Relationship Of EZH2 And DNMT1 Expression With The Methylation Status Of Tumor Suppressor Genes In Triple-negative Breast Cancer

BackgroundsTriple negative breast cancer(TNBC) refers to a non-expression of estrogen receptor ER ,progesterone receptor PR and human epidermal growth factor receptor 2 subtypes Her-2. TNBC accounts for 15%-17% of all breast cancer patients, which is the worst of subtypes human breast cancer. African of non-national America have higher prevalence of young women before menopause.Many scholars have reported that nearly a quarter of Chinese breast cancer patients belongs to TNBC. TNBC has younger, aggressive, high degree of malignancy, poor prognosis, family history and other characteristics.Because it does not express ER, PR and Her-2 and often occur invasion and metastasis early but can not benefit from endocrine therapy and anti-Her-2 targeted therapy. Studies of its pathogenesis has far-reaching significance for its diagnosis and treatment.Aberrant promoter methylation is a frequent early event.It occurs in a variety of tumors. Therefore, tumor-associated gene methylation status is a sensitive indicator of early tumorigenesis. Normal cells into tumor cells and invasion growing of tumor cell along with epigenetic change, aberrant gene methylation plays an important role in tumorigenesis and evolution, especially. Methylation as an epigenetic mechanisms involved in the progression of breast cancer,and breast cancer is closely associated with the invasion,metastasis and clinical prognosis.It’s significance for the treatment of breast cancer. With the study of the methylation mechanism of breast cancer suppressor genes,we can obtain f new diagnostic and treatment methods of TNBC.ABSTRACT 1Objective To study the expression and significance of EZH2 and DNMT1 in triple-negative breast carcinoma (TNBC) and explore the relationship with TNBC clinical pathological significance.Methods The clinical and pathological data of 209 cases of breast cancer collected from Guangzhou Kingmed Center for Clinical Laboratory of January 2008 to May 2013. retrospectively. Immunohistochemistry SP assay was used to determine EZH2 and DNMT1 expression in paraffin-embedded specimens of 209 cases of breast cancer tissues and 65 cases of benign lesions(more than 4cm away from tumor). The differences in protein expressions among tissues were compared, and correlation analysis was performed to determine the relationship between protein expression and clinico-pathological indices. Logistic repressionlysis was applied for the analysis of positive expression-related factor of EZH2 and DNMT1. Spearman rank correlation was used to analyse the interrelation of EZH2 and DNMT1.Results The positive expression rate of EZH2 in triple-negative breast carcinoma、Non-triple-negative breast carcinoma and adjacent breast tissue is 86.3%、89.8% and 40.0% respectively.while the positive expression rate of DNMT1 is 63.4%、6.6% and 44.6%.Compared with adjacent breast tissue, the positive expression rate of EZH2 and DNMT1 increased (40.0% vs 87.6% and 44.6% vs 64.6% respectively P<0.05); There was no relationship between EZH2 expression and age,menstrual status,histologic type and vessel and nerve invasion (P>0.05) but exhibits differences in terms of histological grade、tumor size and lymph node metastasis (P<0.05). Multivariate analysis demonstrated that histological grade and tumor size were independent correlation factors of EZH2 expression in TNBC.The expression of DNMT1 exhibits differences in terms of histological grade and tumor size (P<0.05),logistic regression analysis showed that the histological grade of TNBC was the main factor impacting the expression of DNMT1; There was a positive correlation between EZH2 and DNMT1 expression in TNBC tissues (r=0.34, P< 0.01).Conclusions The expression of EZH2 and DNMT1 have relationship with ocurrance and progress in TNBC.EZH2 is positive related to DNMT1,and combined detection of then maybe valuable for evaluating the progress of TNBCABSTRACT 2Objective The purpose of this section is to detect five tumor suppressor genes 14-3-3sigma, RASSF1A, CDH1, P16,ERα promoter CPG methylation status in triple-negative breast cancer and adjacent tissues. Analysis the relationship of tumor suppressor genes, respectively 14-3-3sigma, RASSF1A, CDH1, P16, ERα CPG methylation status