A Study Of The Role Of HOXA5 In The Carcinogenesis And Treatment Of Human Breast Cancer

To study the expression of HOXA5 gene in primary human breast cancer and breast cancer cell lines and explore the underlying mechanism of gene expression change in order to gain insight into the possible role of HOXA5 in the carcinogenesis and treatment of breast cancer.MethodsPart I:Real-time reverse transcription polymerase chain reaction (Real-time RT-PCR)was used to investigate HOXA5mRNA expression in 60 cases of primary breast cancer and 24 cases of benign breast lesion. Statistical methods were applied to explore the correlation between HOXA5 expression and clinical pathological data.Part II: To study the relationship between HOXA5 promoter methylation and HOXA5 expression, methylation specific PCR(MSP) was firstlyapplied to detect gene promoter methylation status of HOXA5 in breast cancer cell lines MDA-MB-231, SKBR3 and MCF7 and real-time RT-PCR was used to detect HOXA5 mRNA expression of them. Furthermore, MDA-MB-231 was treated with 5-aza-deoxycytidine (5-Aza-CdR), a DNA methylation inhibitor. The promoter methylation status and expression of HOXA5 were examined and compared with that of control without the treatment of this drug. In addition, Semi-quantitive RT-PCR was applied to detect the expression of DNMT1mRNA in MDA-MB-231 and MCF7. DNA obtained from 60 primary breast cancer and 12 benign breast lesions was subjected to HOXA5 promoter methylation study by methylation specific PCR. Statistical methods were used to study the correlation of HOXA5 promoter methylation and clinical pathological data such as patients age,tumor size,lymph node status ,clinical stage and so on.Part III:Immunohistochemical test was used to detect protein expression of HOXA5 and PR. The relationship between HOXA5 protein expression and HOXA5 promoter hypermethylation was statistically examined. The correlation between HOXA5 and PR protein expression was also researched.Part I: A reduced expression of HOXA5mRNA was present in primary breast cancer compared with benign breast lesions. There was a lower HOXA5 expression in lymph node positive cases than in lymph node negative ones, which suggested HOXA5 may play a role in the metastasis of breast cancer.Part II :The promoters of HOXA5 in MDA-MB-231 and SKBR3 were hypermethylated. However, MCF7 had HOXA5 expression. HOXA5mRNA expression could be restored in MDA-MB-231 after the treatment with 5-aza-deoxycytidine. The expression was dose and time dependent. MDA-MB-231 was partially demethylated after the treatment of 5-aza-deoxycytidine. DNMT1 mRNA was much higher in MDA-MB-231 than in MCF7.Of all tumor tissue samples amplified, 55(91.7%) presented HOXA5 methylation. Benign breast lesions were unmethylated in HOXA5 promoter except for 2 cases of fibroadenoma which was partially methylated. These results confirmed that methylation observed in primary breast cancers was a tumor -specific change. There was a much lower HOXA5 mRNA expression in HOXA5 hypermethylated breast cancer than in unmethylated cases. There was a lower expression in lymph node positive samples than in lymph node negative ones.Part III:HOXA5 protein was located in the cytoplasm of mammary epithelium and myoepithelium. Occasionally HOXA5 protein was seen in the cell membrane and a stronger staining was appeared surrouding the nuclear membrane. In general, a strong staining of HOXA5 was seen in benign breast lesions and a reduced HOXA5 protein expression emerged in 44 primary breast cancer. There was an association between decreased expression of HOXA5 protein and HOXA5 hypermethylation. HOXA5 protein expression was distinctively different between lymph node positive and negative cases. No relationship was found between HOXA5 expression and other clinical pathological factors such as the age of patients, the size and location of tumor, clinical stage. PR protein staining was positive in 20(43.5%) primary breast cancer. No relationship was found between HOXA5 and PR expression.ConclusionHOXA5 expression was reduced in primary breast cancer and breast cancer cell lines. We concluded that hypermethylation of HOXA5 promoter was related with the reduced HOXA5 expression. HOXA5 reduction may paly a role in the metastasis ofbreast cancer. No relationship was found between HOXA5 and PR protein expression in this study.