DNA Methylation Status Of Apc And WT1Gene In Intraductal Proliferative Lesions Of Breast And Invasive Cancer

[Objective] By selecting two tumor suppressor genes including the adenomatous polyposis coli (APC) gene and Wilms' tumor suppressor1(WT1) gene as research objects, we analyze the promoter methylation status of APC and WT1gene at different stages of breast tumorigenesis and the mRNA expression level of breast invasive ductal carcinoma.The purpose of this study was to understand the relationship between each target genes' methylation status and breast tumorigenesis and progression, analysis the effect of promoter methylation on mRNA expression, investigate the relationship between aberrant methylation of APC gene in breast invasive ductal carcinoma and clinical pathological features, and to provide a theoretical basis for early diagnosis, prevention and treatment of breast cancer.[Methods] Breast surgical specimens, including46cases of invasive ductal carcinoma(IDC),20cases of ductal carcinoma in situ(DCIS),13cases of atypical ductal hyperplasia(ADH),27cases of usual ductal hyperplasia(UDH) and17cases of normal breast tissue adjacent to cancer, were collected from the Department of Pathology in Kunming General Hospital of People's Liberation Army in2010-2011. All archival haematoxylin and eosin-stained sections were reviewed to confirm the diagnosis according to the WHO tumor classification and pathological diagnostic criteria of the breast tumors in2006. Firstly, We extracted the genomic DNA from all the fresh or paraffin-embedded tissue and detected the promoter methylation frequency and the methylation level of the APC and WT1gene in each case using methylation-specific polymerase chain reaction (MSP) and Quantity One software. Secondly, forty-six cases of IDC samples were grouped according to the clinical data including patient age, tumor size, histological grade, axillary lymph node metastasis, ER, PR and HER-2status to compare the differences in methylation status. Thirdly, the MSP products were sequenced using the method of TA cloning. Lastly, APC and WT1gene mRNA relative expression levels in twenty-six cases of IDC was detected using real-time fluorescent quantitative PCR (qPCR) and the effect of gene methylation on gene expression was analyzed.[Result]1The APC gene methylation frequencies were as follow:0(0/17) for NBT,0(0/27) for UDH,69.23%(9/13) for ADH,90.00%(18/20) for DCIS and80.43%(37/46) for IDC. Pairwise comparisons of APC methylation frequencies between NBT and ADH,NBT and DCIS, NBT and IDC, UDH and ADH, UDH and DCIS, UDH and IDC were significant different(P<0.05); No statistically significant differences were found in APC methylation frequencies between other pairwise groups (P>0.05). The APC gene methylation levels were as follow:0.070±0.147for ADH,0.298±0.496for DCIS,0.557±0.306for IDC. Pairwise comparisons of APC methylation levels between ADH and IDC,DCIS and IDC had significant statistical differences (P <0.05); no statistically significant differences were found in methylation levels between ADH and DCIS (P>0.05).2The APC gene methylation status was independent of clinical pathological data including patient age, tumor size. histological grading, axillary lymph node metastasis, ER, PR and HER-2status.3The median of the APC mRNA relative expression level in IDC group was0.0587750. which was significantly lower than that in NBT group(P<0.05); The median of APC mRNA expression levels in the methylation group of IDC was0.0482350,while in the unmethylation group it was0.3099850. There was significant difference between this two groups(P<0.05). APC gene methylation level and APC mRNA expression in IDC group was negatively correlated (r=-0.469, P<0.05).4The WT1gene methylation frequencies were as follows:0(0/17) for NBT,0(0/27) for UDH,38.46%(5/13) for ADH.45.00%(9/20) for DCIS and0(0/46) for IDC.Pairwise comparisons of WTI methylation frequencies between NBT and ADH. NBT and DCIS, UDH and ADH, UDH and DCIS, IDC and ADH, IDC and DCIS were statistically significant different, P<0.05; no statistically significant differences in the methylation frequencies were found in other groups, P>0.05. The methylation level of WTI gene of ADH and DCIS were0.076±0.674and0.242±0.285respectively. The methylation level between ADH and DCIS was not significantly different,P>0.05.5The median of the WTI mRNA relative expression level in IDC group was3.5621700, which was significantly higher than that in NBT group (P<0.05).[Conclusion]1The degree of methylation of the APC gene in NBT and UDH, ADH and DCIS, IDC samples increased gradually, suggesting aberrant APC gene promoter methylation plays an important role in the breast tumorigenesis and progression.2In IDC, the methylation status of APC gene is independent of clinical pathological data.3In IDC, the mRNA expression of the APC gene is negatively correlated with methylation degree, suggesting that the aberrant hypermethylation of APC gene promoter is one of the important mechanisms leading to gene inactivation.4WTI gene is not found methylated in the NBT and UDH group, however is hypermethylated in both ADH and DCIS groups,which suggests that aberrant WTI gene promoter methylation may play an important role in early stages of breast tumorigensis.5WTI mRNA is highly expressed in the majority of IDC samples,in which the WTI gene was umnethylated at the same time.Thus the unmethylation of WTI gene may promote the gene expression and act as oncogene in breast progression.