The Clinical And Mechanism Study Of Serum MiR-186-5p As Novel Biomarkers For Acute Coronary Syndrome

BACKGROUD:Acute coronary syndrome(ACS)is a serious and potential life-threatening ischaemic heart disease and leads high morbidity and mortality worldwide.Percutaneous coronary intervention(PCI)is the mainstay of therapy to restore coronary perfusion and alleviate myocardial ischemia for ACS,Nevertheless,ACS patients are prone to occur major adverse cardiovascular events(MACE)after PCI yet.There is still a lack of highly sensitive and specific noninvasive biomarkers to monitor clinical condition and assess prognosis of ACS patients.MicroRNAs(miRNAs)are non-coding small RNAs of 19-23 nucleotides in length and have been well-established as pivotal post-transcriptional regulators of target gene expression.The latest studies demonstrate that circulating miRNA can be used as the novel approach for diagnosis and prognosis of ACS patients.Previous studies have revealed that serum miR-186-5p levels are significantly elevated in ACS patients and may be serve as a molecular marker for risk stratification of atherosclerotic cardiovascular diseases.However,the kinetic signatures of circulating miR-186-5p in ACS patients after PCI therapy remain unclear exactly,and the distribution pattern and their association with ACS prognosis still need exhaustive evaluations.Furthermore,the fundamental mechanism of miR-186-5p participated in the process of cardiomyocyte ischemia need to be further explored.OBJECTIVE:(1)To detect the dynamic change of serum miR-186-5p ACS patients before PCI,and to investigate their distribution pattern and clinical values for ACS.(2)To elucidate the underlying molecular mechanism of miR-186-5p participated in the physiological and pathological processes of myocardial ischemia.METHODS:(1)92 ACS patients and 96 healthy subjects were enrolled in this study.Serum miR-186-5p levels in controls and ACS patients on admission(before PCI)and at different time points after PCI treatment within 7 days were detected by quantitative reverse-transcription PCR(qRT-PCR).Exosomes were isolated from serum samples using a commercial kit,and miR-186-5p concentrations were determined and compared the differences in in exosomes and corresponding exosome-depleted supernatant.One-year follow-up were performed to evaluation and calculated the incidence of MACE in ACS patients after PCI.Moreover,ROC curve,Logistic regression analyses,Spearman correlation and Cox regression analyses were applied to assess the clinical values of serum miR-186-5p for ACS.(2)miR-186-5p target gene was predicted by bioinformatics software.In vivo experiments,the acute myocardial infarction(AMI)model of SD rats was induced by permanent left anterior descending(LAD)coronary artery ligation;and the miR-186-5p levels in serum and myocardial tissues of rats were measured and compared,meanwhile related protein levels were also detected.In vitro experiments,oxygen-glucose deprivation(OGD)models were established by combining Anaeropack and glucose-free medium without serum;and the concentrations of miR-186-5p and specific proteins were examined in H9C2 cells;Luciferase reporter gene experiments were used to verify the targeting relationship between miR-186-5p and predicted genes;Western Blot,flow cytometry and cell proliferation experiments were performed to explore and validate the underlying mechanism of miR-186-5p in processes of cardiomyocyte apoptosis induced by OGD.RESULTS:(1)Serum miR-186-5p levels were remarkably increased in patients with ACS(STEMI,NSTEMI,UA)than in the control group(all p<0.05),and there were no significant differences among these three ACS subgroups.The high serum miR-186-5p levels in ACS patients before PCI were significantly decreased at all time points within 7 days after PCI(all p<0.05),and returned to near control levels about 2 days after PCI(p>0.05).The miR-186-5p concentrations in isolated exosomes were obviously lower than those in corresponding exosome-depleted supernatant in controls and ACS patients,and the miR-186-5p contents in exosome-depleted supernatant were positively correlated with the miR-186-5p contents in total serum(r=0.238,p<0.05);unsurprisingly,there was no significant difference of exosomal miR-186-5p levels between ACS patients and control group(all p >0.05).Spearman correlation analyses showed that serum miR-186-5p levels in ACS patients at admission were positively correlated with Gensini scores(r = 0.208,p<0.05);and serum miR-186-5p level after PCI was positively related with GRACE score(r = 0.340,p<0.05)while negatively correlated with LVEF(r =-0.358,p<0.05).ROC curve and logistic analyses indicated that serum and exosome-depleted miR-186-5p can be served as independent risk factors for ACS presence.Within one-year follow-up,the incidence of MACE in ACS patients was 33.7%;and ACS patients with MACE exhibited higher miR-186-5p levels on admission than those without MACE;furthermore,ROC curve and Cox regression analyses validated the favorable prognostic values of serum miR-186-5p in ACS patients after PCI.(2)Bioinformatics software analyses demonstrated that extracellular regulated protein kinase(ERK1/2)may be the target gene of miR-186-5p.In vivo experiments,miR-186-5p levels in serum were elevated in AMI groups compared with sham group;(all p <0.05)however,miR-186-5p concentrations were down-regulated in myocardial tissues of AMI rats(p<0.05)while the levels of ERK1/2 and cleaved caspase-3 were upregulated(all p<0.05).In vitro experiments,when compared with control group,the cellular miR-186-5p levels were significantly decreased(all p<0.05)while ERK1/2 and cleaved caspase-3 levels were elevated(p<0.05)in OGD groups;and in extracellular medium,the levels of miR-186-5p in OGD groups were markedly increased(p<0.05).Relative levels of ERK1/2 in H9C2 cells showed opposite changes with miR-186-5p concentrations after transfected with miR-186-5p mimic or inhibitor.Luciferase reporter assays confirmed that miR-186-5p could down-regulate expression of ERK1/2 through binding to the complementary target sites in 3'-UTR of its mRNA.Results from Flow cytometry analyses showed that OGD promoted apoptosis in H9C2(p<0.05),and up-regulation of miR-186-5p remarkably restrained the apoptosis both in OGD and non-OGD cells(all p<0.05).CCK-8 assay showed that OGD markedly suppressed the growth of H9C2(p<0.05),whereas overexpression of miR-185-5p effectively enhanced the proliferation ability inhibited by OGD(all p<0.05).CONCLUSIONS: Serum miR-186-5p levels were significantly increased in ACS patients and gradually decreased within 7 days after PCI.Serum miR-186-5p was predominantly existed as exosome-free form,which might be passively released from injured cardiomyocytes.The serum levels of miR-186-5p were well correlated with the occurrence of MACE in ACS patients after PCI during one-year follow-up.Moreover,cellular miR-186-5p could regulate the apoptosis and proliferation of cardiomyocytes via targeting ERK1/2.The present study may provide a promising target for for clinical monitoring,prognosis assessment and treatment of ACS.