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目的通过观察乳腺癌发生模型 MCF10中 WT1基因启动子区甲基化状态和 mRNA 表达水平,探讨该基因在乳腺癌发生中的作用。方法应用甲基化特异性 PCR 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MCF10DCIS.com、浸润癌细胞系(MCF10CAla、MCF10CAld、MCF10CAlh)、经典乳腺癌细胞系 MCF7及正常乳腺组织中 WT1基因启动子区甲基化状态,然后用逆转录-聚合酶链反应(RT-PCR)和即时定量 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 MCF10A、癌前细胞系 MCF10AT、导管内癌细胞系 MCF10DCIS.com、浸润癌细胞系(MCF10CAla、MCF10CAld、MCF10CAlh)、经典乳腺癌细胞系 MCF7中,WT1基因启动子均处于高度甲基化状态。与正常乳腺组织相比,WT1基因 mRNA 在 MCF10模型的增生细胞系、癌前细胞系、导管内癌细胞系、浸润癌细胞系和经典乳腺癌细胞系 MCF7中的表达均有不同程度的增加(MCF10A、MCF10AT、MCF10CAla、MCF10CAld、MCF10CAlh、MCF10DCIS、MCF7的 WT1基因 mRNA 表达量分别是正常乳腺组织3.23、1.94、4.20、1.53、4.20、4.35、28.69倍)。结论乳腺癌发生过程中 WT1 mRNA 的表达不被启动子甲基化所抑制;WT1mRNA 过表达出现于乳腺癌发牛的早期阶段,提示该基因在乳腺癌发生中起作用。
OBJECTIVE: To investigate the methylation status and mRNA expression of WT1 gene promoter in breast cancer MCF10 and to explore the role of this gene in breast cancer. Methods MCF10A, MCF10AT, MCF10DCIS.com, MCF10CAla, MCF10CAld and MCF10CAlh were detected by methylation-specific PCR and sodium bisulfite sequencing. , The classic breast cancer cell line MCF7 and normal breast tissue WT1 gene promoter region methylation status, and then reverse transcription - polymerase chain reaction (RT-PCR) and real-time quantitative PCR detection of mRNA expression levels of the above samples. Results In the MCF10 model proliferating cell line MCF10A, the precancerous cell line MCF10AT, the intraductal carcinoma cell line MCF10DCIS.com, the invasive carcinoma cell line MCF10CAla (MCF10CAld, MCF10CAlh) and the classical breast cancer cell line MCF7, the WT1 gene promoter In a highly methylated state. Compared with normal breast tissue, the expression of WT1 mRNA increased to some extent in MCF10 model hyperplasia cell line, precancerous cell line, intraductal carcinoma cell line, invasive carcinoma cell line and classical breast cancer cell line MCF7 ( The mRNA expression levels of WT1 in MCF10A, MCF10AT, MCF10CAla, MCF10CAld, MCF10CAlh, MCF10DCIS and MCF7 were 3.23,1.94,4.20,1.53,4.20,4.35,28.69 times higher than those in normal breast tissues, respectively. Conclusion The expression of WT1 mRNA in breast cancer is not inhibited by promoter methylation. The overexpression of WT1 mRNA appears in the early stage of breast cancer development, suggesting that this gene may play a role in the carcinogenesis of breast cancer.