| BackgroundAcute coronary syndrome(ACS)is a serious threat to human life and health.The vast majority of ACS is the inevitable result of unstable coronary atherosclerotic plaques.ACS treatment selection and prognostic evaluation are closely related to the severity of coronary lesions.However,nowadays the severity of coronary artery lesions is mainly assessed by coronary angiography,an invasive and expensive test.It is difficult to popularize coronary angiography due to its high risk.Therefore,novel biomarkers are needed to evaluate the severity of coronary artery lesions simply and effectively.MicroRNA(miRNA)is a class of endogenous noncoding single stranded smallRNA molecules with a length of about 22 nucleotides and high genetic stability.MiRNA is ubiquitous and plays important roles in life activities,closely related to tumors,metabolic diseases,cardiovascular diseases,cerebrovascular diseases and infectious diseases.Recent studies have shown that miRNA is expected to be a biomarker for diagnosis and evaluation of diseases such as tumors and cardiovascular diseases.As prominent members of the miRNA family,miRNA-221/222 could inhibit the proliferation,migration and differentiation of endothelial cells and promote the proliferation,migration and dedifferentiation of vascular smooth muscle cell(VSMC)to produce the progression of coronary atherosclerosis.It was confirmed that the levels of miRNA-221/222 are elevated in patients with coronary atherosclerotic disease(CAD).However,there is no further verification of associations of circulating miRNA-221/222 with the severity of coronary artery lesions in ACS patients.In this study,we applied real-time fluorescent quantitative PCR(RT-qPCR)to detect the relative expression levels of the circulating miRNA-221/222 in patients with ACS and the control group respectively.The correlation of the circulating miRNA-221/222 and the severity of coronary artery lesions were analyzed in this study.We also investigated the feasibility that the circulating miRNA-221/222 predicts the risk of ACS as biological markers,and discussed its possible molecular mechanism and provided evidence for further basic experiment and clinical application.Objective(1)To compare the relative expression levels of circulating miRNA-221/222 between ACS patients and control subjects in this case-control study.(2)To analyze the correlations between miRNA-221/222 and the severity of coronary artery lesions and to investigate the associations of miRNA-221/222 with occurrence of ACS.(3)To explore the feasibility of miRNA-221/222 as biomarkers for ACS risk prediction.Subjects and methodsThis study collected continuously 500 cases with coronary angiography in Qilu Hospital of Shandong University from October 2016 to October 2017.A total of 338 cases were screened strictly according to inclusion criteria and exclusion criteria,which consisted of 267 patients of ACS(including 191 patients of UA and 76 patients of AMI)and 71 cases of control group.Taking 5 ml artery blood from the right radial artery or right femoral artery before heparinization in coronary angiography and extracting the total RNA from plasma.RT-qPCR was used to test the expression levels of circulating miRNA-221/222.SPSS 19.0,GraphPad Prism 5.0 and MedCalc 15.2 for statistics were applied for the date analysis.The distribution of continuous variables expressed with the mean standard deviation(mean ± SD).The independent sample t test or nonparametric rank sum test was used to compare the differences between two groups.One-way ANOVA was used to compare the differences among more samples.The distribution of classification variables expressed with composition ratio.Chi-square test was used for the comparison of the differences between two groups or among more samples.Pearson or Spearman rank correlation was used to analyze the correlation between two variables.Multivariate linear regression was used to analyze the linear relationship between circulating miRNA-221/222 and Gensini score.Logistic regression model was used to analyze the correlation between circulating miRNA-221/222 and the occurrence of ACS.Receiver operating characteristic curve(ROC curve)analysis was used to evaluate the predictive value of circulating miRNA-221/222 for coronary artery stenosis>50%.Results(1)Comparison of the relative expression levels of circulating miRNA-221/222:The circulating miRNA-221/222 relative expression levels in ACS patients were significantly higher than those in control group(P<0.05).MiRNA-221/222 in the UA group were significantly higher than those in control group(P<0.05),and so did the levels in the AMI group(P<0.05).However,there was no significant difference in miRNA-221/222 between the UA group and the AMI group(P>0.05).