microRNA miR-200b differentially affects proliferation, invasiveness and stemness of endometriotic cells by targeting the transcription factors KLF4, ZEB1 and ZEB2

Introduction: Endometriosis is a hormone-dependent disease characterized by growth of endometrial tissue at ectopic locations. Recent results indicate a dysregulation of microRNAs, small non-coding posttransciprional regulators of gene expression in endometriosis, suggesting a role in the pathogenetic process. In this study, we investigated the functional impact of miR-200b, a microRNA downregulated in endometriosis, on the immortalized endometriotic cell line 12Z and primary endometriotic stroma cells in vitro.

Methods: Endometriotic cells were transiently transfected with miR-200b precursors or negative control miRNAs. Targets of miR-200b were analyzed by quantitative real-time PCR. The cellular phenotype was monitored by Matrigel invasion and MTT cell viability assays as well as flow cytometric methods.

Results: Quantitative real-time PCR showed a significant downregulation of the transcription factors ZEB1 and ZEB2, and an increase of E-cadherin, a target predicted to inhibit epithelial-mesenchymal transition (EMT). This hypothesis was confirmed by the results of invasion-assays which showed a decreased invasiveness of 12Z cells. Furthermore, the stem cell-factor KLF4 was expressed more strongly due to miR-200b overexpression along with an increase in the proportion of cells with high ABC-transporter activity (side population-analysis) known as a functional stem cell property, and an increase in cell viability.

Conclusions: We conclude that the down-regulation of miR-200b in endometriosis tissue may induce EMT and increase invasiveness, possibly facilitating establishment of lesions at ectopic sites. In contrast, miR-200b downregulation may reduce stem cell features via regulation of KLF4 expression.