and clinicopathological parameters, which can further revealed the tumor suppressor gene methylation factors for triple negative breast and provide a theoretical basis for early diagnosis and treatment of TNBC.Methods Genomic DNA was extracted in 131 cases of triple-negative breast cancer and 65 cases of adjacent tissues.Methylation-specific PCR method (MSP) was used to detect promoter area methylation status of tumor suppressor genes P16, CDH1, RASSF1A,14-3-3sigma and ERa.To analysis the relationship of each major clinical parameters of triple-negative breast cancer and gene methylation.SPSS 13.0 was used to analysis all data, Ⅹ2 test or Fisher’s exact test were used to analysis count data, all test are bilatERal, P<0.05 was significant.Results 14-3-3sigma, RASSF1A, CDH1, P16,ERa gene methylation of the promoter region of incidence varies.14-3-3sigma methylation stripe size of 104bp, unmethylated fragment of 106bp. In triple-negative breast cancer incidence 14-3-3 sigma gene methylation in the promoter region of up to 71.4%, in the adjacent tissues methylation rates were 29.3%, the difference was statistically significant triple-negative breast cancer incidence 14-3-3sigma methylation was significantly higher than adjacent tissues (P<0.05)’, RASSF1A methylation stripe size of 94bp, unmethylated fragment of 108bp. Incidence of RASSF1A gene methylation in the promoter region of 42.1% in triple-negative breast cancer in adjacent tissues methylation rate was 19.1%, the difference was statistically significant.The incidence of RASSF1A methylation was significantly higher than adjacent tissues (P<0.05); CDH1 methylation stripe size of 115bp, unmethylated fragment of 97bp. In triple-negative breast cancer CDH1 gene promoter methylation rate was 37.8% in adjacent tissues methylation rates were 16.1%, the difference was statistically significant, triple-negative breast cancer in CDH1 methylation was significantly higher than adjacent tissues (P<0.05); P16 methylation stripe size of 151bp, unmethylated fragment of 150bp. Incidence of P16 gene methylation in the promoter region of 40.3% in triple-negative breast cancer in adjacent tissues methylation rate was 23.5 percent, respectively, the difference was statistically significant, triple-negative breast cancer in CDH1 methylation was significantly higher than adjacent tissues (p<0.05).Conclusions:The four kinds of gene methylation in triple-negative breast cancer methylation were higher than the adjacent tissues. This suggests that in this study the four kinds of tumor suppressor gene methylation may contribute to malignant cells. 14-3-3sigma, CDH1, P16 gene hypermethylation may directly affect the lymph node metastasis of tumor cells. The methylation of RASSF1A gene also correlated with histological grade organization, combined detection of the methylation status of the four in triple negative breast cancer may provide new ideas to develop treatment programs.ABSTRACT3Objective To combine in triple negative breast cancer DNMT1 expression and EZH2, analysis DNMT1 and EZH2 expression and tumor suppressor genes 14-3-3sigma, RASSF1A, CDH1, P16, ERa start relationships promoter methylation status, and further revealed affect tumor suppressor gene methylation factors provide a theoretical basis for the early diagnosis and treatment of triple negative breast cancer.Method Methods of EZH2 and DNMT1 immunohistochemical were same to the first part,14-3-3 sigma, RASSF1A, CDH1, P16, ER3, ER4, ER5 gene methylation status detection method, were same to the second part. All data using SPSS 13.0 statistical software for analysis.x2 test or Fisher’s exact test were used to analysis the relationship between EZH2 DNMT1 expression and tumor suppressor gene methylation status in the triple negative breast cancer, P<0.05 was considered statistically significant.Results The expression of EZH2 was associated with 14-3-3sigma, P16, ERα gene methylation P values were (0.001,0.001,0.003),respectively;The expression of DNMT1 was associated with 14-3-3sigma methylation status in triple negative breast cancer (P=0.036).Conclusions DNMT1 and EZH2 overexpression maybe helpful to the early diagnosis of triple negative breast cancer, anti-EZH2 and DNMT1 treatment and then reverse tumor suppressor gene methylation may be an effective treatment for triple negative breast cancer.