| BackgroundCoronary artery disease(CAD)is the primary cause of death throughout the world.The pathological basis of CAD is atherosclerosis(AS).The studies about AS began 100 years ago,yet its mechanism is not entirely clear.In recent years,the role of inflammatory cells and inflammatory cytokines has been paid more and more attention.CC chemokine ligand 17(CCL17)belongs to the CC chemokine subfamily.CCL17 secreted by macrophages in AS plaques can recruit more inflammatory cells while suppressing the function of regulatory T cells(Tregs)with antiinflammatory effects.Excessive amounts of CCL17 will make the proinflammatory and anti-inflammatory response imbalance within the atherosclerotic plaque,so that the plaque expands and becomes unstable.Hence CCL17 plays an important role in the onset of CAD.However,the relationship between CCL17 and CAD in human beings is unclear.Here,the relationship between serum CCL17 level and CAD incidence was studied.We also investigated the association between the single nucleotide polymorphisms(SNPs)of CCL17 gene and CAD,followed by the functional studies for the disease-associated SNPs.Our study is aimed to identify new biomarkers for detection of high risk population and new treatment methods for the disease.Materials and Methods1.Patients presenting to our center for coronary angiography between January 2013 and December 2013 were recruited for the study.Serum CCL17 levels were determined by enzyme-linked immunosorbent assay(ELISA).Atherosclerosis severity was assessed in each patient according to the Gensini score(GSS).2.Genotype data from the HCB panel(Han Chinese in Beijing,China)of the HapMap Project was used to determine patterns of linkage disequilibrium(LD)and select tag SNPs in CCL17.HAPLOVIEW 4.2 software was used to choose tag SNPs that capture common genetic variation in CCL 17.3.SNP genotyping of the study population was achieved by TaqMan probe method.4.The TRANSFAC software and the Jaspar database were used to perform bioinformatics analysis to identify the potential function of the disease-associated SNP rs223828.5.rs273828 was identified to be able to bind to transcription factor transcription factor-1(AP-1)and have the ability to regulate the transcription.We examed the transcriptional activity of the allele C or allele T of rs223828 by the luciferase reporter activity assay.6.The difference of AP-1 binding ability between allele C and allele T of rs223828 was analyzed by electrophoretic mobility shift assay(EMSA)and Super-shift experiment at the in vitro level.7.The difference of AP-1 binding ability between allele C and allele T of rs223828 was analyzed by chromatin immunoprecipitation assay(ChIP)and real-time quantitative PCR at the in vivo level.Results1.In total,971 consecutive patients were enrolled in this study,including 158 non-CAD patients and 813 CAD patients.CAD patients had higher serum CCL17 levels compared to patients without CAD.Meanwhile,there was a significant linear trend between serum CCL17 levels and the different CAD subtypes.Finally,serum CCL17 levels were positively associated with the GSS.2.According to the criterion for tag SNPs selection,5 tag SNPs(rs223895,rs4784805,rs9302690,rs223899,and rs223828)were selected,which could capture all SNP variation of CCL17.3.The allele T of rs223899 and the allele T of rs223828 were found to be associated with risk of CAD.The allele T of rs223828 was also associated with increased serum CCL17 levels.4.Bioinformatics analysis by Transfac software and the Jaspar database indicated that rs223828 was located at the binding site of AP-1.Furthermore,prediction programs revealed that the binding affinity of AP-1 for the effect allele T of rs223828 was higher than that of the allele C.5.The allele T of rs223828 significantly increased luciferase activity compared with the major allele C.6.EMSA experiment showed that the biotin probe containing allele T had a higher binding ability to one of the nucleoproteins than allele C.The Super-shift and ChIP experiments demonstrated that the polymorphism of rs223828 affected the binding ability of AP-1,in vitro and in vivo,respectively.ConclutionIn this study,we demonstrated that CAD patients had higher serum CCL17 levels compared to non-CAD patients.Serum CCL17 levels were positively associated with the severity of CAD.The allele T of CCL17 SNP rs223828 is associated with increased risk of CAD as well as higher serum CCL17 levels.It could directly influence CCL17 promoter activity and may be a potential AP-1 transcription factor binding site.Our study has certain limitations.The causal inference was verified by a case-control study;however,a prospective cohort study is needed to confirm the findings of this study.Although our in vitro studies evaluated the potential mechanism of the SNP,additional investigations are warranted to fully elucidate the mechanisms that underlie the association between rs223 828 and CAD risk.In the subsequent work,prospective cohort studies and cell or animal experiments would be conducted.CCL17 might be a new biomarker for screening high-risk population of CAD and a new therapeutic target. |
PDF Full Text Request