The Expression And Methylation Status Of RAR-β2 Gene In Breast Cancer And Premalignant Lesion

Objective:To investigate the expression and methylation status of retinoic acid receptor beta 2 (RAR-β2) gene in breast cancer patients, precancerous lesions of breast cancer, benigh tumors and normal breast tissues, then to seek the relationship of promoter hypermethylation of RAR-B2 gene with its mRNA expression and clinicopathologic parameters, and to analyze the role of RAR-B2 playing in breast cancer genesis.Methods:1. Immunohistochemistry method was used to detect the expression of RAR-B2 protein in specimens from 40 cases of breast cancer,40 cases of atypical ductal hyperplasia,40 cases of fibroadenoma and 40 cases of normal breast tissues.2. Semi-quantitative reverse transcription PCR was used to detect the expression of RAR-B2 in 60 cases of infiltrating ductal carcinoma,20 cases of atypical ductal hyperplasia,20 cases of intraductal carcinoma, and 20 cases of adenofibroma. Hemi-nested methylation specific PCR was performed to analyze the methylation status of RAR-B2.Results:1. The positive expression rate of RAR-B2 was 30%(12/40) in breast cancer, 17.5%(7/40) in atypical ductal hyperplasia,87.5%(35/40) in fibroadenoma, and 95%(38/40) in normal breast tissues. The expression of RAR-B2 protein in breast cancer was significantly lower than that in normal breast tissues(P<0.05).The expression of RAR-B2 was not significantly different between atypical ductal hyperplasia and breast cancer(P>0.05). No correlation was found between the expression of RAR-B2 protein and the tumor size, menopausal age, lymph node metastasis, clinical stage, histological grade, or protein expression of ER and PR in breast cancer tissues(P>0.05).2. At the mRNA level,17 out of 60 breast cancer samples showed the expression of RAR-β2 mRNA. The expression of RAR-β2 gene in breast cancer patients was lower than that in normal breast tissues or fibroadenoma samples(P<0.05). 3. The methylation frequency of RAR-B2gene was 0 in normal breast tissues, 10%(2/20) in fibroadenoma tissues, and 30%(6/20) in intraduct atyplasia. With the degree of atypia increase, the trend of methylation frequency had been gradually strengthened, but changes were not significant among light, middle, weight grade(P>0.05). The methylation frequency of ductual carcinoma in situ was 40%(8/20).4. Hypermethylation of the RAR-B2 promoter was detected in 27/60 (45%) cases of breast cancer. The methylation frequency of breast cancer was higher than that of normal tissues (P<0.05). No relationships was found between the methylation status and the tumor size, lymph node metastasis, pathological stage, histological grade, or expression of ER and PR in breast cancer (P>0.05).5.24 out of 43 cases of RAR-β2-negative patients were detected promoter hypermethylation, while 3 out of 17 cases of RAR-β2-positive patients presented with promoter hypermethylation. A negative correlation was found between RAR-B2 expression and its methylation status(P<0.05).Conclution:1. Immunohistochemistry experiments showed that, RAR-B2 protein expression level was reduced in breast cancer and premalignant lesion. RAR-β2 gene may play a repressive role in the initiation of breast cancer, and the loss of the expression of RAR-β2 gene may be the initial step in breast carcinogenesis.2.The MSP experiments showed that, the methylation rate of RAR-B2 trends to increase from atypical ductal hyperplasia to ductual carcinoma in situ and to infiltrating ductal carcinoma, suggesting that RAR-B2 gene promoter hypermethylation may be involved in the initiation and development of breast cancer.3. A negative correlation was found between RAR-β2 expression and its methylation status. DNA methylation is one of mechanisms regulating the expression of RAR-β2.