The Relationship Between MiR-29b And Gestational Diabetes Mellitus And Its Molecular Mechanism

Gestational diabetes mellitus(GDM)is defined as any degree of glucose intolerance with onset or first recognition during pregnancy.It does not exclude the possibility that unrecognized glucose intolerance may have antedated or begun concomitantly with the pregnancy.GDM is one of the most common complications during pregnancy.GDM causes serious harm to maternal and fetal,and even has long-term impact on mothers and babies.At present,the pathogenesis of GDM is still unclear.But some studies have shown that GDM has a variety of pathogenic factors,and may be the result of co-effect between genetic and environmental factors.MicroRNA(miRNA)is a class of single-stranded small-molecule non-coding RNAs with a length of 18-23 nucleotides and highly conserved in the evolutionary process that play a negative regulatory role at the epigenetic level.Recent studies have found that more and more miRNAs play an important role in the pathogenesis of GDM.Placenta is the only interface that connects pregnant women and fetuses.So,it is an important organ to study the pathogenesis of GDM,a type of disease with abnormal glucose metabolism.In our previous study,we used miRNAs chip to screen for differentially expressed miRNAs in GDM and normal placenta to explore the molecular biological regulation mechanism of miRNAs in GDM,in this study,we used qRT-PCR and in situ hybridization to validate the expression patterns of candidate miRNAs in GDM and normal population samples.Bioinformatics was used to predict the target genes of differentially expressed miRNAs,and Dual-Luciferase(?)Reporter Assay System was used to verify miRNAs regulating the expression of candidate target genes by constructing a 3’-untranslated region(3’-UTR)expression plasmid for the target genes.Over expression,knock down and rescue of candidate miRNAs’ target gene were used to investigate the effects of candidate miRNAs on the proliferation,activity,apoptosis,migration and invasion of human chorionic trophoblast cells from the placenta through target genes.According miRNAs chip results,we selected three differentially expressed miRNAs,includelet-7b,miR-1202 and miR-29b,and validated the expression patterns of candidate miRNAs in large populations by qRT-PCR.The results showed that the expression level of miR-29b in GDM is significantly lower than that in normal control,and the expression level of young normal maternal is significantly higher than that of older one.In situ hybridization showed that miR-29b was localized in placental trophoblast cells.MiR-29b overexpression can inhibit cell proliferation,migration and invasion,and promote cell apoptosis.Meanwhile,miR-29b knockdown can promote cell migration and invasion.To study the molecular mechanism of miR-29b play a role,we predicted the target genes for miR-29b by miRBase and TargetScan.Three candidate miR-29b target genes related to diabetes mellitus and GDM were screened:ING2,ING3 and HIF3A.Dual-luciferase reporter system analysis showed that miR-29b mimic with HIF3A 3’-UTR co-transfected cells can reduce luciferase activity,whereas miR-29b inhibitor and HIF3A 3’-UTR co-transfected cells can increase luciferase activity.MiR-29b mimic or inhibitor co-transfected cells with ING2 or ING3 3’-UTR have no significant effect on luciferase activity.These results suggest that HIF3A may be the target gene for miR-29b.To further verify the relationship between miR-29b and HIF3A,the binding site of miR-29b on HIF3A 3’-UTR was mutated,and the ability to bind to miR-29b was significantly reduced after mutation.Meanwhile,the effect of miR-29b on endogenous target gene expression was analyzed by Western blot,and the results showed that miR-29b could negatively regulate the expression of HIF3A.These results confirm that HIF3A is one of the target genes of miR-29b.Overexpression of HIF3A can promote cell proliferation,migration and invasion,but HIF3A knockdown can inhibit cell migration and invasion.To investigate whether HIF3A is a miR-29b functional target gene,the effect of miR-29b and its target gene on the biological behavior of cells was analyzed by genetic compensation.When trophoblast cells HTR8-/SVneo overexpress miR-29b,supplementation of exogenous HIF3A can antagonize miR-29b overexpression-induced cell proliferation,migration and invasion inhibition.When miR-29b was lowly expressed in HTR8_/SVneo cells,inhibition of endogenous HIF3A expression can cause cell migration and invasion to return to normal levels.The results of this study show a decrease in miR-29b expression in GDM placenta,and that promotes proliferation,migration and invasion of placental trophoblast cells by upregulating the expression of HIF3A.This study investigated the correlation between miR-29b and GDM from molecular epidemiological perspective.Meanwhile,the effect of miR-29b on placental-derived trophoblast cell function was also determined.