Gestational diabetes mellitus (GDM) could be identified as dramatic glucose fluctuation at the duration of pregnancy, and this prevailing disease is attributed to maternal overweight, late productivity age and the high incidence of diabetes, eventually leading to increasing cardiovascular risk to both mothers and infants [1]. Nowadays, GDM remains a prevalent complication of pregnant women that debilitates health of a considerable number of pregnancies [2]. Trophoblast cells are the main component of placental development, and their abnormal differentiation and death are the basis for impairing pregnancy health and leading to complications [3]. High glucose (HG) is a pivotal manifestation of GDM, and it could activate trophoblast cell damage and death, which in turn inflict placenta tissue functions and catalyze metabolic disorder [4,5]. As a mechanism of cell programmed death triggered by pro-inflammatory cytokines, pyroptosis results in microenvironmental disturbance and placenta impairment, so as to stimulate pregnancy diseases and their complications [6]. Currently, the possible GDM treatment approaches could cause unfavorable outcomes to maternal placenta and the unborn fetus [7]. Under this background, new prevention targets and possible management for GDM are in urgent need.

As the most common mRNA modification in multiple diseases, N6-methyladenosine (m6A) modification can be installed by the methyltransferase complex and removed by demethylases, thereby mediating GDM progression [8,9]. Methyltransferase 3 (METTL3), a predominant m6A RNA methyltransferase, is most widely discussed in the conditions of inflammation [10]. METTL3 is activated in HG environment, and it retards the development of placenta tissues and trophoblast cells by sabotaging cellular growth, survival, and mobility and accelerating cell death [11]. Functionally, transcription factors are necessarily involved in trophoblast cell biological behaviors of GDM patients [12]. CCAAT enhancer binding protein beta (CEBPB) is strongly expressed in GDM, which predicts increased glucose content and exasperated insulin resistance [13]. Moreover, CEBPB facilitates cell pyroptosis to aggravate cardiovascular symptoms [14]. Notably, CEBPB is regulated by m6A modification to influence immune function and inflammatory reaction [15]. Thus, we hypothesize that CEBPB expression in trophoblast cells might be modulated by METTL3-mediated m6A modification.

microRNAs (miRs) are reliable harbingers of GDM diagnosis, prevention and prognosis, and they are differentially expressed in subjects with GDM and they could mediate cellular metabolic activities [16]. The binding relation between CEBPB and miRs could be an interesting mechanism in type 2 diabetes [17], inspiring us to explore the possible miR binding to CEBPB. miR-96-5p expression is suppressed in HG environment, which disrupts trophoblast cell survival and proliferation [18]. Furthermore, miR-96-5p is negatively related to protein levels of pyroptosis-related indicators as miR-96-5p decelerates cardiomyocyte pyroptosis in sepsis [19]. As a transcription factor, CEBPB binds to the promoter of miRNAs, thereby negatively regulating miRNA expression [20]. Hereby, we assumed that CEBPB might target miR-96-5p promoter and negatively regulate its expression. Subsequently, possible target genes of miR-96-5p were explored via databases, and cyclin D2 (CCND2) was noticed. CCND2 is surprisingly increased in GDM by serving as a downstream target gene of miRs in the transcriptional factor-target mechanism [21]. Collectively, functional assays are carried out to discuss the mechanism of METTL3 in HG-induced trophoblast cell pyroptosis in GDM patients by the CEBPB/miR-96-5p/CCND2 axis via m6A modification, thus broadening the understanding of GDM mitigation.