Single-nucleotide Polymorphisms Inside MicroRNA Target Sites Influence The Susceptibility To Type2Diabetes

Diabetes is a metabolic disorder caused by a variety of causes, which ischaracterized by chronic hyperglycemia and is one of the top five diseases inmorbidity and mortality in the world. It is estimated that by2030the number ofworldwide diabetes will increase to552million and the prevalence of diabetes will bereached9.9%, at an averaged annual growth of2.7%, which is1.7times the annualgrowth of the total world adult population. In China, it was estimated approximately92.4million adults have been suffering from type2diabetes, which accounts formore than90%of the number of diabetes. In addition,148million adults haveprediabetes. Diabetes can cause the acute and chronic complications and especiallychronic complications can injury multiple organs. The high disability and death rateseriously affect the patient’s physical and mental health, and bring enormous burdento patient’s families and society. T2DM has become an important public healthproblem which has seriously injuried heath and lives, so the exploration of T2DMrisk factors and prevention of disease occurrence and development have beenconsidered at imminent.The recent breakthrough discovery of a new family of naturally endogenous,small (~22nucleotides), non-coding but functional RNAs (microRNAs) have beenproven to play a role not only in regulation of normal physiological function, but alsoin the development of the wide spectrum of diseases including T2DM. MiRNAs has a direct role in insulin secretion and production, pancreatic islet development, insulinaction and indirectly control glucose and lipid metabolism and lead to influence thedevelopment of diabetes and its complications. MiRNAs exerts its function throughbinding to specific complementary sequences in the3’ untranslated regions (UTRs) oftarget mRNAs and negatively regulate target gene transcriptional expression. Recentstudies have indicated that single-nucleotide polymorphisms (SNPs) that reside in themicroRNA (miRNA) target site can affect the binding of miRNA to mRNA, whichcan affect gene or protein expression and lead to influence the occurrence of certainhuman diseases. However, gene polymorphisms in microRNA-binding sites have notbeen reported to be associated with susceptibility of T2DM.In this study, we used different algorithms to screen diabetes-related SNPs inmicroRNA target sites and adopted a case-control study design to investigate theassociation of these gene polymorphisms and the susceptibility of T2DM in southeastHan population of Chian. We find the potential risk factors of T2DM in theperspective which miRNAs regulat target gene. The results of the study will providefurther rich molecular mechanisms for the etiology of T2DM and novel geneticsusceptibility markers in molecular diagnostics of T2DM and screening of high-riskpopulation.Part Ⅰ Assiontion study of single miRNA target sitepolymorphisms inside miRNA target site with susceptibilitly oftype2diabetesIn the study, we first obtained diabetes-related miRNAs through an extensivemining of bioinformatics according to previously reported molecular biologyliteratures and public database. Bioinformatics softwares were used to predict theirtarget sites. After screening, we identified diabetes-related miRNAs target gene and polymorphisms within microRNA-binding sites. Finally,10candidate SNPs inmicroRNA target sites were selected. We conducted a case-control study and detectedall subjects (1017T2DM patients,1059health controls) by utilizing TaqMan assaydetection technology. We analyzed the association between different genotypes andT2DM in southeast Han population of Chian. Associations between genotypes andT2DM risk (ORs and95%CI) were estimated by logistic regressions. The mainresults were as follows:1. The distribution in cases and controls of diabetes-related microRNA target sitespolymorphismsWe analyzed ten SNPs of diabetes-related miRNAs target sites respectively. Afteradjustment for age, gender, and waist circumstance, the variants of rs1366600（T>C）of insulin receptor (INSR) gene and rs2292899（G>A）of acyl-CoA synthetase1(ACSL1) gene were positively associated with risk of T2DM, while the variant ofrs11724758（G>A）of fatty-acid-binding protein2(FABP2) gene was negativelyassociated with risk of T2DM. Compared with the wild-type homozygoters1366600TT, the CC homozygote and the combined genotype TC/CC of rs1366600were all associated with a significant risk effect for T2DM [Adjusted ORs (95%CIs)=2.03(1.02-4.01);1.28(1.04-1.57)]. Compared with rs2292899GG wild-type genotype,the variant genotype GA increased the risk of T2DM by22%[Adjusted OR (95%CI)=1.22(1.004-1.49)]. However, the variant genotype GG of rs11724758was associatedwith