Transcriptomic analysis and competing endogenous RNA network in the human endometrium between proliferative and mid-secretory phases

Abstract

Successful embryo implantation is the first step for establishing natural pregnancy and is dependent on the crosstalk between the embryo and a receptive endometrium. However, the molecular signaling events for successful embryo implantation are not entirely understood. To identify differentially expressed transcripts [long-noncoding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs] and competing endogenous RNA (ceRNA) networks associated with endometrial receptivity, the current study analyzed gene expression profiles between proliferative and mid-secretory endometria in fertile women. A total of 247 lncRNAs, 67 miRNAs and 2,154 mRNAs were identified as differentially expressed between proliferative and mid-secretory endometria. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that these differentially expressed genes were significantly enriched for 'cell adhesion molecules.' Additionally, 98 common mRNAs were significantly involved in tryptophan metabolism, metabolic pathways and FoxO signaling. From the differentially expressed lncRNA/miRNA/mRNA ceRNA network, hub RNAs that formed three axes were identified: The DLX6-AS1/miR-141 or miR-200a/OLFM1 axis, the WDFY3-AS2/miR-135a or miR-183/STC1 axis, and the LINC00240/miR-182/NDRG1 axis. These may serve important roles in the regulation of endometrial receptivity. The hub network of the current study may be developed as a candidate marker for endometrial receptivity.

Keywords: RNA sequencing; competing endogenous RNA network; endometrial receptivity; mid-secretory endometrium; proliferative endometrium.

Figure 1

Volcano plots and hierarchical clustering analysis of the DE transcripts in proliferative and mid-secretory endometria. Volcano plots of DE (A) mRNAs and (B) lncRNAs. The top 5 up and down DE transcripts are indicated by red circles. The hub RNAs are indicated by green circles. Hierarchical clustering analysis of the DE (C) mRNAs and (D) lncRNAs. Transcripts were selected using P<0.05 and an FC ≥1.5 or ≤-1.5. Blue boxes indicate proliferative endometrium samples. Red boxes indicate mid-secretory endometrium samples. DE, differentially expressed; lncRNA, long non-coding RNAs; up, upregulated; down, downregulated; FC, fold change.

Figure 2

Volcano plot and hierarchical clustering analysis of DE miRNAs in proliferative and mid-secretory endometria. (A) Volcano plot of DE miRNAs. The top 5 up and down DE miRNAs are indicated by red circles. The hub RNAs are indicated by green circles. (B) Hierarchical clustering analysis of DE miRNAs. miRNAs were selected using P<0.05 and FC ≥1.5 or ≥-1.5. DE, differentially expressed; miRNA, microRNA; up, upregulated; down, downregulated.

Figure 3

GO enrichment and KEGG pathway analysis based on differentially expressed genes in the proliferative and secretory endometria. Top 10 upregulated GO terms enriched for (A) biological processes, (B) cellular components and (C) molecular functions. Top 10 downregulated GO terms enriched for (D) biological processes, (E) cellular components and (F) molecular functions. (G) The top 20 KEGG pathways based on the up- and downregulated genes. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4

Quantitative PCR validation of DE lncRNAs, miRNAs and mRNAs in proliferative and mid-secretory endometria. The expression of (A) five lncRNAs, (B) five miRNAs and (C) five mRNAs were detected by quantitative PCR in proliferative and mid-secretory endometrium samples. Data are presented as the normalized fold change. Experiments were repeated in triplicate, and values are expressed as mean ± SEM. ^*P<0.05 and ^**P<0.01 vs. proliferative. lncRNA, long non-coding RNA; miRNA or miR, microRNA.

Figure 5

Transcriptome and bioinformatics analysis of DEGs from the current study and previous RNA-sequencing results. (A) Venn diagram of the number of DEGs in the four assessed studies. (B) Top 20 KEGG pathways for the 98 common genes of the four studies. Ingenuity pathway analysis network generated from 98 common genes from the four studies. (C) Network related with ‘cell cycle, cellular assembly and organization, DNA replication, and recombination and repair’. (D) Network related with ‘cell death and survival, cellular development, and connective tissue development and function’. DEGs, differentially expressed genes.

Figure 6

ceRNA network related to endometrial receptivity. (A) lncRNA/miRNA/mRNA ceRNA network for proliferative and mid-secretory endometria. (B) ceRNA subnetwork for DLX6-AS1. (C) ceRNA subnetwork for WDFY3-AS2. (D) ceRNA subnetwork for LINC00240. Circles, squares and diamonds represent lncRNAs, miRNAs and genes, respectively. Upregulated genes are indicated in red and downregulated genes are indicated in blue. Dotted lines emphasize important networks related to endometrial receptivity. lncRNA, long non-coding RNA; miRNA, microRNA; ceRNA, competitive endogenous RNA.

Figure 7

RNA expression correlation between the three ceRNA subnetworks in proliferative and secretory endometria. The expression of (A) DLX6-AS1, hsa-miR-141-3p and OLFM; (B) WDFY3-AS2, hsa-miR-183-5p and STC1; and (C) LINC00240, hsa-miR-182-5p and NDRG1 (C) was detected by quantitative PCR in proliferative and secretory endometria. The circles, squares and diamonds represent lncRNAs, miRNAs and genes, respectively. Upregulated genes are indicated in red and downregulated genes are indicated in blue. All data are presented as the mean ± SEM, and experiments were repeated in triplicate. ^**P<0.01. ceRNA, competitive endogenous RNA; lncRNA, long non-coding RNA; miRNA, microRNA>=.