Differential gene expression in decidualized human endometrial stromal cells induced by different stimuli

Abstract

Decidualization can be induced by culturing human endometrial stromal cells (ESCs) with several decidualization stimuli, such as cAMP, medroxyprogesterone acetate (MPA) or Estradiol (E₂). However, it has been unclear how decidualized cells induced by different stimuli are different. We compared transcriptomes and cellular functions of decidualized ESCs induced by different stimuli (MPA, E₂ + MPA, cAMP, and cAMP + MPA). We also investigated which decidualization stimulus induces a closer in vivo decidualization. Differentially expressed genes (DEGs) and altered cellular functions by each decidualization stimuli were identified by RNA-sequence and gene-ontology analysis. DEGs was about two times higher for stimuli that use cAMP (cAMP and cAMP + MPA) than for stimuli that did not use cAMP (MPA and E₂ + MPA). cAMP-using stimuli altered the cellular functions including angiogenesis, inflammation, immune system, and embryo implantation whereas MPA-using stimuli (MPA, E₂ + MPA, and cAMP + MPA) altered the cellular functions associated with insulin signaling. A public single-cell RNA-sequence data of the human endometrium was utilized to analyze in vivo decidualization. The altered cellular functions by in vivo decidualization were close to those observed by cAMP + MPA-induced decidualization. In conclusion, decidualized cells induced by different stimuli have different transcriptome and cellular functions. cAMP + MPA may induce a decidualization most closely to in vivo decidualization.

Figure 1

Differential gene expressions by different decidualization stimuli. (A) Hierarchical cluster analysis comparing the transcriptome of four types of dESCs induced by different stimuli (cAMP, cAMP + MPA, MPA, and E₂ + MPA,) and the corresponding two control samples. Distances of gene expression pattern are indicated as height. (B) Numbers of DEGs in dESCs induced by four types of decidualization stimuli. (C) 4-way Venn diagrams comparing DEGs induced by four types of decidualization stimuli. Genes belonging to each compartment (a–o) are shown in Supplementary Table S2. (D) Venn diagrams indicating the number of specifically and commonly up- or down-regulated genes between decidualization stimuli.

Figure 2

Differences in cellular functions altered in dESCs by different stimuli. The up- or down-regulated genes by each decidualization stimulus were subjected to GO-REVIGO analysis. The GO terms were classified into several groups according to their associated cellular functions (center column). Common cellular functions are indicated with orange rectangles. The cellular functions altered by cAMP-used stimuli and MPA-used stimuli are indicated with blue and yellow rectangles, respectively. The representative GO terms belonging to each cellular function are shown. The ratio of the number of identified genes to all genes in each term is designated as “Gene ratio” and is indicated with the size of the circle. P-values of each term are indicated with colors.

Figure 3

Validation of RNA-sequence data by real-time RT-PCR. ESCs were treated with the decidualization stimuli for 4 days (cAMP or cAMP + MPA) or 14 days (MPA or E₂ + MPA). ESCs cultured without decidualization stimuli for 4 days or 14 days were used as the controls, respectively. We selected 8 genes that are the representative of the uncommon cellular functions. mRNA levels of these selected genes and decidualization markers (IGFBP-1 and PRL) were quantified by real-time RT-PCR. Values were normalized to those of MRPL19. The relative mRNA expression levels of each decidualized cells (cAMP, cAMP + MPA, MPA, E₂ + MPA) were calculated as fold changes to the corresponding control cells. We repeated the three independent experiments on each three independent patient's cells. Then, the mean ± SE of fold change from three different individuals was used as a relative expression value in decidualized cells. Data are mean ± SE of three independent experiments. a, P < 0.05 vs. control; b, P < 0.01 vs. control; c, P < 0.05 vs. cAMP; d, P < 0.01 vs. cAMP.

Figure 4

Cellular functions associated with DEGs by in vivo decidualization. By utilizing public single-cell RNA-sequence data of the human endometrium during the menstrual cycle, 2579 and 3768 genes were identified as the up- or down-regulated genes by in vivo decidualization, respectively. They were subjected to GO-REVIGO analysis. The GO terms were classified into several groups according to their associated cellular functions (center column). The representative GO terms belonging to each cellular function are shown. The ratio of the number of identified genes to all genes in each term is designated as “Gene ratio” and is indicated with the size of the circle. P-values of each term are indicated with colors.

Figure 5

Comparison of DEGs and cellular functions between in vivo decidualization and in vitro decidualization. (A) DEGs by in vitro decidualization were compared with those by in vivo decidualization. The ratios of the number of common DEGs by each decidualization stimuli to DEGs by in vitro decidualization are shown as percentages. (B) Cellular functions associated with common DEGs by cAMP + MPA stimulus. 226 up-regulated genes and 536 down-regulated genes by cAMP + MPA stimulus were identified as common DEGs. They were subjected to GO-REVIGO analysis. The GO terms were classified into several groups according to their associated cellular functions (center column). The representative GO terms belonging to each cellular function are shown. The ratio of the number of identified genes to all genes in each term is designated as “Gene ratio” and is indicated with the size of the circle. P-values of each term are indicated with colors.