Differentially-Expressed miRNAs in Ectopic Stromal Cells Contribute to Endometriosis Development: The Plausible Role of miR-139-5p and miR-375

Abstract

microRNA (miRNA) expression level alterations between endometrial tissue and endometriotic lesions indicate their involvement in endometriosis pathogenesis. However, as both endometrium and endometriotic lesions consist of different cell types in various proportions, it is not clear which cells contribute to variability in miRNA levels and the overall knowledge about cell-type specific miRNA expression in ectopic cells is scarce. Therefore, we utilized fluorescence-activated cell sorting to isolate endometrial stromal cells from paired endometrial and endometrioma biopsies and combined it with high-throughput sequencing to determine miRNA alterations in endometriotic stroma. The analysis revealed 149 abnormally expressed miRNAs in endometriotic lesions, including extensive upregulation of miR-139-5p and downregulation of miR-375 compared to eutopic cells. miRNA transfection experiments in the endometrial stromal cell line ST-T1b showed that the overexpression of miR-139-5p resulted in the downregulation of homeobox A9 (HOXA9) and HOXA10 expression, whereas the endothelin 1 (EDN1) gene was regulated by miR-375. The results of this study provide further insights into the complex molecular mechanisms involved in endometriosis pathogenesis and demonstrate the necessity for cell-type-specific analysis of ectopic tissues to understand the interactions between different cell populations in disease onset and progression.

Keywords: EDN1; HOXA10; ectopic stroma; endometriosis; miR-139-5p; miR-375; microRNA; small RNA sequencing.

Figure 1

Relative miRNA expression levels (log2 scale) in (A) paired uncultured eutopic (n = 6) and ectopic (n = 6) stromal cells and (B) paired cultured eutopic (n = 6) and ectopic (n = 6) stromal cells. The ΔCt values were calculated as follows: miRNA Ct value − average Ct value of reference genes (RNU44 and RNU48). * p-value < 0.05. Outliers (defined as datapoints outside 1.5 times the interquartile range above the upper quartile and below the lower quartile) are pointed out with black dots. For illustrative purposes, relative expression levels (ΔCt) were multiplied by −1.