| Background and Objective: Breast cancer is one of most frequent malignant tumors among the female, and its incidence rises up year by year. Now, the treatment to breast cancer is still traditionary exairesis in clinic, but post-opradiotherapy,chemotherapy,and endocrine therapy are only subsidiary means. Although the therapeutic plan can improve survival rate of patients, it significantly influences patients'long-dated quality of life. Therefore, to discover a pristine tumor molecular marker and match reasonable therapy, is so imperative to reduce hazardness of breast cancer. The aim of study was to investigate the hypermethylation status of CpG-island in RASSF1A promoter region, protein expression of RASSF1A and B-RAF in breast cancer and premalignant lesion, and to analyze their role in early diagnosis and therapy of breast cancer.Methods: Samples of surgically resected breast cancer and premalignant lesion, were collected from 2005 to 2007, among them 20 cases of benign hyperplastic tissue(4 cases intraductal hyperplastic tissue, 16 cases fibroadenoma), 10 cases of intraductal atypical hyperplasia,10 cases of cancer in situ,60 cases of primarily invasive ductal cancer and 20 cases corresponding adjacent cancer tissues. The routine H&E-stained sections were reviewed to verify the morphologic diagnosis. Hypermethylation status of the RASSF1A promoter region was analyzed by methylation specific polymerase chain reaction(MSP). Western-blot analysis was used to detect the expression of RASSF1A and B-RAF. Statistical analysis was performed using the SPSS 11.5 software.Results:1. The methylation frequency of RASSF1A gene is 0 in benign breast hyperplasia and adjacent cancer tissues; intraductal atypical hyperplasia's frequency is 20.0%, with the degree of atypia increase, the trend of the frequency has been gradually strengthened, but changes was not significant among light,middle,weight grade (P>0.05); breast cancer in situ organizations methylation frequency of 40.0%, compared with dysplasia, the difference was not significant(P>0.05).2. Invasive breast cancer, RASSF1A gene methylation frequency was 61.7%, its frequency was higher than adjacent tissues, and benign hyperplasia and intraductal atypical hyperplasia (P<0.05). With the increase of the degree of malignancy of breast cancer, RASSF1A promoter methylation level accordingly strengthened, and there were significant differences betweenâ…¢-grade andâ… +â…¡-grade breast cancer (P<0.05). 3.Invasive breast cancer, there were correlated among RASSF1A gene methylation level and breast cancer tumor grade and clinical stage (P<0.05), but methylation level had nothing to do with tumor tissue lymph node metastasis (P>0.05).4.RASSF1A protein expression rate were 100%,100%,80.0%,80.0% in the adjacent cancer tissues, and benign breast hyperplasia, intraductal atypical hyperplasia and carcinoma in situ tissue; the breast cancer's negative expression rate was 36.7%,there were significant difference during tumor grade and clinical stage (P<0.05).5. There were correlated between RASSF1A gene methylation and its expression in breast cancer (P<0.01).6. B-RAF had only expressed in breast cancerâ…¡,â…¢-grade, a positive rate of 18.3%, there were significant difference tumor grade(P<0.05). there were not correlated among B-RAF protein and RASSF1A gene methylation and its protein expression (P>0.05).Conclusion:1. The MSP experimental results show that, RASSF1A promoter methylation level rised up in breast cancer and premalignant lesion, suggesting that RASSF1A gene promoter hypermethylation may be involved in the the occurrence of breast cancer development. It is expected to monitor pathological changes in breast tissue effective molecular markers.2. Western blot experiments showed that, RASSF1A gene expression level was reduced in breast cancer and premalignant lesion, and that the incidence of breast cancer, development and RASSF1A gene expression may be related to the silent.3. There was related between RASSF1A gene methylation and its protein expression, suggesting RASSF1A gene promoter hypermethylation may be participated in the progress of breast cancer,and it may lead to reduce expression of protein.4. B-RAF protein have a higher level of expression in the breast cancer tissue differentiation lower (gradeâ…¡,â…¢), there were statistical significance between the expression of B-Raf and breast cancer classification difference (P <0.05), it can help clinical pathology of breast tumors.5. There were no statistical significance among super RASSF1A gene methylation,its protein expression and B-RAF protein expression (P> 0.05), two genes may play a independent role in the course of breast tumor formation. |
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