The expression and ovarian steroid regulation of endometrial micro-RNAs

Abstract

MicroRNAs (miRNAs) which regulate gene expression stability displayed an aberrant expression profile in ectopic endometrium (ECE) as compared to eutopic (EUE) and normal endometrium (NE). We assessed the expression of miR-17-5p, miR-23a, miR-23b and miR-542-3p, their predicted target genes, steroidogenic acute regulatory protein, aromatase and cyclooxygenase-2, and influence of ovarian steroids on their expression in endometrial stromal (ESC) and glandular epithelial cells (GEC). The results indicated a lower expression of miR-23b and miR-542-3p and higher level of miR-17-5p in paired ECE and EUE as compared with NE. These levels were elevated and inversely correlated with the level of expression of their respective target genes in ECE. The expression of these miRNAs and genes was differentially regulated by 17beta- estradiol, medroxyprogesterone acetate, ICI-182780 and RU-486, or their respective combinations in ESC and GEC. We concluded that altered expression of specific miRNAs in ECE, affecting the stability of their target genes expression, has direct implications in pathogenesis of endometriosis.

Figure 1

Bar graphs show the mean ± SEM of relative expression of miR-23a, miR-23b, miR-17-5p, and miR-542-3p as well as CYP19A1, steroidogenic acute regulatory protein (StAR), and cyclooxygenase 2 (COX-2) in the endometrium of women without endometriosis (EN) and paired eutopic (Eutopic Endo, EU) and ectopic (Ectopic-Endo, EC) endometrium. The level of expression of these micro-RNAs (miRNAs) and messenger ribonucleic acids (mRNAs) was determined relative to control using ΔΔCT method and their mean values in EN were independently set at 1. Asterisks ** and *** are significantly different from *, and ** are different from *** (P < .05) analyzed using either a 2-tailed Student t test for 2 group comparisons or AN OVA with Tukey’s test for multiple comparisons.

Figure 2

Bar graphs show the expression of miR-17-5p, miR-23a, miR-23b, and miR-542-3p as well as steroidogenic acute regulatory protein (StAR), cyclooxygenase 2 (COX-2), and CYP19A1 in endometrial stromal cells (ESCs) isolated from women without endometriosis and the effect of ovarian steroids on their expression. The ESCs (1 × 10⁶/well in 6-well plates) were cultured and following treatments with 17β-estradiol (E₂), ICI-182780 (ICI), E₂ + ICI, medroxyprogesterone acetate (MPA), RU-486 (RU), and MPA + RU, their total RNA was isolated and subjected to real-time polymerase chain reaction (PCR). The data were expressed as relative to control using ΔΔCT method and following independent normalization the expression level of each micro-RNA (miRNA) and messenger RNA (mRNA) in untreated control (Crtl) was arbitrarily set as 1. Asterisk * is significantly different from Ctrl, with arrows showing the differences between corresponding treatment groups, ie, E₂, ICI, E₂ + ICI. These experiments were preformed using 3 independent cell cultures and results were analyzed using either a 2-tailed Student t test for 2 group comparisons or ANOVA with Tukey’s test for multiple comparisons with P < .05 was considered significant.

Figure 3

Bar graphs show the expression of miR-17-5p, miR-23a, miR-23b, and miR-542-3p as well as steroidogenic acute regulatory protein (StAR), cyclooxygenase 2 (COX-2) and CYP19A1 in endometrial glandular epithelial cells (GEC) isolated from women without endometriosis and the effect of ovarian steroids on their expression. The endometrial stromal cells (ESCs; 1 × 10⁶/well in 6-well plates) were cultured and following treatments with 17β-estradiol (E₂), ICI-182780 (ICI), E₂ + ICI, medroxyprogesterone acetate (MPA), RU-486 (RU) and MPA + RU, their total RNA was isolated and subjected to real-time polymerase chain reaction (PCR). The data were expressed as relative to control using ΔΔCT method and following independent normalization, the expression level of each micro-RNA (miRNA) and messenger RNA (mRNA) in untreated control (Crtl) was arbitrarily set as 1. Asterisk * is significantly different from Ctrl, with arrows showing the differences between corresponding treatment groups, ie, E₂, ICI, E₂ + ICI. These experiments were preformed using 3 independent cell cultures and the results were analyzed using either a 2-tailed Student t test for 2 group comparisons or ANOVA with Tukey’s test for multiple comparisons with P < .05 considered significant.