Methylation of microribonucleic acid Let-7b regulatory regions in endometriosis.

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.