The Relationship Between Progesterone Receptor Methylation And Clinicopathological Factors In Endometrial Cancer

Endometrial carcinoma is the hormone dependent-cancer, and it counts for about 20-30% of the female reproductive tract cancer, and it becomes a threat to the women health. Progesterone plays an important role in the regulation of normal endometrium function by binding to progesterone receptor (PR). But in endometrial cancer, PR is always down-regulated. And the downregulation of PR is always related to the clinicalpathological factors in endometrial carcinoma. Patients with PR down-regulation or silencing always have bad prognosis and high metastasis rate, resistant to hormone treatment, so PR is very important to the endometrial cancer. But the mechanism under the downregulation of PR is still unknown. Previous reports showed that epigenetics play an important role in gene downregulation and silencing, so in our research, from the epigenetic point of view, we study the relationship between the aberrant methylation of PR gene and clinicopathological factors of endometrial cancer.The first part:the methylation of PR gene in different differentiated endometrial cancerObjectives: to understand if there is any aberrant methylation in different differentiated endometrial cancer cell lines and specimens and whether methylation density is different in different differentiated endometrial cancer, and also to examine the relationship between DNMT. expression levels and clinicopathological factors.Methods: In 43 endometrial cancer specimens, Realtime PCR was performed to test the expression level of PR, DNMT1, 3A, 3B. MSP was used to screen the PR methylation in different differentiated endometrial cancer samples; in endometrial cancer cell lines, MSP and BSP analysis were performed to measure the PR methyation, to further test the PR methylation repress its expression, DNMT inhibitors ADC and histone deacetylase inhibitor TSA were used to treat the cell lines, and compare the different expression of PR before and after the treatment. MTT and flow cytometry were used to measure the inhibition rate and apoptosis after the treatment of ADC and TSA.Results: We found in endometrial cancer samples and cell lines there is aberrant methylation in the PR promoter and the first exon region, after treatment of the cell line with DNMT inhibitors ADC and HDAC inhibitors TSA, we found PR is upregulated, and at the same time, aberrant methylation sites are decreasing, the growth of cell line is repressed, flow cytometry showed that ADC and TSA could induce the apoptosis of endometrial cancer cells, P could enforce this effect.Conclusion: In endometrial cancer cell lines, there is aberrant methylation in the promoter and exon region of PR, and these methylation is closely related with PR repression. The methylation density of exon region is related with PR expression in different differentiated endometrial cancer, DNMT overexpression may play a significant role in endometrial cancer development.The second part:PR methylation is associated with histone modification in endometrial cancerObjectives: Based on the results of the first part, we further study the histone modification after PR methylation, and clarify the mechanism under PR repression. HDAC1, 6 were also studied to understand the relationship between HDAC1, 6 expression and clinicopathological factors.Methods: ChIP was performed to study the histone acetylation in PR methylation and demethylation status, Realtime PCR was employed to measure the mRNA levels of the HDAC1, 6 and the relationship between the expression and clinicopathological factors.Results: In endometrial cancer cell line, histone H3 and H4 are deacetylated in PR promoter and exon region, when PR is demethylated by ADC and TSA, histone H3 and H4 association is increasing. And in endometrial cancer tissues, the expression of HDAC1 and HDAC6 is higher in Grade I endometrial cancers than in Grade II and III tissues, and serous and clear cell endometrial cancers expressed low levels of HDAC1, 6 than endometriod cancer tissues.Conclusion: Histone modification may take part in PR repression after PR methylation, HDAC expression level is consistent with PR expression in endometrial cancer, it may work as an activator of PR transcription.