The Effect And Mechanism Of SF-1 Methylation Abnormality In Endometrial Carcinoma

Endometrial carcinoma is one of the three most common malignant tumors of female genital tract. In recent years the incidence of endometrial carcinoma is high and the mortality rate is on the rise. The occurrence and development of endometrial carcinoma is the result of interaction of internal and external multiple factors, and long-term estrogen stimulation is the main risk factor. Studies have indicated that there is abnormality of DNA methylation and hypomethylation is a early epigenetic change during the carcinogenesis and development of endometrial carcinoma.SF-1 gene is an important transcription factor in estrogen biosynthesis. It can activate the expression of genes involved in cholesterol transfer and steroidogenesis, and it plays a key role in the biosynthesis of steroid hormones. So, does SF-1 gene play a role and be regulated by methylation in the development of endometrial carcinoma? And what is the molecular mechanism?Therefore, this study is designed firstly to detect the expression of SF-1 and its gene promoter methylation status in endometrial carcinoma tissues, and then to investigate the effect of SF-1 gene methylation abnormality on the biological activity of endometrial cancer cell lines and its molecular mechanism. The aim of this study is to explore a new molecular marker and to provide the experimental basis for the effective diagnostic and therapeutic strategies in endometrial carcinoma.Part1 Analysis of the status of SF-1 gene promoter methylation and its clinical significance in endometrial carcinomaObjectiveTo detect the expression of SF-1 protein and methylation related protein DNMTs, and the methylation level of SF-1 gene promoter in endometrial cancer tissues, paracancerous endometrial tissues and normal endometrial tissues. To explore the methylation status of SF-1 gene promoter and its association with clinical pathological characteristics in endometrial carcinoma.Methods1. The expression of SF-1 protein was detected in normal endometrium, endometrial carcinoma and adjacent tissues by immunohistochemical staining.2. The expression level of SF-1 protein and DNMTs protein were detected in normal endometrium, endometrial carcinoma and adjacent tissues by western blot analysis.3. Analysis the relationship between SF-1 protein expression and clinical pathological characteristics in endometrial carcinoma.4. The methylation status of SF-1 gene promoter was detected in endometrial carcinoma and paracancerous tissues by bisulfite sequencing PCR method.Results1. Immunohistochemical staining showed that SF-1 protein in endometrial carcinoma tissues was expressed significantly higher than that in normal endometrial tissues (P<0.05). In the same cases, the expression of SF-1 protein in endometrial carcinoma tissues was higher than that in paracancerous endometrial tissues, the difference was statistically significant (P< 0.05).2. Western blot analysis showed that SF-1 protein was significantly higher expressed in endometrial carcinoma tissues compared with samples from normal endometrium (P<0.01), and paracancerous endometrial tissues (P<0.05). The expression of DNMT3a and DNMT3b protein in endometrial carcinoma tissues were significantly lower than those in paracancerous tissues (P<0.05).3. In endometrial cancer of different pathological types and different pathological grades, the expression level of SF-1 protein was different, the difference was statistically significant (P<0.05). Pathological grade higher, the expression level of SF-1 was significantly higher; SF-1 protein was significantly higher expressed in special types of endometrial carcinoma than that in endometrioid adenocarcinoma.4. BSP results showed that the percentage of methylated cytosine in endometrial carcinoma tissues (8.2%) was significantly lower than that in paracancerous tissues (40.9%) (P<0.05), indicating that methylation level of SF-1 gene promoter in endometrial carcinoma was low.Conclusions1. The SF-1 may play a role in the occurrence and development of endometrial carcinoma. The abnormal high expression of SF-1 is associated with poor prognosis in patients with endometrial carcinoma.2. Abnormally low methylation in promoter region of SF-1 gene may be the cause of the elevated level of SF-1 protein expression. Hypomethylation status of SF-1 gene promoter is intensively related to the progression of endometrial carcinoma.Part2 Effect of SF-1 promoter methylation abnormality on the growth of endometrial cancer cellsObjectiveTo study the expression of SF-1 and the effect on the cell biological behavior in endometrial cancer cell lines treated with DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine.Methods1. Endometrial carcinoma cell lines HEC-1-A and RL95-2 as the study object, respectively treated with methyltransferase inhibitor 5-Aza-CdR of different concentrations, each cell line was divided into two groups: 5-Aza-CdR group and control group without any treatment.2. The cell proliferation was determined by EdU fluorescence labeling method in endometrial cancer cells treated with 5-Aza-CdR of different concentrations.3. The expression level of SF-1 protein was detected in control group and 5-Aza-CdR groups of different concentrations by western blot analysis.4. The methylation level of SF-1 gene promoter was detected in control group and 5-Aza-CdR group by bisulfite sequencing PCR method.5. The mRNA expression of SF-1 downstream target genes including StAR, HSD3β2 and CYP19A1 were detected by qPCR method in endometrial cancer cell lines after treated with 5-Aza-CdR.6. The cell apoptosis was observed by flow cytometry, the expression of apoptosis related protein Bcl-2, Bax, Caspase-3 and cell cycle protein Cyclin D3 were detected by western blot method in 5-Aza-CdR group and control group.Results1. According to the results of EdU detection of cell proliferation, 5-Aza-CdR of low concentration promoted the endometrial cancer cell proliferation, otherwise,5-Aza-CdR of high concentration inhibited cell proliferation. due to cell toxicity(P<0.05).2. Western blot analysis showed that SF-1 protein was expressed in endometrial cancer cells lines HEC-1-A and RL95-2. Compared with the control group, the expression of SF-1 protein was increased significantly after treated with 5-Aza-CdR (P<0.05).3. BSP sequencing results showed that 5-Aza-CdR of low concentration decreased the methylation level of SF-1 gene promoter CpG significantly (P< 0.05).4. Quantitative real-time PCR demonstrated that the mRNA expression of gene StAR, HSD3(32 and CYP19A1 were increased significantly after treatment of 5-Aza-CdR (P< 0.05).5. Flow cytometry analysis demonstrated that compared with control, cell apoptosis was significantly reduced in 5-Aza-CdR group of low concentration, while cell apoptosis was increased obviously in 5-Aza-CdR group of high concentration (P<0.05).6. The results of the expression of apoptosis related protein Bcl-2, Bax, Caspase-3 demonstrated, Bcl-2 expression was significantly increased, the ratio of Bcl-2/Bax increased, or Caspase-3 expression was significantly decreased after treated with 1.0 uM 5-Aza-CdR (P<0.05). After treated with 5.0 uM 5-Aza-CdR, Bax expression was significantly increased (P<0.01), the ratio of Bcl-2/Bax was decreased, and Caspase-3 expression was significantly increased (P<0.05).7. In both HEC-1-A and RL95-2 cells, the expression of cell cycle protein Cyclin D3 were significantly increased after treated with 5-Aza-CdR in concentration of 1.0 uM and 2.5 uM (P<0.05).Conclusions1. Inhibition of SF-1 gene promoter methylation by 5-Aza-CdR promoted the expression of SF-1 in endometrial cancer cells, indicating that the promoter demethylation leads to high expression of SF-1.2. Demethylation of SF-1 gene promoter and then up-regulation of SF-1 and its downstream target genes, enhanced cell proliferation and inhibited apoptosis in endometrial cancer cells, indicating SF-1 gene has the characteristics of oncogene.3. SF-1 may be intensively related to the development of endometrial carcinoma. The level of SF-1 methylation may become a novel molecular target predicting cell proliferation ability.