The Promoter Methy Lation Of Tumor Suppressor Genes And Epigenetic Regulation By UHRF1 Complex In Endometrial Carcinoma

Purpose:To search for new methylation biomarkers of endometrial carcinoma (EC), and to further explore the epigenetic regulation mechanism of tumor suppressor genes.Design:One handred and fifty-five endometrium samples, including normal endometrium (n= 27), simple hyperplasia (n= 22), complex hyperplasia (n=29), atypical hyperplasia (n=18) and endometrial adenocarcinoma (n= 59), were evaluated for the methylation of six tumor suppressor genes (CDH13, SHP1, HIN1, DKK3, CTNNA1 and PCDH8) by methylation specific PCR (MSP). The human endometrial cancer cell lines Ishikawa and Kle were treated with 5-aza-2’-deoxycytidine (5-Aza-CdR) and trichostatin A (TSA) either alone or in combination. Changes of methylation status and mRNA expression of CDH13 and SHP1 were investigated by MSP and reverse transcription real-time quantitative PCR (qRT-PCR) respectively. Co-Immunoprecipitation (CO-IP) was used to detect expression of UHRF1/PRMT5 complex.Results:The methylation rate of CDH13 and SHP1 was very high (81.36% and 86.44%, respectively) in EC, but that of HIN1, DKK3, CTNNA1 and PCDH8 genes was low (8.47%,15.25%,20.34% and 16.95%, respectively). The high frequent methylation of CDH13 gene was found not only in EC but in complex hyperplasia and atypical hyperplasia (51.72% and 50.00% respectively), and the methylation was correlated with the age, cancer differentiation and tumor infiltration depth (p< 0.05). SHP1 gene was only frequently methylated in EC samples while the methylation rate was much lower in the other groups, and the methylation was correlated with the age, cancer differentiation (p< 0.05). Both CDH13 and SHP1 complete methylation were detected in untreated Ishikawa and KLE cell lines and were partially reversed by 5-Aza-CdR or TSA, consistently, mRNA was increased (p < 0.01). After treatment by the combination of the two inhibitors, the methylation of CDH13 and SHP1 was completely reversed and mRNA was significantly increased (p < 0.01). Arginine methyltranslcrasc PRMT5 can bond with UHRF1 and was down regulated by treated with 5-Aza-CdR and TSA treatment either alone or in combination compared with untreated group (p< 0.05).Conclusions:The promoter of CDH13 and SHP1 were hypermethylation in endometrial tumors. Methylation of CDH13 is also observed at the earlier stages of endometrial tumorigenesis, including complex and atypical hyperplasias. Our findings also suggest that UHRF1 comes into play through the way forming complex with PRMT5 to contribute to EC.