study hypothesis: DNA methylation is regulated by hypoxia in endometriosis. study finding: Hypoxia causes global hypomethylation through AU-rich element binding factor 1 (AUF1)/microRNA-148a (miR-148a)- mediated destabilization of DNA methyltransferase 1 (DNMT1) mRNA. what is known already: Eutopic endometrial and ectopic endometriotic stromal cells have the same genetic background, but differ in several cellular and molecular responses. Both hypoxia and DNA methylation regulate several genes involved in the development of endometriosis. study design, samples/materials, methods: This laboratory study included 15 patients of reproductive age with endometriosis or normal menstrual cycles. Paired endometrial and endometriotic tissues were collected for assaying the levels of DNMT1, 3a and 3b using quantitative RT-PCR, western blot and immunohistochemical (IHC) staining. Primary cultured endometrial stromal cells maintained in normoxia/ hypoxia (1% O2) or treated with hypoxia-mimetic compounds were also assayed. The levels of DNA 5-methylcytosine were assayed by using IHC in clinical specimens and murine tissues, and by ELISA in cultured stromal cells. The 3'-untranslated region reporter assay was used to evaluate the effect of hypoxia, microRNAs (miRNAs) and human antigen R (HuR)/AUF1 on DNMT1 mRNA stability. RNA immunoprecipitation was used to assess the interaction of HuR/AUF1 and miR-148a/DNMT1 mRNA under hypoxia. Finally, a transplant-induced mouse model of endometriosis using 20 mice was used to elucidate the alteration of Dnmt1 levels and DNA methylation in the endometriotic tissues. main results and the role of chance: Levels of DNMT1 mRNA and protein and 5-methylcytosine were lower in the ectopic stromal cells (P , 0.05) than in the eutopic cells. Treatment with hypoxia and its mimetic compounds recapitulated the reduced levels ofDNMT1 and 5-methylcytosine levels (P < 0.05 versus control). Hypoxia treatment destabilized DNMT1 mRNA through recruitment of miR-148a and AUF1. Mutations introduced to the miR-148a targeting site or AU-rich element (ARE) restored the hypoxia-suppressedDNMT13'-untranslated region (3'-UTR) reporter activity (P < 0.05 versus control). Levels of proteins of three hypermethylated genes in endometrial stroma cells, GATA6, HOXA3 and SLC16A5, were elevated after 72 h of hypoxia treatment (P < 0.05 versus control). Finally, a transplant-induced model of endometriosis demonstrated the down-regulation of DNMT1 and a decrease in 5-methylcytosine in the endometriotic tissues (P , 0.05, eutopic versus ectopic). limitations, reasons for caution: Primary human cell cultures and a murine model were used in this study, and thus the results may not fully represent the situation in vivo. wider implications of the findings: This is the first study to elucidate how microenvironmental hypoxia links to the epigenetic effects ofDNAmethylation in the endometriosis, and to delineate the molecular mechanism of hypoxia-coordinated AUF1/miR-148a interaction and recruitment to DNMT1mRNAduring the pathogenesis of endometriosis. The development of future therapeutics in endometriosis may aim at disrupting this specific interaction and eventually restore the epigenetic regulation. study funding and competing interest(s): This workwas supported by the National Science Council of Taiwan (NSC101- 2320-B-006-030-MY3). The author declares that there are no conflicts of interest.
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