObjective:Acute myocardial infarction(AMI)is the primary cause of cardiac death.Searching novel diagnostic markers and therapeutic targets has always been the focus of clinicians.Previous studies have found that a variety of exosomal miRNAs increased after AMI and may be related to ventricular remodeling,suggesting that exosomal miRNAs have the potential to become a diagnosis and prognosis biomarker for AMI.Due to the large number of miRNAs and its complex regulatory network,this study aims to screen serum exosomal miRNAs and key genes in AMI patients by bioinformatics methods,so as to provide evidence for subsequent clinical trials and mechanism research.Methods:1.Consult GEO database and related literatures,screen the relevant data of exosomal miRNAs in AMI patients,and extract the differential miRNAs;2.Combined with miRDB and TargetScan websites,predict the target genes of selected miRNAs,and perform GO and KEGG enrichment analysis;3.Select the up-regulated miRNAs,combine with the literatures,further narrow the miRNA range,predict the target genes of the finally selected miRNAs,perform protein interaction network analysis of target genes by STRING software,and select the key genes by PPI analysis;4.Perform GO and KEGG enrichment analysis of the finally selected key genes,and use Cytoscape software to establish the network diagram of miRNA and target gene interaction.Results:1.A total of 7 studies were included,and 77 miRNAs with expression differences were extracted for bioinformatics analysis,of which 32 miRNAs were up-regulated and 45 miRNAs were down-regulated.2.A total of 12335 target genes of 77 miRNAs were predicted by the database,with a total of 2588 GO entries,mainly enriched in biological processes such as growth and development and signaling,and enriched in 150 KEGG pathways,mainly signaling pathways such as endocytosis,MAPK,PI3K-Akt,and Ras.3.Combined with literature reading,12 miRNAs were finally selected,followed by:miR-17-3p,miR-21-3p,miR-29a-3p,miR-30a-5p,miR-122-5p,miR-125a-5p,miR-126-3p,miR-146a-5p,miR-146b-5p,miR-150-5p,miR-199a-5p,and miR-214-3p.4.The target genes were predicted by miRDB database,and PPI network was constructed.After removing discrete genes,90 key genes were finally selected,enriched to 1571 GO entries,mainly enriched in extracellular matrix and structural assembly,forward regulation of cell adhesion and other biological processes,enriched to 154 KEGG pathways,mainly focal adhesion,PI3K-Akt,MAPK,Toll-like receptor,TNF and other signaling pathways.Conclusion:Twelve differentially expressed miRNAs and 90 key genes in exosomes of AMI patients were screened by bioinformatics,which were mainly involved in signal transduction,PI3K-AKT,MAPK,Toll-like receptor,TNF and other signaling pathways.Part Ⅱ Serum Exosomal MiR-146a-5p as a Diagnositic and Prognostic Biomarker for Acute Coronary SyndromeObjective:Acute coronary syndrome(ACS)is a clinical syndrome of acute ischemia caused by unstable plaque rupture of coronary artery,partial or complete blockage of blood vessels by thrombus.AMI is the most important clinical classification.Early diagnosis and timely treatment of ACS are very important to reduce its mortality and improve its long-term prognosis.Combined with the results of bioinformatics screening in the first part,we found that the expression level of plasma miR-146a-5p in AMI patients was significantly higher than that control group.After emergency interventional surgery,the expression level of plasma miR-146a-5p decreased and gradually increased after 72 hours,suggesting that miR-146a-5p may be involved in the occurrence and development of AMI.Therefore,this part aims to explore the value of serum exosomAL miR-146a-5p(exo-miR-146a-5p)as a diagnositic and prognostic biomarker for acute coronary syndromeMethods:1.Blood samples and general clinical data were collected from the selected patients;2.Exosomes were extracted from the serum of