Because the symptoms of GDM mostly occur in late pregnancy, women with first-time GDM often do not have enough time for clinical intervention. Since accumulated evidence has demonstrated that GDM can be prevented by diet, medicine and exercise intervention started in early pregnancy, early detection of women with a high risk of GDM is essential. To identify noninvasive plasma molecular markers of GDM, we first screened DEmiRNAs and DEmRNAs by comparing serum samples of GDM patients and those of healthy pregnant women. A total of 264 DEmiRNAs and 1217 DEmRNAs were identified, and these differentially expressed markers might serve as noninvasive plasma molecular markers for predicting GDM in early and mid-pregnancy. Further research is needed to evaluate their predictive value. To date, there have been two studies on identifying noninvasive markers of GDM in early and mid-term pregnancy based on miRNAomics. The expression of several plasma miRNAs including miRNA-29a and miRNA-16 was found to be associated with GDM, and both were also differentially expressed in our present study (miRNA-29a, P = 0.003; miRNA-16, P = 0.008)[15, 17].
The results of the present study also provide insights into the molecular mechanisms that underlie GDM. miRNAs are involved in many biological processes through posttranscriptional regulation. They regulate gene expression and transcription by complementary binding to the 3' end of target genes. However, to date, little is known about the regulatory process between miRNAs and mRNAs in GDM. Thus, we performed an integrative analysis to construct functional miRNA-gene regulatory networks by combining the expression relationships between miRNAs and targets and computational predictions. First, enrichment analysis of the involved signaling pathways was carried out based on DEmiRNAs. The metabolic pathways glypican pathway, proteoglycan syndecan-mediated signaling events, and syndecan-1-mediated signaling events, were the core pathways regulated by miRNAs in GDM. Glypicans belong to the family of membrane-bound heparan sulfate proteoglycans that are linked to the cell surface and are involved in the regulation of growth factor activity. Proteoglycan families are involved in a wide range of diseases, including obesity and diabetes. Impaired glucose metabolism in GDM may alter the expression of proteoglycans, which may impair the biological functions of the placenta. A previous study demonstrated that the expression of a member of the proteoglycan family, endocan, was increased in the human placenta from obese women with GDM and in response to proinflammatory stimuli. In addition, the metabolic effect of high glucose and high osmotic pressure of GDM patients may contribute to the increased proteoglycan perlecan expression in diabetic placentas. Leelalertlauw et al. found that serum glypican 4 levels were increased with increasing degrees of obesity. Furthermore, Ussar et al. demonstrated that circulating glypican-4 levels correlate with BMI and insulin sensitivity in humans. Moreover, glypican-4 interacts with the insulin receptor, enhances insulin receptor signaling, and increases adipocyte differentiation. These results suggest that abnormal expression of proteoglycan family genes in GDM is helpful for understanding the pathogenesis of GDM.
In the present study, hsa-miR-146a-3p, ranked first in the altered DEmiRNAs of GDM because of its lowest P value, was downregulated with a log (fold change) of -12.76 in the blood samples of GDM patients. Changes in miR-146a in diabetic patients and animals were reported in a previous study. Liu et al. demonstrated that treatment of diabetic mice with miR-146a mimics robustly reduced diabetic peripheral neuropathy. Then, the target genes of hsa-miR-146a-3p were identified based on the parallel DEmRNAs in the present study. The results indicated that TRAF6 was not only identified as a target of hsa-miR-146a-3p but also a hub gene involved in the pathophysiological process of GDM. TRAF6 has been demonstrated to be a target gene of miR-146a at the experimental level in previous immune studies. Regarding diabetes, Kamali et al found that miR-146a antagomir significantly increased TRAF6 mRNA levels in human umbilical vein endothelial cells under hyperglycemic conditions. In addition, previous research revealed that culture of aortic endothelial cells in high glucose medium significantly increased TRAF6 expression in a time-dependent manner. In addition, TRAF6 mediated high glucose-induced endothelial dysfunction via NF-KB and AP-1-dependent signaling. To date, the role of miR-146a and its target gene TRAF6 in the pathogenesis of GDM has never been explored. In the present study, GSA revealed that several metabolic processes were enriched in the mechanism of TRAF6 in GDM. Moreover, TRAF6 was involved in all three core pathways regarding glypican metabolism in GDM based on pathway analysis of DEmiRNAs. Furthermore, we confirmed the altered expression of TRAF6 in 8 pairs of GDM blood samples to be higher than that in healthy pregnant blood samples in another independent patient group. Thus, serum TRAF6 may serve as a novel biomarker for GDM.
Other core genes, such as CASP8 and CHD3, were confirmed to be higher in 8 pairs of GDM blood samples than in healthy pregnant women blood samples. At present, there is no research on the relationship between CASP8 or CHD3 and GDM. Wu et al. found that maternal type 2 diabetes mellitus causes heart defects in the developing embryo manifested by excessive apoptosis in heart cells, with increasing apoptosis markers of cleaved caspase 3 and 8. Whether CASP8 can be used as a marker of fetal heart defects in pregnant women with GDM needs to be further studied.
There are limitations to this study. The serum GDM markers screened from the GSE19649 and GSE98043 datasets were independently externally validated using the GSE19649 dataset. The sample size was relatively small. However, the clinical information, such as age and BMI, of GDM and healthy pregnant women were matched in the GSE19649 dataset, which improved the test effectiveness. In addition, other GDM noninvasive markers explored in this study still need to be further verified by expanding the sample size in the future.
In summary, a series of potential GDM noninvasive serum markers were identified by integrated miRNA-mRNA expression profile analysis. Enrichment analysis indicated that GDM-related miRNAs were mainly enriched in the glypican pathway, proteoglycan syndecan-mediated signaling events, and syndecan-1-mediated signaling events. In addition, miR-146a-3p/TRAF6 might play a central role in the pathogenesis of GDM through the above pathways.