Association And Mechanism Study Of The Untranslated Region Genetic Variations In TRIB1, SLC22A3 Gene With Coronary Heart Disease In The Chinese Han Population

PartⅠ Association study of the untranslated region genetic variations in TRIB1, SLC22A3 gene with lipids and coronary heart disease in the Chinese Han populationPurpose: To investigated the association of the untranslated region genetic variations in TRIB1(rs3201475), SLC22A3(rs539298 、 rs1810126 、rs3088442 and rs2076828) with lipids level, risk and severity of coronary heart disease(CHD) in the Chinese Han population.Methods: This study employed the case-control study method. We collected the CHD patients’ and controls’ blood, epidemiological and clinical data in the hospitals with strictly obeying the advanced established inclusion and exclusion criteria. A total of 300 CHD patients and 300 controls were enrolled in the study. We got the lesion site and lesion number information and then calculated the Gensini score according to the results of coronary angiography in CHD patients. Five SNPs(rs3201475 in TRIB1, rs539298、rs1810126、rs3088442 and rs2076828 in SLC22A3) were genotyped by the Sequenom mass array system. The Haploview program was applied to estimate the linkage disequilibrium(LD) measures(D’ and r2) between paired SNPs. Haplotype analysis was performed with Phase 2.0 software. The SPSS 20.0 statistical software was applied to complete the general information, the Hardy-Weinberg test, and the association of SNPs with lipids level, risk and severity of CHD.Results: 1. In the case control analysis, the CHD group had higher prevalence of smoking, hypertension and diabetes, higher triglyceride and lipoprotein(a)(Lp(a)) levels and had a higher using rate of blood lipid lowering drug, but lower high-density lipoprotein cholesterol level, and all the above differences were statistically significant(P<0.05).2. The results of lipids correlation analysis showed that there was a significant difference among total cholesterol levels for different genotypes of rs3201475 in TRIB1 gene,and the total cholesterol level of TT genotype carriers was significantly higher than that of CC genotype(P<0.05). The Lp(a) levels for different genotypes of rs3088442 in SLC22A3 gene were significantly different in the general population group, the control group, the CHD without using lipid-lowering drugs group, and the lipoprotein(a) levels of AA genotype carriers were significantly lower than that of GG genotype, what is more, the lipoprotein(a) levels of GA+AA genotype carriers were significantly lower than that of GG genotype(P<0.05).3. The frequency distribution of rs3088442 genetype in the CHD group and the control group were statistically significant with or without adjustment for age, sex, smoking, and body mass index, and AA genotype carriers’ CHD risk was lower than GG genotype carriers’(P<0.05). we also found that the A allele of the rs3088442 can reduce the risk of CHD than the G allele(odds ratio(OR)=0.78, 95%CI: 0.64-0.94, P=0.011).In the stratified analysis of risk factors, rs3088442 AA genotype carriers’ CHD risk was lower than GG genotype carriers’ in the groups like the male group, the age≤60 years group, BMI≥25 kg/m2 group, no history of hypertension group, no history of diabetes group(P<0.05). However, no significant association was found between rs3201475 in TRIB1 gene, rs539298、rs1810126 and rs2076828 in SLC22A3 with CHD in the analysis(P>0.05). When stratified analysis was carried out in the rs3201475 locus, there was no significant difference in the genotype frequency distribution in CHD group and control group(P>0.05).4. The association analysis between rs3088442 in SLC22A3 gene and CHD severity showed that: Compared with GG genotype, AA carriers of SNP rs3088442 had higher risk in major coronary occurrence risk of disease(OR= 1.44, 95%CI=1.44-1.88; P=0.006). Correlation coefficient between rs3088442 genotype and left main coronary artery disease was statistically significant(Ptrend=0.046). In addition, the correlation coefficient between the rs3088442-G allele and the Gensini score was statistically significant(Ptrend=0.012).Conclusions: 1. Smoking, hypertension, diabetes mellitus, triglyceride and Lp(a) may be risk factors of CHD, and the high density lipoprotein cholesterol may be the protective factor of CHD in the Chinese Han population.2. Rs3201475 