Relationship Between Inflammatory Factors And Gestational Diabetes Mellitus And The Combination Of MiRNA With PAX4, KCNB1, SLC30A8 And INSR Genes

ObjectiveTo explore the associations of inflammatory markers--hite blood cell (WBC) count、 alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the first trimester of pregnancy and high sensitivity C-reactive protein (hsCRP) in the second and third trimester with gestational diabetes mellitus (GDM)Methods725 women with GDM and 935 women who remained euglycemic throughout pregnancy were enrolled in this study. Pre-pregnancy weigh、 height and blood pressure were recorded, WBC count、 ALT、 AST levels were detected between 8 and 12 weeks of pregnancy, at 24 to 28 weeks of pregnancy, the hsCRP、 glucose and insulin levels were detected. The associations of WBC count、 ALT, AST and hsCRP levels with the blood glucose and insulin levels were retrospectively analyzed, meanwhile, we also researched the associations of those factors with the occurrence of GDM.Results1. WBC count、 ALT、 AST levels in the first trimester and GDM1) White blood cell count and ALT levels in the first trimester of pregnancy were significantly increased in GDM subjects compared with normal glucose tolerance (NGT) subjects (p<0.01), there was no significant difference of AST levels between two groups.2) Logistic regression analysis illustrated that elevated white blood cell count was an independent risk factor for GDM (OR=1.119, p<0.05). The ROC curve revealed that threshold of WBC count was 7.965X×109/L (AUC 0.566, p1<0.01)3) Multiple linear regression analysis:HOMA-IR was associated with white blood cell count (B=0.051, P<0.05),1 h blood glucose after oral 50 grams of sugar was related to white blood cell count (B=0.044, P<0.05), fasting plasma true insulin was related to white blood cell count (B=0.214, P<0.05),1 h true insulin after 100 grams OGTT was related to AST(B=0.616, P<0.05),2 h true insulin after 100 grams OGTT was related to ALT and AST (B=0.148 and B=0.936, p<0.05),3h true insulin after 100 grams OGTT was related to ALT and AST (B=0.189 and B=0.688, P<0.05)2. hsCRP in the second and third trimester and GDM1) The hsCRP levels in the second trimester of pregnancy were significantly increased in GDM subjects compared with subjects with normal glucose tolerance (NGT) (p<0.01)2) Logistic regression analysis illustrated that elevated hsCRP was an independent risk factor for GDM (OR=1.064, p<0.05)3) Fasting blood glucose was associated with hsCRP (B=0.018, P<0.05);1 h blood glucose after oral 50 grams of sugar was related to hsCRP (B=0.022, P<0.05); 1,2 and 3h blood glucose after 100 grams OGTT were related to hsCRP (B=0.064、 0.054、0.052, P<0.05); glycosylated hemoglobin(HbAlc) was related to hsCRP(B=0.016, P<0.05); fasting plasma insulin was related to hsCRP (B=0.303, P<0.05); 1,2 and 3h true insulin after 100 grams OGTT were related to hsCRP (B=1.257.1.547,1.626, P<0.05);HOMA-IR was associated with hsCRP (B=0.075, P<0.05)Conclusions1. The WBC count in the first trimester of pregnancy could significantly increase the risk of GDM, which is expected to become one of the predictors of GDM.2. The elevated hsCRP levels in the second and third trimester of pregnancy could significantly increase the risk of GDM, which is expected to become one of markers for the diagnosis of GDM.3. GDM is a chronic low-grade inflammatory disease, in which the low-grade inflammation has always existed.ObjectiveTo explore the combinability of miRNA with polymorphism in 3’UTR of gene PAX4, KCNB1、 SLC30A8 and INSR and the effect of miR-223 on expression of PAX4 gene.MethodsWe select SNPs which the genotype or allele frequency distribution has statistically significant differences between GDM and the control group in our previous experiment, including rs71269 (PAX4)、 rs1051295 (KCNB1)、 rs2466293 (SLC30A8) and rs1366600 (INSR). We predict the miRNA siting with SNPs by SNP Function Prediction、 MicroRNA.org. PolymiRTS Database 3.0、 MiRDB, Targetscan 6.2 and Hoctar and Dual-Luc if erase Reporter Assay System was used to confirm the binding between SNPs and predicted miRNA. We demonstrated the effect on expression of PAX4 gene after miR-223 siting with rs712699 by real-time PCR and western blot.Results1. The results of Dual-Luciferase Reporter Assay System showed that rs2466293 and miRNA489-3p, rs1051295 and miRNA216a-5p, rs712699 and miRNA-374b、 miRNA-369-3p、 miRNA-20a、 miRNA-513b-3p, rs1366600 and miRNA-520b、 miRNA-302e、 miRNA-17、miRNA-20a、miRNA-93 failed to combine and the expression of luciferase gene has no difference between minor alleles and major alleles2. Real-time PCR revealed that compared with containing gene PAX4 rs712699 loci GG genotype plasmid group, the mRNA in containing gene PAX4rs712699 loci AA genotype plasmid group was significantly reduced (2-△△Ct was 0.523).3. Western blot indicated that the protein bands between containing gene PAX4 rs712699 loci GG genotype plasmid group and containing gene PAX4 rs712699 loci AA genotype plasmid group have no difference.ConclusionsThe miR-223 inhibited the expression of PAX4 gene after miR-223 harboring the AA genotype of rs712699. However, the inhibitory effect is weak, protein bands showed no difference in western blot.