(2)Comparison of the relative expression levels of circulating miRNA-221/222 after controlling confounding factors:The circulating miRNA-221 relative expression level was positively correlated with alanine aminotransferase(ALT),diuretics,nitrates and low molecular weight heparin(LMWH)and negatively correlated with high density lipoprotein(HDL),?-blocker and statin(P<0.05).Controlling those confounding factors,miRNA-221 in ACS patients was still significantly higher than those in control group(P<0.05).MiRNA-221 in UA group was still significantly higher than those in control group(P<0.05).However,there were no significant differences in miRNA-221 between the control group and the AMI group as well as between the UA group and the AMI group(P>0.05).The circulating miRNA-222 relative expression level was positively correlated with hypertension,nitrates and LMWH and negatively correlated with male,smoking,HDL and P-blocker(P<0.05).Controlling those confounding factors,miRNA-222 in ACS patients was still significantly higher than those in control group(P<0.05).MiRNA-222 in AMI group was still significantly higher than those in control group as well as than in UA group(P<0.05).However,there was no significant difference in miRNA-222 between the control group and the UA group(P>0.05).(3)The correlations between circulating miRNA-221/222 relative expression levels and Gensini score:Stepwise multivariate linear regression analysis was performed to determine the predictive variables of Gensini score among clinical baseline,CAD risk factors,laboratory tests,medication and miRNA-221 which showed that age,previous myocardial infarction,white blood cell(WBC),Clopidogrel/Ticagrelor,angiotensin converting enzyme inhibitor(ACEI)/angiotensin receptor blocker(ARB),?-blocker and miRNA-221 entry regression equation,indicating that miRNA-221(?=0.100,P=0.041)was positively linear correlated with Gensini score.Similarly,miRNA-222(?=0.100,P=0.047)was positively linear correlated with Gensini score.(4)The associations of the circulating miRNA-221/222 relative expression levels and diseases occurrence:?Stepwise multivariate Logistic regression analysis was performed to determine the predictive variables of ACS among sex,age,systolic blood pressure(SBP),diastolic blood pressure(DBP),smoking,hypertension,diabetes,CAD family history,total cholesterol(TC),triglyceride(TG),HDL,low density lipoprotein(LDL),fasting blood glucose(FBG),homocysteine(Hcy)and miRNA-221,which showed that age,smoking,diabetes,CAD family history and miRNA-221 were the independent predictors,indicating that miRNA-221 was the risk factor for ACS(OR:1.159,95%CI:1.033-1.299,p=0.012).Similarly,miRNA-222 was the risk factor for ACS(OR:1.132,95%CI:1.024-1.251,P=0.015).?Stepwise multivariate Logistic regression analysis was performed to determine the predictive variables of UA and AMI,indicating that miRNA-221 was the risk factor for UA(OR:1.154,95%CI:1.031-1.290,P=0.012)and AMI(OR:1.153,95%CI:1.030-1.290,P=0.013).Similarly,miRNA-222 was the risk factor for UA(OR:1.120,95%CI:1.018-1.232,P=0.020)and AMI(OR:1.132,95%CI:1.028-1.245,P=0.011).(5)The diagnostic value of the circulating miRNA-221/222 for coronary artery stenosis?50%:ROC curve analysis was used to evaluate the diagnostic value of circulating miRNA-221/222 for coronary artery stenosis?50%.It showed that the area under curve(AUC)and the 95%confidence interval(CI)of miRNA-221/222 were respectively 0.605(0.550-0.657)and 0.643(0.589-0.694),and the differences were statistically significant.Conclusion(1)The circulating miRNA-221/222 relative expression levels in ACS patients were significantly higher than those in control group.(2)The circulating miRNA-221/222 relative expression levels were significantly positively associated with the severity of the coronary artery lesions.(3)The circulating miRNA-221/222 relative expression levels were significantly associated with the occurrence of ACS,and may become biomarkers for ACS risk prediction.(4)The circulating miRNA-221/222 relative expression levels were expected to be novel biomarkers for the diagnosis of coronary artery stenosis>50%. |
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