High-Throughput Sequencing Approach Uncovers the miRNome of Peritoneal Endometriotic Lesions and Adjacent Healthy Tissues

<div><p>Accumulating data have shown the involvement of microRNAs (miRNAs) in endometriosis pathogenesis. In this study, we used a novel approach to determine the endometriotic lesion-specific miRNAs by high-throughput small RNA sequencing of paired samples of peritoneal endometriotic lesions and matched healthy surrounding tissues together with eutopic endometria of the same patients. We found five miRNAs specific to epithelial cells – miR-34c, miR-449a, miR-200a, miR-200b and miR-141 showing significantly higher expression in peritoneal endometriotic lesions compared to healthy peritoneal tissues. We also determined the expression levels of miR-200 family target genes E-cadherin, ZEB1 and ZEB2 and found that the expression level of E-cadherin was significantly higher in endometriotic lesions compared to healthy tissues. Further evaluation verified that studied miRNAs could be used as diagnostic markers for confirming the presence of endometrial cells in endometriotic lesion biopsy samples. Furthermore, we demonstrated that the miRNA profile of peritoneal endometriotic lesion biopsies is largely masked by the surrounding peritoneal tissue, challenging the discovery of an accurate lesion-specific miRNA profile. Taken together, our findings indicate that only particular miRNAs with a significantly higher expression in endometriotic cells can be detected from lesion biopsies, and can serve as diagnostic markers for endometriosis.</p></div

Receiver operating characteristic (ROC) curve analysis for combined signature of miR449a/200a/200b.

<p>The miRNA expression signature of miR-449a/200a/200b discriminated endometriotic lesions (n = 22) from non-diseased tissues (n = 24) with 95.8% sensitivity and 95.4% specificity. Area under the ROC curve (AUC) is 0.95 (95% CI, 0.83–0.99).</p

The expression of selected miRNAs in endometriotic lesions (n = 22), histologically unconfirmed lesions (n = 10) and healthy tissues (n = 14) using qRT-PCR.

<p>The fold changes for endometriotic lesions and unconfirmed lesions are calculated relative to healthy tissues using the 2^−ΔΔCT method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112630#pone.0112630-Livak1" target="_blank">[26]</a>. The error bars denote SEM (standard error of the mean). * Denotes comparison of endometriotic lesions with healthy tissues, <i>P</i><0.0001.</p