Analysis Of The Status Of DACH1Gene Promoter Methylation In Endometrial Carcinoma And The Clinical Significance Of The DACH1Gene Promoter Methylation

BackgroundEndometrial carcinoma (EC) is one of the most common female malignant tumors occoured in the female reproductive tract, which accounts for about20%～30%of malignant tumor. Endometrial carcinoma becomes the serious damage to the women's health. In recent years incidence and mortality of the endometrial cancer are rising all over the world, even in some of the developed countries. Reviewing of the global data,1880000cases of endometrial carcinoma were diagnosed and410000patients died of endometrial carcinoma all over the world. In the United States, about40000new cases of endometrial carcinoma were diagnosed and more than7000patients died of endometrial carcinoma each year. According to the new data,46470new cases of endometrial carcinoma were diagnosed and8120patients died of endometrial carcinoma in2011. An increasing endometrial cancer incidence (which is even equivalent to the cervical cancer) was observed in China recent years. Endometrial cancers were classified into two different subtypes:the estrogen-related type(type I endometrioid adenocarcinoma) and the non-estrogen-related type(typeⅡ, non endometrioid adenocarcinoma such as uterine papillary serous carcinoma and clear cell adenocarcinoma). Compared with patients with type Ⅰ, most patients with type Ⅱ are women at an older age (or after the menopause) who are diagnosed late. The tumor cells of those patients are poorly differentiated. Because the higher recurrence and metastasis rates, type Ⅱ has a poor prognosis and low5-year survival rate. The prognosis of endometrial cancer is better than malignant tumors occurred in other reproductive organs compared with ovarian cancer and cervical cancer, but the5-year survival rate of endometrial cancer is not optimistic. The5-year survival rate of endometrial carcinoma in stage Ⅰ～Ⅱ is about83%, while stage Ⅲ is about73%and stage IV is about73%respectively.For the past few years, with the development of molecular biology research, the i mportant role of apparent genetics in the generation and development of carcinoma ha s been realized. Apparent genetics modification, a class of genetic regulation mechani sm higher than the level of gene is the ties linking genotype and phenotype.Epigenetic alterations, defined as heritable changes in gene expression but without DNA sequence changing, including DNA methylation, histone modification, microRNAs and chromatin remodeling, are increasingly focused on recently, for their reversible nature DNA methylation involved in tumor occurrence and development is considered to be an important mechanism of tumor formation, and its main characteristic is the coexistence of the wide range of oncogene hypomethylation and some regional hypermethylation of tumor suppressor genes. It is reported that there is DNA methylation of a variety of tumor-associated gene promoter (including cell cycle regulation, angiogenesis, apoptosis, and gene promoters) in endometrial cancer, which directly affects the cell cycle and signal transduction pathways causing cell growth, differentiation and apoptosis abnormal. DNA methylation plays an important role in tumor occurrence and development. Endometrial cancer gene promoter hypermethylation, resulting in the expression of the gene silencing, suggesting that gene promoter methylation status may be an important molecular mechanism of endometrial cancer occurrence and development.The DACH1found by Hammond in mammals is homologous with Drosophila dac gene. The dac gene which can induce the formation of the Drosophila ectopic eyes and plays a decisive role in the direction of cell differentiation is main member of the Drosophila retinal determination (RD) signaling pathway (including ey/Pax6, So/Six, eya/Eya etc.) The study found that the DACH1gene's lack of expression or low expression in breast cancer, prostate cancer, endometrial cancer, suggesting that the DACH1gene may be a new tumor suppressor gene.This study was designed to evaluate the methylation status of DACH1promoter in different endometrial carcinoma cell lines, normal endometria and endometrial carcinoma tissue, aiming at investigating the relationship between DACH1gene promoter methylation and clinical significance of endometrium carcinoma. Consequently, this may help to study the pathogenesis, diagnosis, treatment and prognosis of endometrial carcinoma.Part One Analysis of the methylation status of DACH1gene promoter in different endometrial carcinoma cell lines and the correlation between DACH1gene promoter methylation and the expression of DACH1protein.Objects:Analysis of the status of DACH1gene promoter methylation in different endometrial carcinoma cell line, including Ishikawa, ECC-1, SPEC-2, HEC-1-A, KLE Ishikawa and SPEC-2were perserved in the local lab,while KLE, ECC-1and HEC-1-A were donated by professor Wang Chen-guang.Methods:1. Identification the status of DACH1gene promoter methylation in endometrial carcinoma cell lines, endometrial carcinoma tissues and normal endometriums were detected by Methylation specific PCR (MSP).2. Identification of the expression of