Association Between The Polymorphisms In The 3’ UTR Of CORIN And Cardiovascular Diseases And MiR-302c Regulates Corin Expression By Binding To The CORIN 3’UTR

Backgrounds Cardiovascular disease(CVD) is a worldwide problem and life threatening. At 2008, about 17.3 million people died of CVD in the world and the mortality of CVD is the first palce in all-cause death. Essential hypertension(EH) and atrial fibrillation(AF) are common CVD. Both of them are ascertained as polygenic inheritance diseases, which are the interaction between environmental and genetic factors. Some external factors or diseases such as high-salt-fat diet, smoking, lack of exercise, obesity and diabetes can increase the risk of EH/AF. By positional cloning of family linkage analysis, it has found some mutations in MR, WNK4, AME, NR3C2, KLHL3 and CUL3 in the hypertension pedigrees, and SCN5 A, SCN10 A, KCNQ1 and NPPA in the AF pedigrees. However, the vast majority of patients with EH/AF are from sporadic population. Therefore, it is limited to discuss the genetic mechanisms of complicated diseases using family linkage analysis. Genome-wide association study(GWAS) is a new strategy, which is based on the tens of millions of single nucleotide polymorphisms(SNPs) as markers to find the genetic characteristics of complicated diseases in the case-control sporadic population. Currently severval GWAS have found some loci and genes are associated with EH/AF. ATP2B1, CYP17A1, PLEKHA7, SH2B3, TBX3-TBX5, CACNB2, MTHFR, ANF652, NPPA, CDH13 and RYR2 polymorphisms are associated with EH. Chromosome 4q25,1q21, ZFHX3 and NPPA polymorphisms are associated with AF. Corin is a trypsin-like protease highly expressed in cardiomyocytes. The primary function of Corin in the heart is to activate pro-atrial natriuretic peptide(pro ANP) and pro-brain natriuretic peptide(pro BNP). Pro ANP and pro BNP are important cardiac hormones, which are essential to regulate salt-water balance, blood pressure/volume and resist renin-angiotension-aldosterone system(RAAS). Recent studies have found that CORIN polymorphisms were associated with EH and left ventricular hypertrophy(LVH). However, most of them focused on the SNPs in CORIN coding area. The associations between SNPs in CORIN 3’ untranslated region(3’ UTR) and EH/LVH are unclear. In the study, we select four SNPs in CORIN 3’ UTR(rs3749585, rs3749584, rs4695253 and rs12641823) and to study the correlations between the four SNPs and EH/LVH. In addition, based on the previous results that NPPA(encoding pro ANP protein) mutations and polymorphisms were associated with AF, and plasma N-terminal pro ANP(NT-pro ANP) levels were increased in AF patients, we speculate that Corin, as the pro ANP convertase, may also play an important role in AF. In the study, the correlation between Corin and AF is discussed from two aspects-- SNPs in CORIN 3’ UTR and AF; plasma Corin levels and NT-pro ANP levels in patients with AF. Micro RNAs(mi RNAs) are highly conserved noncoding small RNA molecules that regulate a large fraction of the genome. Mi RNAs regulate gene expression of messenger RNA(m RNA) at the posttranscriptional level through transcript degradation or translational repression by binding to the 3’ UTR of the target m RNA. The specific mi RNA that can regulate CORIN m RNA expression by binding to the CORIN 3’UTR is unknown. Therefore, we find the mi RNA by bioinformatics technology and test whether the mi RNA can regulate Corin expression.Materials and methods The study was a case-control study. All patients were recruited from the First Affiliated Hospital of Dalian Medical University between the period of December 2012 and March 2014. Subjects were from northeastern China and self-declared to be of ethnicChinese Han origin. The cases were patients with CVD including EH and AF. We collected the peripheral blood samples of all subjects and extracted genomic DNA using potassium iodide method. The four SNPs in CORIN 3’UTR ——rs3749585、rs3749584、rs4695253 and rs12641823 were genotyped by fluorescent dye-based high resolution melting(HRM) analysis on the Roche Light Cycler 480 II system. Statistical analysis was subsequently performed to evaluate the association of the variants with EH/AF under different genetic models. We measured the plasma Corin and NT-pro ANP in AF patients(n=141) and controls(n=139) by enzyme-linked immunosorbent assay(ELISA). AC16 cells were transfected with mi R-302 c and the Corin expressions of m RNA and protein were detected by real-time PCR(RT-PCR) and ELISA/western blot(WB), respectively. Then we