RESUMO
OBJECTIVE: To evaluate whether Let-7b regulatory regions are methylated in endometriosis and whether there are specific CpG methylation sites that can be identified as key epigenetic regulatory locations. DESIGN: Laboratory study. SETTING: Academic Medical Center. PATIENT(S): Twenty-one women with (n = 12) and without (n = 9) endometriosis. INTERVENTION(S): Laboratory investigation. In vitro assessment of Let-7b methylation. MAIN OUTCOME MEASURE(S): Four targeted regions upstream of Let-7b predicted to be the regulatory regions of this microribonucleic acid (miRNA) were amplified using bisulfite-specific polymerase chain reaction. Deoxyribonucleic acid sequences were analyzed to determine methylation status at each predicted regulatory region and CpG island. RESULT(S): Regions were chosen on the basis of percent (%) GC content and data from Ensembl/ENCODE databases, which predict locations of promoters, enhancers, CTCF, and transcription factor binding sites as well as candidate cis-regulatory elements. A region 1,161 base pairs upstream of the Let-7b coding region was significantly differentially methylated in ectopic samples compared with eutopic endometrium from patients with endometriosis. Four specific CpG islands within this region 2 were further analyzed individually, and 1 was found to be significantly methylated in endometriosis. We identified that transcription factor SP1 was predicted to bind to a sequence that contained this specific methylated CpG in endometriosis. CONCLUSION(S): We identified differential Let-7b methylation in endometriosis, demonstrating that the epigenetic nature of the disease extends to the regulation of miRNAs. Methylation of this novel Let-7b regulatory region explains the decreased levels of this miRNA in endometriosis and is distinct from the regions implicated in regulating Let-7b in cancer. Understanding of the disease-specific mechanisms leading to diminished expression may allow for better understanding of the etiology of endometriosis as well as development of new treatment options.