DACH1protein in endometrial carcinoma cell line was detected by western blotting.Results:1. The methylation status of DACH1gene promoter with Ishikawa and ECC-1cell lines were negative, while SPEC-2,KLE and HEC-1-A were positive by MSP.2. The DACH1protein was detected in Ishikawa, ECC-1, SPEC-2, HEC-1-A and KLE via western-blot. The low expression of DACH1protein was observed in SPEC-2and KLE, which promoter of DACH1was methylated.Conclusions:1. There are differences with methylation of DACH1gene promoter among different endometrial cancer cell lines. The high methylation status of the DACH1gene promoter in typeⅡ endometrial carcinoma suggests that DACH1gene promoter hypermethylation status may play an important role in the molecular mechanisms of the occurrence and development in human endometrial cancer, especially the type Ⅱ of endometrial cancer.2. It indicates that the DACH1gene promoter methylation may be associated with low expression of DACH1protein. Part Two Analysis of the status of DACH1gene promoter methylation in endometrium carcinoma and its relationship with pathological featuresObjective:To analyze the status of DACH1gene promoter methylation and explore its association with clinic pathological factors and prognosis of endometrium carcinoma (EC)Method:We selected80cases of endometrial cancer confirmed by pathological examination in Qilu Hospital of Shandong University and Second Affiliated Hospital of Shandong University from February2004to August2008. All the patients have accepted the comprehensive staging surgery, and the fresh carcinoma tissues were treated with10%formalin, paraffin-embedded and made into serial sections, one of them was re-examined by HE staining. All the rest of the tissues were preserved in-70℃refrigerator. Patients aged31to72years with average of54.6years. Pathological grading:G121cases, G218cases, G341cases. Pathological stage based on2000FIGO staging criteria:stage Ⅰ51cases, stage Ⅱ15cases, stage Ⅲ11cases, stage Ⅳ3cases. Pathological types:Endometrial adenocarcinoma53cases, Special type of carcinoma27cases (16cases of endometrial serous papillary carcinoma and11cases of endometrial clear cell carcinoma). Depth of myometrial invasion:≤1/261cases,>1/219cases. Lymph node metastasis:positive in14cases and66cases of negative. All endometrial cancer patients had not received radiotherapy, chemotherapy or hormone therapy.Identification the methylation status of DACH1gene promoter in endometrial carcinoma tissues and normal endometriums were detected by MSP.Twenty normal endometrium tissues obtained from the fractional curettage because of dysfunctional uterine bleeding in the corresponding period (2008) were regarded as control. Each specimen has a histopathological diagnosis. Identification of the expression of DACH1protein in endometrial carcinoma and normal endometrium was detected by western blotting.χ2test and correction χ2test were uesd for the comparison of enumeration data.Correlation analysis of DACH1protein expression and the status of DACH1gene promoter methylation in endometrial carcinomawas performed by Pearson.Results:1. The methylation rate of the DACH1promoter in the EC tissues (30%,24/80) was significantly higher(P<0.05) than that in the normal endometrium tissues (5%,1/20).2. The methylation rate of the DACH1promoter in G1-2carcinoma tissues is18%(7/39), significantly lower(P<0.05) than that in G341%(17/41). The methylation rate of the DACH1promoter in endometrioid adenocarcinoma is23%(12/53), significantly lower (P<0.05) than that in special types of endometrial carcinoma44%(12/27). The methylation status of DACH1promoter was independent of the age of patients, surgical-pathologic staging, myometrial invasion, lymph node metastasis(P>0.05,).3. The DACH1expression was rarely detected in the endometrial carcinomawhich promoter of DACH1was methylated, while the expression was positive in the endometrial carcinoma with unmethylated promoter via western-blot. The analysis of24endometrial carcinomawith methylated DACH1promoter shows that there is a negative correlation between the intensity of DACH1expression and the methylation status of DACH1gene promoter (r=-0.30, P=0.008).Conclusions:1. High methylation rate in DACH1gene promoter in low-level endometrial cancer indicates that the DACH1gene promoter methylation may be associated with poor prognosis of patients with endometrial cancer.2. The high methylation status of the DACH1gene promoter in a special type of endometrial carcinoma suggests DACH1gene promoter hypermethylation status may play an important role in the molecular mechanisms of the occurrence and development in human endometrial cancer, especially the Ⅱ type of endometrial cancer, which can be a new breakthrough to study this type of tumor.3. In the endometrial tissue of methylated DACH1gene, the DACH1protein expression and DACH1gene promoter methylation was significantly correlated, indicating that low expression or loss of expression of DACH1gene in endometrial carcinoma may be related to the DACH1gene promoter methylation status. DACH1gene promoter methylation can lead to transcriptional silencing and inactivation, also confirming the DACH1gene could act as a tumor-suppressor gene in another way.