Analysis of key genes and their functions in placental tissue of patients with gestational diabetes mellitus

Expectant mothers with gestational diabetes mellitus (GDM), a common pregnancy complication, have an increased risk of developing type 2 diabetes mellitus [1]. Over the past 20 years, the prevalence of GDM has doubled, affecting approximately 10% of pregnancies in the USA [2, 3]. Babies born to mothers with GDM are typically at a high risk for macrosomia, neonatal cardiac dysfunction, neonatal hypoglycemia, stillbirth, childhood obesity, and type 2 diabetes mellitus [4‐6]. Given the worldwide prevalence and adverse outcomes of GDM, there is an urgent need to grasp the pathophysiology and pathogenesis of the disease [2].

Previous studies have suggested that GDM is caused by enhanced insulin resistance and pancreatic beta (β)-cell dysfunction [7], involving genes that are related to insulin signaling, insulin secretion, maturity-onset diabetes of the young, and lipid and glucose metabolism, to name a few [8, 9]. Subsequently, it was found that inflammatory pathways [10], metabolic disorder [11], oxidative stress [12], and vitamin D concentrations [13] were also related to GDM. Furthermore, some genetic alterations, such as those of the genes encoding β3-adrenergic receptor [14] and transcription factor 7-like 2 polymorphism [15], were also found to be associated with GDM. Moreover, GDM results in major changes in the expression profiles of placental genes, with a significant increase in markers and mediators of inflammation [10]. Recently, several microarray studies have verified that the cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), estrogen receptor 1 (ESR1) [16], fibronectin 1 (FN1), and leptin (LEP) [17] genes were essential for the pathogenesis of GDM. However, because the genes related to GDM have not yet been fully identified, the biological processes underlying the pathogenesis of this disease remain unclear.

In this study, the gene expression profiles of placental tissue from women with GDM were compared with those of matched normal placental tissue by microarray analysis, to screen out differentially expressed genes (DEGs) in GDM. The identified DEGs were then submitted to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses to explore the crucial pathways of GDM. Additionally, a protein-protein interaction (PPI) network was constructed and subnetwork module mining was performed to seek out the candidate disease genes. Finally, microRNAs (miRNAs) and transcription factors (TFs) that target the candidate DEGs were identified and analyzed. The results from this study may lay the groundwork for future research on the pathogenesis of GDM.

GDM describes the condition of abnormal sugar metabolism or potential decreased glucose tolerance before pregnancy and is confirmed during pregnancy [30‐32]. It is a complex disease, being influenced by many factors such as the environment, society, and genes [33]. Moreover, genetic studies have suggested that multiple genes are involved in the disease [8]. In our study, DEGs in GDM and their enriched functions were screened out via bioinformatic analysis, and four key genes (viz., HLA, CXCL9, CXCL10, and PTPRC) were identified to be crucial to the disease. Moreover, miR-223-3p, miR-520, and TBP were found to be strongly linked to those DEGs, indicating their importance in GDM.

CXCL9 and CXCL10 are categorized as “inflammatory” chemokines. Shimada and coworkers postulated that the binding of CXCL10 to CXCR3 played a crucial role in the suppression of pancreatic β-cell proliferation [34]. Besides this, CXCL10 could interact with Toll-like receptor 4 to continuously activate c-Jun N-terminal kinases and protein kinase B (Akt), induce the cleavage of p21-activated protein kinase 2, and switch the Akt signal from proliferation to apoptosis, resulting in the suppression of pancreatic β-cell proliferation [35]. The present study demonstrated that CXCL10 was significantly enriched in the Toll-like receptor signaling pathway, leading us to speculate that it is a key gene that participates in the pathogenesis of GDM by regulating the progress of the Toll-like receptor signaling pathway. Although CXCL9 has similar functional and structural characteristics as CXCL10, it was reported that CXCL9 could not bind to Toll-like receptor 4 [36]. In this study, CXCL9 was significantly enriched in the cytokine signaling pathway and may thus play a critical role in the pathogenesis of GDM by regulating the inflammatory pathway.

HLA, the gene for the human MHC, plays a pivotal role in the antigen presentation of extracellular and intracellular peptides and the regulation of immune responses [37]. Compared with other regions of the human genome, the MHC genes on chromosome 6 are more associated with the susceptibility to common diseases like diabetes, and indeed many reports have shown that HLA gene variants are related to the predisposition to type 1 diabetes mellitus [38]. Additionally, although type 2 diabetes mellitus is not an autoimmune disease or associated with the HLA gene, there is evidence that genes in the HLA region might have an influence on the genetic susceptibility to this metabolic disorder [39]. Importantly, Steinborn and colleagues found that GDM was related to an increased humoral immune response against HLA-class II antigens [40]. Our study highlights the importance of HLA in the progression of GDM, during which the gene is downregulated, and emphasizes that the autoimmune response is significantly associated with the disease pathogenesis.

PTPRC (CD45) has an essential role in lymphocyte development, antigen receptor signal transduction, and modulation of the signals emanating from integrin and cytokine receptors [41]. In diabetes mellitus, protein tyrosine phosphatases act as negative regulators of insulin signal transduction [42]. A previous study demonstrated that the homozygous deletion of protein tyrosine phosphatase 1B (PTP1B) in myocytes enhanced both the insulin-dependent activation of insulin receptor autophosphorylation and the tyrosine phosphorylation of insulin receptor substrates, and increased insulin sensitivity [43]. Moreover, it was shown that the expression of PTPRC was related to residual β-cell function in type 1 diabetes mellitus [44]. Our results reveal that PTPRC is likely to be a key gene that impacts GDM.

Because miR-223 was found to be significantly dysregulated in GDM, it was selected as a potential circulating biomarker for this disease [45]. Additionally, as a stress-related miRNA, miR-223 negatively regulated the cryopyrin-encoding gene NLRP3 and subsequently interleukin-1 beta production [46]. In our study, production of the TFs zinc finger E-box binding homeobox 1 (ZEB1) and Forkhead box O1 (FOXO1) was regulated by miR-223-3p. FOXO1, a target of insulin signaling, regulates metabolic homeostasis in response to oxidative stress. The interaction of FOXO1 with β-catenin could attenuate the WNT signaling pathway, which is involved in lipid metabolism and glucose homeostasis [47]. Besides this, FOXO1 was targeted by miR-520 h and miR-520 g-3p, which were speculated to influence insulin sensitivity in human white adipose tissue through their predicted effects on glucose metabolism [48]. ZEB1, a zinc finger TF, is associated with placental development. It was reported that ZEB1 cooperated with FOXO members to suppress B-lymphocyte proliferation [49]. TBP is a universal eukaryotic TF. It was found that the enhancement of TBP-2 expression caused impairment of glucose-induced insulin secretion and insulin sensitivity [50]. In the present study, TBP was found to regulate many HLA genes (HLA-DQA1, HLA-F, and HLA-DQA2), implying its indispensable role in GDM.

In conclusion, four immune-related DEGs of GDM (viz., HLA, CXCL9, CXCL10, and PRPTC) appeared to be associated with not only the autoimmune process but also residual β-cell function. miR-223-3p, miR-520 (i.e., miR-520 h and miR-520 g-3p), and TBP regulated most of the DEGs, especially cellular metabolism-related genes (FOXO1 and ZEB1). These results provide new insights into the mechanisms of GDM pathogenesis.