Tumor Suppressor Of MiR-137 Is Hypermethylated In Endometrial Cancer

Background:Endometrial cancer is the most common gynecological cancer. Endometrial cancer is the fourth common malignancy in women and the most common gynecologic cancer in the U.S. This disease can be broadly classified into two subtypes, endometrioid endometrial cancer(EEC, type I), and non-EEC(type II) with primarily serous histology. In addition to the causal role of genetic amplification, mutation and translocation, we reported the emerging role of micro RNA(mi RNA) leading to oncogenic activation. mi RNAs are a class of small noncoding RNAs(18-25 nucleotides), known to result in m RNA degradation or translational inhibition.Dysregulation of mi RNA through epigenetic mechanisms contributes to tumorigenesis.This study seeks to identify epigenetic aberrance within micro RNAs(mi RNAs) that are DNA hypermethylated in endometrial primary tumors.Methods:Endometrial normal cells(EM-E6/E7/TERT) and cancer cells(AN3CA, HEC1 A, KLE, RL-95-2 genomic DNA were bisulfite-treated by the EZ DNA Methylation kit(Zymo Research, Irvine CA), and amplified by PCR, then digested with the methylation-sensitive enzyme.The inhibition effect of HEC1 A,HEC1A-mi R137, HEC1A-PCMVMIR was observed for 3h,48 h,96 h by MTT assays and colony formation assay.For Western blot analysis,briefly, protein lysates were loaded on 4-20% Mini-PROTEAN TGX Gel(Bio-Rad, Hercules CA) and then transferred to polyvinylidene difluoride(PVDF) membranes. Antibodies against LSD1(EPR6825),EZH2,and b-actin were obtained from Abcam or Cell Signaling. Tumors were generated by subcutaneous injection of cancer cells(2x106) into 6-week-old female athymic nude mice.Results:Hypermethylation of mi R-137 in both endometrioid and serous subtypes of endometrial cancer led to lost expression of mi R-137.Treatments of hypermethylated endometrial cancer cells with epigenetic inhibitors reactivated mi R-137. Genetic overexpression of mi R-137 suppresses cancer cell proliferation and colony formation in vitro. When transfected cancer cells were implanted in nude mice, cancer cells with overexpressed mi R-137 grew more slowly and formed smaller tumors when compared with vector transfectants.Western blotting showed that mi R137 ecreased anti-apoptotic protein EZH2?H3K23me3?B-Caternin?EGFR?c-Myc expression in endometrial cancer cell lines.Conclusion:We have identified hypermethylated mi RNAs in human endometrial primary tumors and verified that mi R-137 is epigenetically silenced by DNA methylation in both EEC and serous subtypes of endometrial tumors, as well as in endometrial cancer cell lines. Our data showed that mi R-137 is a tumor suppressor and is hypermethylated in endometrial cancer.