miR-196b targets c-myc and Bcl-2 expression, inhibits proliferation and induces apoptosis in endometriotic stromal cells

STUDY QUESTION: What is the global expression pattern of microRNAs (miRNAs) in endometriotic stromal cells and is miR-196b involved in the pathogenesis of endometriosis? SUMMARY ANSWER: Several miRNAs are aberrantly expressed in endometriotic cyst stromal cells (ECSCs), including miR-196b whose expression is repressed in endometriotic stromal cells. WHAT IS KNOWN ALREADY: Although, histologically, endometriotic tissues and normal proliferative endometrium are similar, a number of distinct molecular differences have been reported to date. The anti-apoptotic and excessive proliferative properties of endometriotic cells are considered to be involved in the development and progression of endometriosis. STUDY DESIGN AND SIZE DURATION: ECSCs and normal endometrial stromal cells (NESCs) were isolated from ovarian endometriotic tissues and eutopic endometrial tissues, respectively and compared. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Aberrantly expressed miRNAs in ECSCs were identified by a global miRNA microarray technique. The roles of miR-196b in ECSC proliferation, apoptosis, and c-myc and B-cell lymphoma/leukemia (Bcl)-2 mRNA expression were investigated with precursor hsa-miR-196b transfection. The methylation status of the miR-196b gene in ECSCs and the effect of a DNA demethylating agent on miR-196b expression were also examined. MAIN RESULTS AND THE ROLE OF CHANCE: miRNA microarray analysis identified eight down-regulated miRNAs (including miR-196b) and four up-regulated miRNAs in ECSCs. Compulsory expression of miR-196b directed the inhibition of proliferation and the induction of apoptosis in ECSCs. miR-196b was found to suppress c-myc and Bcl-2 mRNA expression in ECSCs, and there was a significant correlation between miR-196b and HOXA10 expression in ECSCs and NESCs. The miR-196b gene was hypermethylated in ECSCs when compared with NESCs, and the treatment with a DNA demethylating agent restored the expression of miR-196b in ECSCs. LIMITATIONS AND REASONS FOR CAUTION: miRNA expression profiles were investigated only in the stromal component of ectopic and eutopic endometrium samples. In addition to miR-196b, the roles of other miRNAs aberrantly expressed in ECSCs should be examined. WIDER IMPLICATIONS OF THE FINDINGS: The present findings suggest that aberrant miRNA expression plays an important role in the pathogenesis of endometriosis as a part of epigenetic mechanisms, that expression of miR-196b in ECSCs is repressed by DNA hypermethylation of the miR-196b gene and this repression may be involved in the development of proliferative and anti-apoptotic characteristics of endometriosis. STUDY FUNDING: This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 20591920 to K.N. and no. 23592407 to H.N.) and The Uehara Memorial Foundation (to K.N.).