a significantly reduced risk of T2DM compared to the AA genotype withadjusted OR and95%CIs of0.76(0.58-0.997). All the significances remained after the1000permutation test. No evidence suggested other seven SNPs were associated withT2DM.2. Stratified analysis of SNPs in microRNA target sitesThe stratified analysis showed that the risk rs1366600C allele of INSR gene in the additive model significantly increased risk of T2DM among males, the elderly(age>55), abdominal obesity people, non-smokers and non-drinkers [Adjusted ORs(95%CIs)=1.31(1.00-1.70);1.41(1.11-1.80);1.55(1.17-2.06);1.29(1.03-1.61);1.31(1.06-1.63)]. The risk of T2DM increased by21%among males carrying the riskrs2292899A allele of ACSL1gene [Adjusted OR (95%CI)=1.21(1.01-1.46)]. Therewas more pronounced decrease in T2DM risks among males, elderly (age>55),smokers and drinkers carryinghe risk rs11724758A allele of FABP2gene [AdjustedORs (95%CIs)=0.83(0.69-0.99);0.75(0.63-0.89);0.78(0.61-0.99);0.70(0.53-0.93)].There was no significant difference in the magnitude of the associations between theremaining seven SNPs and T2DM in subjects with different age, gender, abdominalobesity, smoking, drinking.Part Ⅱ Assiontion study of multiple miRNA target sites andtheir combined effects with environment and the susceptibilitlyof type2diabetesIn the view of type2diabetes is a multifactorial diseases caused by the combinedeffects of genes and environmental factors. In this study, we used crossover analysis,logistic regressions and BPANN methods to explore the joint-action of gene-gene andgene-environment with T2DM risk in in southeast Han population of Chian. The mainresults were as follows:1. The joint-action of SNPs in microRNA target sites with T2DM riskRs1366600, rs2292899and rs11724758which had significant associations with therisk of T2DM based on the single-locus analysis in the first part, assessed thegene–environmental joint-action with T2DM risk. A cumulative effect was observedbetween the joint-action of three SNPs and T2DM risk. With the number of risk allelescarry increased, the prevalence of T2DM also significantly increased. The stratified analysis showed that the cumulative effect was still observed among males, elderly(age>55), abdominal obesity, smokers, non-smokers and drinkers. Compared with thereference group (0–1), individuals carrying two or more risk alleles still had asignificant increased risk for T2DM.2. The joint-action of gene and environment factors with T2DM riskRs1366600of INSR gene, rs2292899of ACSL1gene and rs11724758of FABP2gene were respectively assessed the gene–environmental (abdominal obesity)interaction by crossover analysis. Compared with normal weight person not carryingrisk genotypes (rs1366600TC/CC), the risk of T2DM was significant increasedamong the abdominal obesity person not carrying risk genotypes[Adjusted OR(95%CI)=2.17(1.75-2.68)]. Compared with normal weight person not carrying riskgenotypes (rs2292899GA/AA), the risk of T2DM was significant increased amongthe abdominal obesity person not carrying risk genotypes and normal weight personcarrying risk genotypes [Adjusted ORs=2.73,1.32;95%CIs=(2.04-3.67)，(1.01-1.72)].However, those who exposed to both environmental factor and genotype factors(putative risk genotypes) had the highest risk of T2DM [Adjusted ORs=3.56,3.04;95%CIs=(2.61-4.87),(2.31-3.99) respectively]. Nevertheless, the subjects withabdominal obesity exposed to rs11724758GA/AA variant genotype (putativeprotective genotype) had an increased risk of T2DM [Adjusted OR=2.24;95%CI=(1.71-2.95)], while the abdominally obese subjects exposed to rs11724758GGgenotype (putative risk genotype) had the highest risk for T2DM [Adjusted OR=2.62;95%CI=(1.92-3.58)].3. Multiple logistic regression analysis for the associations between the related factorsand T2DMWe used multiple logistic regression analysis to analysis multiple logisticregression analysis for the associations between the related19factors and T2DM, inculing ten SNPs of diabetes-related miRNAs target sites. With stepwise regressivemethod,7variables were statistically significant in the associations of diabetesoccurrence (P<0.05).4. BPANN multiple analysis for the associations between the related factors andT2DMUsing the19related factors as input variables and T2DM diagnosis as outputvariables, we established BPANN model with all available samples. The transferfunction was logsig function. Learning rate was0.1. Training error was0.01.Maximum training steps set to1000steps. After the termination of training, the MIVwas obtained. And then according to the absolute value of MIV, we sequenced therelated factors in the order of diabetes family historyl, hypertension history, waist,drinking, hyperlipoproteinemia, smoking and rs1366600, which were the top tenfactors.