the patients and identified by transmission electron microscopy,nanoparticle tracking analysis,and Western blot(WB);3.Real-time PCR(RT-qPCR)was used to detect the expression levels of serum exo-miR-146a-5p;4.Enzyme-linked immunosorbent assay(ELISA)was used to determine the expression levels of serum interleukin 1β(IL-1β),interleukin 6(IL-6),and tumor necrosis factor-α(TNF-α)in the ACS control group;5.Receiver operating curve(ROC)was used to analyze the diagnostic value of serum exo-miR-146a-5p for ACS.6.Patients with ACS were followed up to record the incidence of major adverse cardiovascular events(MACE)within one year.The follow-up indicators included:cardiac death,heart failure,recurrent myocardial infarction,and re-revascularization events;7.ROC curve was used to analyze the value of serum exo-miR-146a-5p in the prognostic evaluation of ACS.Results:1.A total of 88 patients(63 in ACS group and 25 in control group)were enrolled.Except white blood cell,troponin I,N-terminal pro-B-type natriuretic peptide and creatinine score were statistically significant,there was no significant difference in other clinical indicators.2.Transmission electron microscopy revealed that the exosomes were round or round vesicular structures;nanoparticle tracking analysis suggested that the average diameter of the vesicles in the extracted samples was 83.18 nm;WB revealed that the exocrine body surface compliance marker proteins CD9,CD63,CD81,and HSP70.3.Serum exo-miR-146a-5p expression levels were significantly higher in ACS patients than in controls.4.The serum levels of IL-6,IL-1β,and TNF-α expression in ACS patients were significantly higher than those in the control group.5.Serum exosome miR-146a-5p has some diagnostic value in ACS patients.6.The one-year MACE event rate in ACS patients was 17.5%.7.Serum exo-miR-146a-5p has some predictive value for the occurrence of MACE events in ACS patients.Conclusion:Serum exosome miR-146a-5p can be used as a biomarker for the diagnosis and prognostic evaluation of ACS.Part Ⅲ Effect of Serum Exosomal MiR-146a-5p on Inflammatory Response and Apoptosis of H9C2 cells in Hypoxia/reoxygenation ModelObjective:Studies have shown that exosomal miRNA are involved in inflammatory response,cell migration,proliferation and apoptosis,and plays an important role in ACS and myocardial ischemia-reperfusion injury.The purpose of this study was to explore the effect of serum exo-miR-146a-5p on the biological behavior of H9c2 cardiomyocyte hypoxia reoxygenation(H/R)model.Methods:1.The H/R model of H9C2 cells was constructed,and the expression levels of miR-146a-5p were detected by RT-qPCR;the viability of cardiomyocytes was detected by CCK-8;the expression levels of IL-6,IL-1β,and TNF-α in the medium were detected by ELISA;and the apoptosis levels of cardiomyocytes were detected by flow cytometry and TUN EL immunofluorescence.2.Exosomes were extracted from the serum of healthy volunteers,and the expression levels of exosomes miR-146a-5p were modified using miR-146a-5p mimics,inhibitors,or negative control group(NC),and the expression levels of exosomes miR-146a-5p were detected by RT-qPCR after the end of treatment.3.The characteristics of PKH26-labeled serum exosomes taken up by H9C2 cells were observed under a fluorescence microscope.4.Serum exosomes from healthy volunteers or modified exosomes were co-cultured with H9C2 cells,myocardial cell injury was induced by H/R,and myocardial cell viability was measured using CCK-8 after the end of treatment;IL-6,IL-1β,and TNF-α expression levels in the medium supernatant were measured by ELISA;and myocardial apoptosis levels were measured by flow cytometry and TUNEL immunofluorescence.Results:1.Compared with normal cultured cells,the expression level of miR-146a-5p in H9C2 cells treated with H/R was significantly decreased,the cell viability was decreased,the expression level of inflammatory factors was increased,and the apoptosis