Transcriptome analysis of eutopic endometrial stromal cells in women with adenomyosis by RNA-sequencing

Abstract

This study aimed to identify differentially expressed genes (DEGs) and molecular pathways in eutopic endometrial stromal cells (EuESCs) from adenomyosis (AM) patients and to provide a new insight into the disease mechanisms. The gene expression profiles in adenomyotic EuESCs (A-EuESCs) and normal ESCs (N-ESCs) were analyzed by RNA-sequencing (RNA-Seq) and validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses were performed to obtain insights into the functions of DEGs. The protein-protein interaction (PPI) network was constructed using the STRING database and visualized by Cytoscape software, and their hub genes were identified. A total of 458 up-/363 down-regulated genes were identified in A-EuESCs versus N-ESCs. The GO enrichment analysis showed that these genes were significantly enriched in calcium-dependent cell-cell adhesion. The most significant term of the KEGG pathway analysis was cytokine-cytokine receptor interaction. There were 145 nodes in the PPI network of the 157 DEGs, which were identified in significant enrichment pathway by the KEGG pathway analysis in N-ESCs and A-EuESCs. The PPI network revealed that IL-6 was a central hub gene. Besides, IL-6 was found as a central hub gene in the pro-inflammatory/chemotactic subnetwork, and EGF was noted as a central hub gene in the angiogenesis subnetwork. Our study indicated the alterations of transcriptomic profiles in A-EuESCs and provided new insights into the pathogenesis of AM. The A-EuESCs in women with AM have fundamental abnormalities that may predispose to pro-invasion/migration and angiogenesis.

Keywords: Adenomyosis; RNA sequencing; angiogenesis; chemokines; eutopic endometrial stromal cells; inflammatory cytokines; transcriptome.

Graphical abstract

Figure 1.

Differential expression analysis in N-ESCs and A-EuESCs (a) Volcano plot shows DEGs in N-ESCs and A-EuESCs. (b) Bar chart showed the number of upregulated and downregulated DEGs in A-EuESCs compared with N-ESCs. (c) Unsupervised principal component analysis (PCA) of different cell lines (3 N-ESCs and 3 A-EuESCs). (d) Hierarchical clustering analysis of the DEGs in N-ESCs group (3 N-ESCs are abbreviated as N1, N2, and N3, respectively) and A-EuESCs group (3 A-EuESCs are abbreviated as A1, A2, and A3, respectively). Yellow and blue bar charts represent significantly upregulated and downregulated genes, respectively.

Figure 2.

Verification of the expression levels of DEGs by qRT-PCR qRT-PCR of top 10 upregulated (a) or downregulated (b) DEGs by RNA-Seq in A-EuESCs (n = 15) compared with N-ESCs (n = 15). GAPDH was used as an internal control for normalization. At least three independent experiments were performed for statistical evaluation. Error bars represent standard deviation (SD). *P < 0.05, *P < 0.01, *P < 0.001, A-EuESCs vs. N-ESCs; ns, not statistically significant.

Figure 3.

Functional annotation analysis of DEGs (a) GO annotation was performed using 821 DEGs in N-ESCs and A-EuESCs to obtain insights into their biological functions. The DEGs were classified into three functional groups, including biological process, cellular component and molecular function. (b) KEGG annotation was performed using 821 DEGs in N-ESCs and A-EuESCs to obtain insights into their pathways involved.

Figure 4.

GO and KEGG pathway enrichment analyses of the significant DEGs. (a) The top 25 enriched GO biological processes of the upregulated DEGs. (b) The top 25 enriched GO biological processes of the downregulated DEGs. (c) The top 25 enriched GO biological processes of all DEGs. (d) The top 25 enriched KEGG pathways of the upregulated DEGs. (e) The top 25 enriched KEGG pathways of the downregulated DEGs. (f) The top 25 enriched KEGG pathways of all DEGs.

Figure 5.

Chord plot analysis of GO terms and KEGG pathways. (a) GO chord plot of 4 designated GO terms belonging to the Biological Process (BP) sub-ontology for A-EuESCs. (b) KEGG chord plot of 2 designated KEGG pathways for A-EuESCs. The genes are linked to their assigned terms or pathways via colored ribbons. Genes are ordered according to the observed log2 fold-change (log2FC), which can be shown in descending intensity of red squares next to the selected genes.

Figure 6.

The constructed PPI networks and subnetworks of the DEGs in N-ESCs and A-EuESCs. (a) PPI network of the 157 DEGs identified in significant enrichment pathway (adjusted P < 0.05) by the KEGG pathway analysis in N-ESCs and A-EuESCs. (b) PPI subnetwork of the DEGs related to inflammatory cytokines and chemokines. (c) PPI subnetwork of the DEGs related to the regulation of angiogenesis. The red circular nodes represent the upregulated DEGs and the blue circular nodes represent the downregulated DEGs; black edges indicate the interaction between two proteins. The larger the circular nodes, the greater the centrality in the PPI network.

Figure 7.

ESCs-secreted protein levels of hub genes in the cell culture supernatants ESCs-secreted protein levels in the cell culture supernatants from N-ESCs (n = 15) and A-EuESCs (n = 15) were determined using ELISA kits. (a) The concentration of IL-6 in the cell culture supernatants. (b) The concentration of EGF in the cell culture supernatants. *P < 0.05, *P < 0.01, *P < 0.001, A-EuESCs vs. N-ESCs.