constructed CORIN-3’ UTR-psi CHECK2 recombinant plasmid, which transiently co-transfected human embryonic kidneys cells(HEK293) with mi R-302 c. Using a dual-luciferase reporter system to experimental validation whether or not mi R-302 c regulates the CORIN m RNA expression by binding to the CORIN 3’ UTR.Results 1. In the Chinese Han population from northeastern China, we found the polymorphisms rs3749585、rs3749584、rs4695253 and rs12641823 in CORIN 3’ UTR. 2. EH-control study included 1332 EH patients and 1552 controls. The CC/CT/TT genotype frequencies of rs3749585 were 24.9%/52.2%/22.90% and 29.9%/47.49%/ 22.62% in EH group and control group, respectively. Allelic analysis demonstrated that rs3749585 was related to EH before or after adjusting for age, gender, AF, heart failure(HF) and T2DM(P=0.041 and P-adj=0.032). A decreased risk of EH was specifically associated with the minor allele C(OR=0.897). 3. The CC/CT/TT genotype frequencies of rs4695253 were 72.6%/24.32%/3.08% and 74.74%/23.90%/1.35% in EH group and control group, respectively. Allelic analysis demonstrated that rs4695253 was related to EH(P=0.036). An increased risk of EH was associated with the minor allele T(OR=1.172).4. No association was detected between rs3749584/rs12641823 and EH under any allelic or genotypic model(P > 0.05). 5. EH patients were divided into EH-LVH group(n=403) and EH-non LVH group(n=929). rs3749585 was associated with LVH before or after adjusting for age, gender, AF, HF and T2 DM in dominant model(P-adj=0.034,OR=0.744); a decreased risk of LVH was associated with the minor allele C. rs4695253 was related to LVH after adjusting for covariates in recessive model(P=0.033); the minor allele T could increase the risk of LVH. No association was detected between rs3749584/rs12641823 and LVH under any allelic or genotypic model(P > 0.05). 6. AF-control study included 539 AF patients and 2535 controls. The CC/CT/TT genotype frequencies of rs3749585 were 29.68%/49.54%/20.78% and 27.65%/ 49.39%/22.96% in AF group and control group, respectively. rs3749585 was associated with AF after adjusting for age, gender, EH, LVH, HF and T2 DM in allelic, additive and recessive models(P-adj=0.025, 0.023 and 0.014, respectively). An increased risk of AF was specifically related to the minor allele C. 7. The CC/CG/GG genotype frequencies of rs3749584 were 10.95%/25.6%/63.45% and 0.51%/22.52%/74.28% in AF group and control group, respectively. rs3749584 was significantly associated with AF before or after adjusting for covariates in any allelic or genotypic model. 8. No association was detected between rs4695253/rs12641823 and AF under any allelic or genotypic model(P>0.05). 9. In patients with AF, plasma Corin levels were 1209±510 pg/ml, which were significantly higher than that in the controls(973±528 pg/ml, P < 0.001). In patients with AF, plasma NT-pro ANP levels were significantly higher than that in the controls(3.1±2.42 vs. 1.77±1.04 nmol/l,P<0.001). 10. In the mi R-302 c transfection group, the expressions of endogenous CORIN m RNA and Corin protein of AC16 cell lysis were significantly lower than the mi R-NC transfection group(P < 0.001);Corin levels in AC16 cell lysis of mi R-302 c transfection group were 0.60±0.072 ng/ml, which were significantly lower than thatof mi R-NC transfection group(0.79±0.030 ng/ml, P<0.001). 11. In the mi R-302c+CORIN-3’UTR-psi CHECK2 recombinant plasmid group, relative luciferase activity was lower than that of the mi R-NC+CORIN-3’UTRpsi CHECK2 group(P<0.001).Conclusions 1. In the Chinese Han population from northeastern China, we found the polymorphisms rs3749585、rs3749584、rs4695253 and rs12641823 in CORIN 3’ UTR. 2. rs3749585 is associated with EH/LVH and a decreased risk of EH/LVH was associated with the minor allele C. 3. rs4695253 is associated with EH and an increased risk of EH was associated with the minor allele T. rs4695253 is not correlated with LVH. 4. No association was found between rs3749584/rs12641823 and EH/LVH. 5. rs3749585 is associated with AF and an increased risk of AF was associated with the minor allele C. 6. rs3749584 is associated with AF and an increased risk of AF was associated with the minor allele C. 7. No association was detected between rs4695253/rs12641823 and AF. 8. Plasma Corin and NT-pro ANP levels were increased in patients with AF. 9. mi R-302 c could downregulate the expressions of endogenous CORIN m RNA and Corin protein in AC16. 10. mi R-302 c could bind to CORIN 3’ UTR specifically. 11. mi R-302 c could downregulate the expression of exogenous CORIN m RNA in HEK293.