Analysis Of The Methylation Status Of Multiple Tumor Suppressor Genes And Their Epigenetic Regulation In Endometrial Carcinoma

Purpose:To search for new methylation biomarkers of endometrial carcinoma and explore the epigenetic mechanisms of their transcriptional regulation.Design:(1)166endometrium samples, including normal endometrium (n=27), simple hyperplasia (n=25), complex hyperplasia (n=30). atypical hyperplasia (n=24) and endometrial adenocarcinoma (n=60), were evaluated for the methylation of four tumor suppressor genes (TSGs)(SOCS1, SOCS3,3OST2, and DLC1) by methylation-specific PCR (MSP).(2) The human endometrial cancer cell line Ishikawa were treated with5-aza-2’-deoxycytidine (5-Aza-CdR) and trichostatin A (TSA) either alone or in combination, and obtained four groups of Ishikawa cell (including untreated group,5-Aza-CdR group, TSA group and5-Aza-CdR+TSA group). Changes of methylation status and mRNA expression of SOCS3and3OST2in the four groups of cells were investigated by MSP and reverse transcription real-time quantitative PCR (qRT-PCR) respectively.(3) Changes of protein and mRNA expression of UHRFl were detected by immunohistochemistry (IHC), western blot (WB) and qRT-PCR in the four groups of Ishikawa cell.(4) In the four groups of Ishikawa cell, WB was used to detect expression of UHRF1and different H3R8methylation states; the association of which and the promoters of the hypermethylated SOCS3and3OST2were analyzed by chromatin immunoprecipitation (ChIP)-qPCR. Results:(1) In EC, the methylation rate of SOCS3and3OST2was very high (88.3%and78.3%, respectively), and that of SOCS1and DLC1genes was low (88.3%and78.3%, respectively). The high frequent methylation of3OST2gene was found not only in EC but in complex hyperplasia and atypical hyperplasia (53.3%and54.2%, respectively), and there was no statistical difference between the two groups, but significant differences between each group of the two groups and the other groups were found (p<0.05).3OST2gene was only frequently methylated in endometrial cancer samples and the methylation rate was much lower in the other groups.3OST2methylation was related with the age and cancer differentiation (p<0.05).(2) Both SOCS3and3OST2complete methylation were detected in untreated Ishikawa cells and were partially reversed by5-Aza-CdR or TSA, consistently, mRNA was increased (P<0.01) and TSA was more efficient than5-Aza-CdR in inducing gene expression. After treatment by the combination of the two inhibitors, SOCS3and3OST2methylation was completely reversed and mRNA was significantly increased (P<0.01).(3) The mRNA and protein levels of UHRF1in untreated Ishikawa cells were very high, and were moderately reduced after treatment with5-Aza-CdR, whereas TSA was much more efficient than5-Aza-CdR. The combination treatment had a synergistic effect.(4) In untreated group, UHRF1and H3R8me2s were enriched on both hypermethylated SOCS3and3OST2promoters, but the enrichment of H3R8me2a was less than them. After5-Aza-CdR and/or TSA treatment, the UHRF1and H3R8me2s enrichment was decreased while H3R8me2a enrichment was increased.Conclusions:SOCS3and3OST2methylation may play an important role in endometrial carcinogenesis, and UHRF1can directly repress their expression. In the same amino acid site of histone, different methylation states can exert different functions:H3R2me2s acts as a repressive mark, while H3R2me2a correlates with transcriptional activity.