in TRIB1 gene is related to the total cholesterol level, and rs3088442 in SLC22A3 gene is strongly associated with Lp(a) level in the Chinese Han population.3. In the Chinese Han population, rs3088442 in SLC22A3 gene is significantly associated with the CHD risk. However, Relevant to the SLC22A3 gene rs3088442 sites and coronary heart disease risk in Chinese Han population, rs3201475 in TRIB1 gene and rs539298, rs1810126, rs2076828 in SLC22A3 gene have no association with the risk of CHD.4. Rs3088442 in SLC22A3 gene may be involved in the severity of CHD.PartⅡ Mechanism study of the untranslated region genetic variations in TRIB1, SLC22A3 gene with coronary heart diseasePurpose: After exploring the regulation effect of the untranslated region genetic variations in TRIB1, SLC22A3 gene, and analysising of SLC22A3 and LPA m RNA level with the occurrence and development of CHD, and analysising the variation in SLC22A3 gene affect SLC22A3 and LPA m RNA level, to clarify the mechanism of genetic variation in the untranslated regions of TRIB1 and SLC22A3 genes on the occurrence and development of CHD.Methods: We firstly constructed the wild type plasmid and mutation type plasmid of rs3201475 and rs3088442 using molecular cloning techniques, then the dual luciferase report gene system was used to detect the expression differences of relative luciferase of the two plasmid of rs3201475 in two cell lines. Also we used the dual luciferase report gene system to detect the expression differences between the rs3088442 plasmid co-transfected with hsa-mi R-147 a mimic and mi R-Negative control in both two plasmids and in two cell lines. Using systematic sampling method to randomly select the inpatients of the Cardiology Department in the second Affiliated hospital of Chongqing Medical University in October 2015, using real-time fluorescence quantitative PCR to detect the SLC22A3 and LPA gene m RNA expression levels in peripheral blood lymphocytes of the selected populations, and the Taq Man probe technology was selected to genotype the rs3088442 loci of the selected populations. We analyzed the differences of SLC22A3 and LPA m RNA expression levels in CHD patients and controls, and then analyzed the SLC22A3 and LPA m RNA expression levels among different genotypes of rs3088442. SPSS 20.0 was used for above analyses.Results: 1. There was no significant difference in the luciferase activity of the two plasmids carrying rs3201475-C and rs3201475-T in Hep G2 and He La cell lines(P=0.335 and P=0.559, respectively).2. We found no significant difference in luciferase activity in carrying rs3088442-G allelic gene plasmid co-transfected with hsa-mi R-147 a mimic and mi R-Negative control in both 293 T and He La cell lines(P=0.537 and P=0.144). While the plasmid carrying the rs3088442-A allele, the luciferase activity was significantly lower in the hsa-mi R-147 a mimic group than in the Control mi R-Negative group in both 293 T and He La cell lines(P=2.2×10-4 and P=2.0×10-3).3. In this study, 67 blood samples were randomly selected for real-time fluorescence quantitative PCR analysis. There were 8 cases in the control group, 59 cases of CHD group(including 12 cases of stable angina, 17 cases of unstable angina, 11 cases of myocardial infarction and 19 cases of non-classification of CHD blood samples). Compared with the control group, the m RNA expression levels of SLC22A3 and LPA gene in peripheral blood of patients with were significantly higher than that of the control group(Ptrend=0.003 and Ptrend=0.012).4. We grouped the samples according to the rs3088442 genotypes. The results showed that compared with the control group of AA in population, with the increase of GA, GG degrees, in peripheral blood of patients with SLC22A3 and LPA gene m RNA expression levels were significantly increased(Ptrend=0.012 and Ptrend=7.0×10-6).Conclusions: 1. Rs3201475 in the 5′-UTR of TRIB1 has no regulatory effect in TRIB1 gene.2. The A allele of rs3088442 in the 3′-UTR region of SLC22A3 is able to regulate the expression of SLC22A3 gene by binding to hsa-mi R-147 a.3. The LPA and SLC22A3 gene m RNA expression levels in peripheral blood may be associated with the occurrence and development of CHD in the Chinese Han population.4. SLC22A3 and LPA gene m RNA expression levels in peripheral blood of patients with CHD are related to the polymorphism of rs3088442.