目的：检测食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）细胞株TE13中（bridge integration factor 1，Bin1）甲基化状态，分析去甲基化药物5-Aza-dC去甲基化前后Bin1表达水平、甲基化状态及细胞生物活性的变化，探讨ESCC发生的可能机制及治疗策略。方法：采用qRT-PCR方法检测去甲基化前后TE13细胞Bin1 mRNA的表达，MSP法检测ESCC细胞株TE13中Bin1启动子区甲基化状态，划痕实验及Transwell实验分别检测去甲基化对TE13细胞迁移及侵袭能力的影响，Western blotting法检测Bin1与基质金属蛋白酶2(MMP-2)及基质金属蛋白酶9(MMP-9)蛋白的表达变化。结果： TE13细胞株中Bin1表现为完全甲基化状态，Bin1mRNA呈低表达，经5-Aza-dC处理后Bin1 mRNA表达显著升高（P<0.01）；划痕试验及Transwell试验显示去甲基化处理可明显降低TE13细胞迁移、侵袭能力（P<0.01）；经5-Aza-dC处理后Bin1蛋白表达显著升高（P<001），MMP-2和MMP-9蛋白表达显著降低（均P<0.01）。结论： DNA甲基化是Bin1低表达或缺失的重要机制之一，并可能通过调节MMP-2和MMP-9蛋白的表达，影响食管鳞状细胞癌细胞株TE13迁移、侵袭能力。

Objective:To detect methylation status of Bin1 in esophageal squamous cell carcinoma (ESCC) TE13 line cell, to analyze changes of expression level of Bin1, its methylation status and bioactivity of the TE13 cell before and after demethylation with demethylation agent 5-Aza-dC, and to explore possible mechanism and therapeutical strategies of ESCC. Methods: Using qRT-PCR, expression of bridge integration factor 1 (Bin1) mRNA in the TE13 cell at pre- and post-demethylation of Bin1 was detected. Methylation status of Bin1 promoter region in the TE13 cells were detected by SDP assay. Effects of demethylation on abilities of migration and invasion of the TE13 cell were respectively tested by scratch and Transwell assays. To use Western blotting assay, expressions of Bin1, matrix metalloproteinases-2 (MMP-2) and MMP-9 proteins in the TE13 cell were detected. Results: Bin1in the TE13 cell was presented as a complete methylation. Expression of Bin1 mRNA was low, and after the treatment of 5-Aza-dC, expression of Bin1 mRNA was significantly increased (P<0.01). Results of scratch and Transwell assays showed that treatment of demethylation evidently decreased migration and invasion abilities of the TE13 cell (all P<0.01). After the treatment with 5-Aza-dC, expression of Bin1 protein was markedly increased, expressions of MMP-2 and MMP-9 proteins were significantly decreased (all P<0.01). Conclusion: DNA methylation could be one of important mechanisms for low expression or absence of Bin1, and the methylation could affected migration and invasion abilities of the TE13 cell by regulating expressions of MMP-2 and MMP-9 proteins.

国家自然科学基金资助项目（No.81201607）；河北省杰出青年基金资助项目（No.H2014206320）；河北省高层次人才项目资助（A201401040）