乳腺癌发生模型MCF10各细胞系中APC基因启动子区甲基化及mRNA表达检测

Promoter methylation and mRNA expression of APC gene in MCF10 breast cancer model

目的探讨乳腺癌发生模型MCF10中抑癌基因APC启动子区甲基化状态及其对mRNA表达的影响。方法应用甲基化特异性聚合酶链反应(MSP)及双亚硫酸钠基因测序技术检测MCF10模型的乳腺增生细胞系MCF10A、癌前细胞系MCF10AT、导管内癌细胞系MCF10DC IS.com、浸润癌细胞系MCF10CA1 a、MCF10CA1d、MCF10CA1h及经典乳腺癌细胞系MCF-7和正常乳腺组织中APC基因启动子1A甲基化状态,然后用逆转录聚合酶链反应(RT-PCR)和实时PCR技术检测上述样品的mRNA表达水平。结果在MCF10模型的增生细胞系、癌前细胞系、导管内癌细胞系、浸润癌细胞系中,APC基因启动子1A处于低甲基化状态;与正常乳腺组织相比,各细胞系APC基因mRNA表达无明显减少,MCF10AT、MCF10CA1d、MCF10CA1h、MCF10DC IS.com的mRNA表达分别减少0.27、0.96、1.78、2.70、2.03倍,MCF10A和MCF-7分别增加0.02和0.33倍)。结论MCF10模型中乳腺癌的发生发展过程与APC基因启动子区异常甲基化无关。

Objective To investigate the promoter methylation status and mRNA expression of APC gene in MCF10 model of breast cancer progression. Methods Methylation specific PCR and sodium bisufite genomic sequencing were employed to detect the methylation status of APC promoter 1A in normal breast tissues, conventional breast cancer cell line MCF-7 and MCF10 model cell lines including MCF10A （breast hyperplastic cell line, non-tumorigenic）, MCF10AT （pre-malignant cell lines, producing slowly progressing hyperplastic and dysplastic lesions）, MCF10DCIS. eom （breast ductal carcinoma in-situ cell line, producing ductal carcinoma in-situ）, MCF10CA1a, MCF10CMd, MCF10CA1h cell lines（ invasive breast carcinoma cell line, forming aggressive tumors of different morphology and metastatic potential）. In addition, mRNA expression of APC was determined by reverse transcriptase PCR and real-time PCR assays. Results Hypomethylation of APC promoter 1A was identified in hyperplastic cell line MCF10A, pre-malignant cell line MCF10AT, ductal carcinoma in-situ cell line MCF10DCIS. corn, invasive carcinoma cell lines MCF10CA1a, MCF10CA1d, MCF10CA1h and normal breast tissue. MCF-7 showed partial methylation at the promoter. Statistically significant reduction of APC mRNA expression was not found in all MCF10 cell lines and MCF-7, compared with that of normal breast tissue （MCF10AT, MCF10CA1a, MCF10CA1d, MCF10CA1h and MCF10DCIS. corn showed reduced mRNA expressions of APC at 0. 27, 0. 96, 1.78, 2. 70, and 2. 03 times respectively. MCF10A and MCF-7 even showed an increase of APC mRNA expression at 0. 02 and 0. 33 times, respectively）. Conclusion The aberrant promoter methylation of APC is not related to the breast cancer progression, at least in the MCF10 model system.