Combined Oral Contraceptive Use, Single-nucleotide Polymorphisms In MicroRNA Binding Sites Of RAAS Genes And The Risk Of Stroke In Chinese Women

As an effective and convenient method for birth control, Combined Oral Contraceptive (COC) is widely used all over the world. It is estimated that nearly100million women of childbearing age prefer COC. There are many reports about the effects of COC in the past50years, for example, it can prevent endocrine, anaemia, bone demineralisation besides non-contraceptive benefits. However, people pay more attention to potential health hazards caused by the COC, such as elevating blood pressure and increasing the risk of myocardial infarction, stroke and venous thromboembolism.Stroke is a rare and serious side effect of COC use, which has caught a great number of researchers’ attention. The results of the studies about the association between COC use and stroke carried out among different populations were not consistent. Moreover, only a few of COC users suffer stroke when they take the same pills. These studies suggested that genetic factors may play an important role in the susceptibility of stroke.The renin angiotensin aldosterone system (RAAS) has become a promising candidate of genetic pathway for the stroke study. MicroRNAs (miRNAs) are a class of small, non-coding, single-stranded RNAs with a length of18-22nucleotides. They are important post-transcriptional regulators of RAAS gene expression. In general, miRNAs bind the3’-untranslated region (3’UTR) of the mRNA of their target genes, causing inhibition of translation or even promoting degradation of the mRNA. Recent studies have indicated that single-nucleotide polymorphisms (SNPs) in3’UTRs targeted by miRNAs alter the strength of miRNA binding, with consequences on regulation of target genes which may affect the individual’s diseases. It has been demonstrated that the variant of rs5186(A>C) of angiotensin II type-1receptor (AGTR1) in RAAS can affect the binding of miR-155to mRNA, which can influence the expression of AGTR1and then lead to the occurrence of hypertension and cardiovascular complications. However, gene polymorphisms in miRNA binding sites in3’UTRs of RAAS have not been reported to be associated with the susceptibility of stroke.In this study, we used different bioinformatics methods to screen SNPs in miRNA binding sites of3’UTRs of RAAS, and analyzed the association between these SNPs and the susceptibility of stroke. We find the potential risk factors of stroke in the perspective which miRNAs regulat target gene. Furthermore, the joint effects of gene-gene, gene-environment were also discussed. The results from this "study will be helpful in preventing stroke, guiding contraceptive use, and promoting the health of women.[Objective] The objective of this study is to investigate the role of COC use and its interaction with the5SNPs in miRNA binding sites of RAAS genes in stroke risk among Chinese women. Furthermore, the joint effects of gene-gene, gene-environment were also discussed.[Methods] A nested case-control study was conducted based on the female cohort of contraceptives in China. The first-ever stroke cases, and controls matched for age and region were recruited from the cohort. The5SNPs in miRNA binding sites of RAAS genes were detected by Tagman allelic discrimination technology.[Results] 1. Stroke risk with COC use and other environmental factors1.1The association between COC use and stroke riskThe risk of stroke associated with COC use was slightly elevated. The risk of hemorrhagic stroke among COC users compared with the non-users was significantly increased (OR=1.83,95%CI:1.25-2.66). Along with the cumulative time of COC use increasing, the risk of stroke also significantly increased (Ptrend<0.001). The odds ratios for hemorrhagic stroke risk and ischemic stroke risk among women with the cumulative time≥20years of COC use compared with non-users were2.89and1.95, respectively.1.2Analyses of risk factors for strokeWomen with hypertension had an increased risk of hemorrhagic stroke by6.90-fold (OR=1.90,95%CI:5.12-12.18), and ischemic stroke by15.80-fold (OR=16.80,95%CI:10.84-26.05). Hyperlipidemia was significantly associated with the risk of ischemic stroke (OR=1.97,95%CI:1.48-2.63), but not hemorrhagic stroke.2. Association between the SNPs in miRNA binding sites of RAAS genes and stroke risk in Chinese women2.1. The distribution of SNPs in miRNA binding sites of RAAS genes in cases and controlsAfter adjustment for age, area, hypertension, BMI, hyperlipidemia, the results showed that the variant of rs5186of AGTR1was positively associated with hemorrhagic stroke risk, while the variant of rs7079of angiotensinogen (AGT) gene was negatively associated with risk of ischemic stroke. Compared with rs5186AA wild-type genotype, rs5186CC or AC/CC genotypes were signicantly associated with the risk of hemorrhagic stroke (OR=1.83,95%CI:1.10-2.97, and OR=1.74,95%CI:1.06-2.87, respectively).A significant reduction of ischemic stroke risk was found among women with rs7079CA or CA/AA genotypes compared with women with rs7079CC wild-type genotype (OR=0.62,95%CI:0.43-0.90, and OR=0.58,95%CI:0.56-0.85, respectively).2.2Stratified analyses of SNPs in miRNA binding sites of RAAS genes by age, BMI, hypertension, hyperlipidemiaThe stratified analyses demonstrated that the risk of hemorrhagic stroke associated with rs5186AC/CC genotype was significantly increased among women aged≥55years (OR=1.93,95%CI:1.02-3.646) or women with BMI<24(OR=1.88,95%CI:1.01-3.52), and the association between ischemic stroke risk and rs5186AC/CC genotype was also significant among women with BMI<24(OR=1.94,95%CI:1.11-3.38).Rs7079CA/AA genotypes reduced significantly the risk of ischemic stroke among the women with hypertension, hyperlipidemia, BMI<24or age≥55years.3. Analyses of joint effects3.1The joint effect of rs5186and rs7079with stroke riskSNPs rs5186and rs7079were confirmed to be associated with the risk of stroke. A cumulative effect was observed between the joint effect of two SNPs and stroke risk. The risk of stroke, especially ischemic stroke, gradually increased with the increasing number of risk alleles in Chinese women (Ptrend=0.0037).3.2Joint effects between COC use and genetic variants on stroke riskCompared with women who didn’t take COCs and didn’t carry the risk genotype (rs5186AA genotype), the odds ratios for hemorrhagic stroke risk among COC users without carrying the risk genotype and COC users carrying the risk genotype were1.71(95%CI:1.18-2.48) and2.81(95%CI:1.45-5.64), respectively.[Conclusion] COC use, hypertension, hyperlipidemia are the risk factors for stroke in Chinese women. The variant of rs5186of AGTR1was positively associated with risk of hemorrhagic stroke, while the variant of rs7079of AGT gene was negatively associated with risk of ischemic stroke.The risk of stroke associated with the joint effect of rs5186and rs7079was significantly increased. COC use combined with the variant of rs5186was significantly associated with the risk of hemorrhagic stroke.