FXYD1 was identified as a hub gene in recurrent miscarriage and involved in decidualization via regulating Na/K-ATPase activity

Abstract

Purpose: Recurrent miscarriage (RM) is a distressing and complicated adverse pregnancy outcome. It is commonly recognized that insufficient decidualization could result in RM, but the molecular mechanisms of decidual impairment are still not fully understood. Thus, this study aimed to identify novel key genes potentially involved in RM and explore their roles played in endometrial decidualization.

Methods: Initially, a combinative analysis of decidual and mid-secretory endometrial transcriptomes was performed to discover hub genes involved in the etiology of RM. And the expression levels of hub genes were evaluated in both primary decidual stromal cells (DSCs) and decidual tissues. Subsequently, the immortalized human endometrial cell line, T-HESCs, was used to investigate whether FXYD1 overexpression affects decidualization by regulating Na/K-ATPase activity.

Results: FXYD domain containing ion transport regulator 1 (FXYD1) was identified as a hub gene in the pathogenesis of RM through various bioinformatic methods. Abnormally increased FXYD1 expression was observed in DSCs and decidual tissues from RM patients compared to that of the normal group. Furthermore, in vitro decidualization was obviously inhibited by the overexpression of FXYD1. Additionally, Na/K-ATPase activity was significantly elevated during decidualization, whereas overexpression of FXYD1 reduced Na/K-ATPase activity. Bufalin, a Na/K-ATPase inhibitor, showed an effectively inhibitory effect on decidualization.

Conclusions: Collectively, FXYD1 was discovered as a hub gene associated with RM, and its expression levels in RM patients were significantly upregulated. Increased FXYD1 expression might lead to decidualization defects by reducing Na/K-ATPase activity, of which presented a novel prospective treatment target for RM.

Keywords: Decidualization; FXYD; Hub genes; Na/K-ATPase; Recurrent miscarriage.

Fig. 1

Identification of DEGs in the decidual tissues and mid-secretory endometrial tissues from RM patients. A Volcano plot of all identified DEGs in the decidual tissues, blue dots represent the downregulated genes, and red dots represent the upregulated genes (3 RM patients vs 3 normal early pregnant women). B An expression heat plot of the top 50 DEGs in the decidual tissues. C Volcano plot of all DEGs in the mid-secretory endometrial tissues, blue dots represent the downregulated genes, and red dots represent the upregulated genes (24 RM patients vs 24 control women). D An expression heat plot of the top 50 DEGs in the mid-secretory endometrial tissues

Fig. 2

Combinative analysis of decidual DEGs and mid-secretory endometrial DEGs from RM patients. A The common upregulated DEGs between decidual and mid-secretory endometrial transcriptomes in RM. B The common downregulated DEGs between decidual and mid-secretory endometrial transcriptomes in RM. C A bar plot shows the GO terms of common DEGs. D The PPI network of common DEGs based on the STRING database. E The top six small molecular compounds with potential therapeutic effects for RM identified by the CMAP database

Fig. 3

Identification of hub modules correlated with RM in the GSE165004 dataset. A Cluster dendrogram of samples. B The scale independence and mean connectivity for various soft threshold powers. C The gene clustering dendrogram obtained from average linkage hierarchical clustering and each color represents a co-expression module. D Heatmap of the correlation between ME and traits (each cell contains correlation coefficients and corresponding p-values). E A bar plot shows the 15 significant GO terms of the genes within the green module, including the top 5 BPs, CCs, and MFs

Fig. 4

Identification of RM-related hub genes. A The Venn diagram of the genes within the green module and the common DEGs obtained from the endometrial and decidual transcriptomes. B The expression level of FXYD1 mRNA in the GSE161969 (decidua). C The expression level of FXYD1 mRNA in the GSE26787 (endometrium)

Fig. 5

The expression of FXYD1 was aberrantly increased in the decidua from RM patients. A The FXYD1 mRNA expression in the decidua of RM patients (RM, n = 8) and normal early pregnant women (CON, n = 8) by RT-qPCR. B The FXYD1 protein expression in the decidua of RM patients (RM, n = 8) and normal early pregnant women (CON, n = 8) by Western blotting. C Densitometric quantification of FXYD1 protein expression in the decidua normalized to GAPDH. D The FXYD1 protein expression in the primary DSCs of RM patients (RM, n = 4) and normal early pregnant women (CON, n = 4) by Western blotting. E Densitometric quantification of FXYD1 protein expression in the primary DSCs normalized to GAPDH. *p < 0.05. **p < 0.01. ***p < 0.001. DSC, decidual stromal cells

Fig. 6

FXYD1 overexpression attenuated decidualization in vitro. A–E The FXYD1, PRL, IGFBP1, FOXO1, and WNT4 mRNA expression in T-HESCs transfected with either control vector or FXYD1-overexpression’ plasmids at 0, 2, and 4 days during decidualization. oe-NC, control vector; oe-FXYD1, FXYD1-overexpression. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001

Fig. 7

FXYD1 modulates decidualization by influencing Na/K-ATPase activity. A The Na/K-ATPase activity in T-HESCs transfected with either control vector or FXYD1-overexpression plasmids. B The Na/K-ATPase activity in T-HESCs at 0, 2, and 4 days during decidualization. C The cell viability in T-HESCs treated with different concentrations of bufalin for 48 h. D–G The PRL, IGFBP1, FOXO1, and WNT4 mRNA expression in T-HESCs treated with bufalin for 4 days during decidualization. con, control; dc, decidualization. *p < 0.05. **p < 0.01. ****p < 0.0001