Screening Of Biomarkers Related To Coronary Artery Disease Based On Bioinformatics

Background:Coronary artery disease（CAD）is a global medical problem.The prevalence of CAD in China is about 11.396 million,and the mortality continues the upward trend since 2012.Therefore,the prevention and treatment of the disease are facing enormous challenges.Studies have shown that the inflammatory markers have becoming the important indicators for predicting the occurrence and development of CAD,and the levels are associated with genetic and environmental factors.Using bioinformatics to identify differentially expressed genes（DEGs）related to CAD and explore the potential biological functions will be helpful to discover new biomarkers and treatments.However,the causal association between biomarker phenotypes and diseases cannot be confirmed due to the potential confounding factors.Mendelian randomization（MR）studies use genetic variants closely related to exposure factors as instrument variables（IV）to evaluate the potential causal relationships between exposures and CAD risks,overcoming the defects of causal timing and confounding factors in the traditional epidemiological study designs.In this study,key genes related to CAD risks were screened by bioinformatics analysis,and then the MR study design was used to explore the causal association between the key gene（Matrix metalloproteinase 9,MMP-9）and CAD risk,and the mediator effect of MMP9 in the relationships between blood cell count and CAD risk was also evaluated.Objectives:The key gene MMP-9 related to CAD risk was screened out based on bioinformatics analysis methods.Then,MR was used to design the MMP-9 gene rs3918242 locus as an instrumental variable to infer the association between MMP-9 levels and CAD risk.And the mediator effect analysis method explored the mediator effect of MMP-9 in the association between white blood cell count and CAD risk.Methods:1.Bioinformatics analysis:The target datasets were downloaded from the GEO online database,and the original data were read in R software（version 4.0.1）.Then,some preprocessing was performed,such as gene probe conversion,data normalization and data integration.Differential expression analysis was performed using the LIMMA package,and the DEGs were screened with|log2FC|>1 and P<0.05 as the threshold.Functional enrichment analysis was conducted by DAVID.The online database STRING was used to predict the protein interaction network of common DEGs.Then,the Cytoscape software was conducted for visualization analysis and screened out key genes.2.Mendelian randomization study design:A total of 1009 patients with CAD diagnosed and1014 controls were included for epidemiological investigation and blood sample collection.The serum MMP-9 level was determined by ELISA.Then,DNA was extracted,and the RFLP-PCR was used to detect the polymorphism of the MMP-9 rs3918242（C/T）.MMP-9rs3918242（C/T）was used as an instrumental variable,combined with MMP-9 levels,to verify the association between genotype-phenotype and genotype-disease outcome,and then infer the causality between related MMP-9 levels and CAD risk association.3.Mediation analysis:The relationship between WBC（X）and CAD（Y）and the relationship between MMP9 level（M）and CAD（Y）were analyzed.Linear regression was then used to analyze whether the white blood cell count（X）was associated with MMP-9 levels（M）.Finally,based on the Process 3.3 of SPSS 23.0 software,the mediation of MMP-9 in the association between white blood cell count（X）and CAD（Y）was analyzed.Results:1.A total of 283 differentially co-regulated genes were identified in this study,including 122up-regulated genes and 161 down-regulated genes.In the functional enrichment analysis,these differential genes were mainly enriched in the inflammatory response and immune response.KEGG analysis found 25 significant pathways,most of which were in the following categories:TNF signaling pathway,infectious diseases,and hematopoietic cell lines.Two key modules were identified through PPI network construction,and KEGG analysis showed that DEGs in the two key modules were mainly enriched in immune response and inflammatory response.Finally,seven key genes（JUN,MMP9,CXCL8,IL1B,PTGS2,ICAM1 and FN1）were screened out as possible new diagnostic and therapeutic targets for CAD.2.A total of 1009 cases of CAD and 1014 samples were finally included in the study.The results showed that the association between MMP-9 levels and the risk of CAD was significant.With the 1ng/ml increased of MMP9 level,the subjects had an 8.1%increased risk of CAD(e^0.078=1.081).The association between MMP-9 levels and CAD risk directly obtained by traditional case-control study design was also statistically significant.For every1ng/ml increased in MMP-9 levels,the risk of CAD had been increased by 1.5%among the subjects（OR=1.015,95%CI=1.01-1.02）.3.The study suggested that diabetes,hypertension,dyslipidemia,smoking,increased WBC,and increased MMP-9 level were the risk factors for CAD（P<0.05）.While,alcohol consumption,increased HDL-C,and increased red blood cell count,the increase in hemoglobin level was the protective factor of CAD（P<0.05）.After avoiding confounders,the risk of CAD was increased by 13%according to per 1.0×10⁹/L increased in WBC（OR=1.13,95%CI=1.07-1.20）.For every 1 ng/ml increased in MMP-9 level,the risk of CAD was increased by 2%among the subjects（OR=1.02,95%CI=1.01-1.02）.Then,WBC was significantly associated with increased MMP9 levels（β=0.346,P=0.039）.After avoiding confounders,mediation analysis showed that the total effect of MMP-9 was 0.127（95%CI=0.063-0.192）,OR=1.135（95%CI=1.065-1.212）.It indicated that the total risk of CAD increased by 14%for each 1.0×10⁹/L increased in WBC count.The direct effect was 0.121（95%CI=0.063-0.178）,OR=1.129（95%CI=1.065-1.195）.It suggested that each 1.0×10⁹/L increased in WBC count directly led to a 13%increase in the risk of CAD.The mediating effect was 0.007（95%CI=0.001-0.014）,OR=1.007（95%CI=1.001-1.014）.It suggested that in the risk of CAD caused by increased WBC,the risk of CAD increased by 0.7%for 1ng/ml increase in MMP-9.In conclusion,after avoiding confounders,MMP-9 still played a partial mediating role in the relationship between WBC count and CAD,with an effective ratio of5.18%.Conclusions:1.DEGs associated with CAD risk were mainly enriched in the inflammatory response and immune response pathways,and seven key genes（JUN,MMP9,CXCL8,IL1B,PTGS2,ICAM1,FN1）might be served as the potential diagnostic and therapeutic targets for CAD.2.A causal relationship between MMP-9 levels and CAD risk was found in the MR study.3.Mediation analysis found that MMP-9 played a partial mediation in the association between WBC count and CAD.