was increased.2.After miR-146a-5pmimic or inhibitor transfection into serum exosomes,miR-146a-5p expression levels were significantly increased or decreased.3.Serum exosomes can be taken up by H9C2 cells.4.Compared with the H/R group,both the exosome group and the miR-146a-5pmimics exosome modification group had a protective effect on the damage of H9C2 cells after H/R,which enhanced cell viability,reduced inflammatory response,and reduced apoptosis,and the miR-146a-5pmimics exosome modification group had a more significant effect.Conclusion:Serum exosome miR-146a-5p has a protective effect on H/R injury in H9C2 cells,and the mechanism may be related to the alleviation of inflammatory response and the reduction of apoptosis.Part Ⅳ The Mechanism of miR-146a-5p Ameliorating H/R Injury in H9C2 Cells by Targeting IRAK1Objective:In the third part,we found that serum exo-miR-146a-5p had a protective effect on H/R injury of H9c2 cells,but the specific mechanism was unclear.Bioinformatics results suggest that IRAK1 is a key target gene of exo-miR-146a-5p and can be enriched in NF-κB signal pathway.Therefore,this part will combine IRAK1 and NF-κB signaling pathway to explore the molecular mechanism of serum exo-miR-146a-5p improving H/R injury of H9C2 cells.Methods:1.The potential target genes of miR-146a-5p were predicted using miRNA database,and the target genes were selected for validation in combination with literature reading.2.The direct targeting relationship between miR-146a-5p and IRAK1 was validated using a dual-luciferase gene reporter assay.3.IRAK1-overexpressing lentivirus was stably transferred into H9C2 cell line and validated by qPCR and WB.4.IRAK1-overexpressing lentivirus(ov-IRAK1)or unloaded lentivirus(NC)was co-transfected with miR-146a-5pmimic or inhibitor into H9C2 cells,and after H/R treatment,TLR-4/IRAK1/TRAF6/NF-κB signaling axis messenger RNA(mRNA)and protein expression levels were detected by qPCR and WB,and the protein expression levels of classical molecules(CleavedCaspase-3,Bcl-2,Bax)in the apoptosis pathway were detected by WB.Results:1.IRAK1 is one of the target genes of miR-146a-5p.2.Dual-luciferase gene reporter assay confirmed that:miR-146a-5p can directly target IRAK1.3.IRAK1-overexpressing lentivirus could be stably transferred into H9C2 cells,and after transfection,IRAK1 was significantly higher at both mRNA and protein levels.4.TLR-4/IRAK1/TRAF6/NF-κB signaling axis mRNA and protein expression in each group:①TLR-4 mRNA and protein expression levels in each group did not show significant differences;②Compared with the NC group,IRAK1 mRNA and protein expression levels were significantly decreased and NF-κB signaling pathway activation was inhibited in the miR-146a-5pmimics group;miR-146a-5pmimics group had the opposite biological effect;③miR-146a-5pmimics and IRAK1 overexpression lentiviral vector(mimics+ov-IRAK1 group)could reverse the protective effect of miR-146a-5pmimics on H/R in H9C2 cells after simultaneous transfection.5.Classical molecule(Cleaved Caspase-3,Bcl-2,Bax)protein expression in the apoptosis pathway in each group:① Compared with the NC group,the CleavedCaspase-3 and Bax protein expression levels were significantly decreased and the Bcl-2 protein expression level was significantly increased in the miR-146a-5pmimics group;the CleavedCaspase-3 protein expression level was significantly increased and the Bcl-2 protein expression level was significantly decreased in the miR-146a-5pinhibitor group;② Compared with the mimics group,the Bcl-2 protein expression level was significantly decreased in the ov-IRAK1+mimics group.Conclusion:1.MiR-146a-5p can directly target IRAK1,inhibit NF-κB pathway activation,and reduce the inflammatory response,thereby improving cell injury after H/R in H9C2 cells 2.MiR-146a-5p has an indirect regulatory effect on apoptosis after H/R in H9C2 cells,and the specific mechanism is unknown. |
PDF Full Text Request