miR-199a Is Upregulated in GDM Targeting the MeCP2-Trpc3 Pathway

Abstract

Gestational diabetes mellitus (GDM), the most common medical pregnancy complication, has become a growing problem. More and more studies have shown that microRNAs are closely related to metabolic processes. The purpose of this paper is to investigate the role of up-regulation of miR-199a-5p expression in GDM. We found that miR-199a-5p was significantly up-regulated in the placenta of GDM patients compared with normal pregnant women, and expressed in placental villi. miR-199a-5p can regulate the glucose pathway by inhibiting the expression of methyl CpG-binding protein 2 (MeCP2) and down-regulating canonical transient receptor potential 3 (Trpc3). This suggests that miR-199a-5p may regulate the glucose pathway by regulating methylation levels, leading to the occurrence of GDM.

Keywords: MeCP2; MiR-199a; TRPC3; gestational diabetes mellitus; placenta.

Figure 1

The expression of miR-199a is up-regulated in the placenta of GDM patients. (A, B) In situ hybridization of miR-199a-specific DIG-labeled LNA probes to detect the localization and expression of miR-199a in the placenta. (C) Real-time PCR detection of miR-199a expression in placenta. n=10. The scale bar indicates a distance of 1000μm. *P<0.05; **P<0.01.

Figure 2

miR-199a mimic upregulates cellular methylation levels. (A, B) 5-aza immunohistochemical detection of the effects of miR-199a mimic and miR-199a inhibitor on the overall methylation level of JEG-3 cells. Brown is a positive signal. n=3. (C) Real-time PCR detection of miR-199a expression in JEG-3 cells treated with different concentrations of 5-aza. n=3. The scale bar indicates a distance of 100μm. *P<0.05; **P<0.01.

Figure 3

Target gene verification of miR-199a. (A) Prediction of the binding site of miR-199a and the 3’-UTR of MeCP2 mRNA and dual-luciferase detection of the target gene interaction relationship between miR-199a and the 3’-UTR of MeCP2 mRNA. n=3. (B) The inhibitory effect of miR-199a on MeCP2 was significantly reduced after induction of point mutations to the binding site. n=3. (C, D) Western-blot verification of the regulatory effect of miR-199a on the protein level of the target gene MeCP2. *P<0.05, **P<0.01.

Figure 4

The expression of MeCP2 is down-regulated in the placenta of GDM patients. (A, B) Immunohistochemical assay to detect the expression of MeCP2 in the placenta of GDM patients. Brown is a positive signal. *P<0.05.

Figure 5

miR-199a regulates MeCP2-TRPC3 expression. (A) The expression of Trpc3 was detected by real-time PCR. n=10. (B) The expression of SFRP4 was detected by real-time PCR. n=10. (C, D) Western blot experiments to detect the expression levels of Trpc3 and SFRP4 in JEG3 cells transfected with miR-199a mimics. n=3, *P<0.05, **P<0.01.