目的：分析桥接整合因子1（bridging integrator-1, Bin1）去甲基化对Bin1 基因表达和食管鳞状细胞癌EC109 细胞增殖能力的影响，并初步探讨其可能的作用机制。方法：甲基化特异性PCR（methylation specific polymerase chain reaction, MSP）法检测去甲基化药物5-氮杂-2'脱氧胞苷（5-Aza-2'-deoxycytidine, 5-Aza-dc）处理后EC109 细胞Bin1 启动子区域的甲基化状态，用qPCR、Western blotting 和MTT法分别检测单独5-Aza-dc 和5-Aza-dc 加转染Bin1 基因干扰片段（Bin1 siRNA）处理对EC109 细胞Bin1 mRNA及其蛋白表达和细胞增殖能力的影响，流式细胞术和Western blotting 检测5-Aza-dc 处理后EC109 细胞周期和细胞周期相关蛋白（Cyclin D1 与CDK4）表达的变化。结果：去甲基化药物5-Aza-dc 处理后，EC109 细胞Bin1 基因启动子区域发生去甲基化。5-Aza-dc 处理的Bin1 去甲基化EC109 细胞Bin1 mRNA和蛋白表达明显上调（均P<0.05），细胞增殖能力明显下降（P<0.05），细胞阻滞在G0/G1 期，表现为S 期细胞比例显著减少，细胞周期相关蛋白Cyclin D、CDK4 表达均明显下调（均P<0.05）。Bin1 去甲基化EC109 细胞转染Bin1 siRNA后，Bin1 mRNA和蛋白表达明显下调，细胞增殖能力增强（均P<0.05）。结论：Bin1 基因启动子区域在EC109 细胞中呈完全甲基化状态，5-Aza-dc 去甲基化可使食管鳞癌细胞EC109 细胞Bin1 表达升高，通过降低细胞周期相关蛋白表达诱导细胞周期阻滞抑制EC109 细胞增殖。证实表观遗传学变化可能与食管癌细胞恶性增殖有关，可为食管癌治疗提供新的思路。

Objective: To analyze effect of demethylation of bridging integrator- 1 (Bin1) on expression of Bin1 gene and proliferation ability of the esophageal squamous cell carcinoma (ESCC) EC109 cell, and preliminarily to explore its possible action mechanism. Methods: Methylation specific Polymerase Chain Reaction (MSP) assay was used to detect methylation status of Bin1 promoter region in the EC109 cell after treatment with demethylation drug, 5-Aza-2’-deoxycytidine (5-Aza-dc). qPCR, Western blotting and MTT assays were used to detect effect of treatment with 5-Aza-dc only and treatment with 5-Aza-dc plus transfection with Bin1 gene interference fragments (Bin1 siRNA) on expressions of Bin1 mRNA and its protein in the EC109 cell as well as proliferation ability of the EC109 cell respectively. Cell cycles of the EC109 cell and expressions of cell cycle-related proteins (Cyclin D1 and CDK4) in the EC109 cell were tested by flow cytometry assay and Western blotting respectively. Results: Demethylation of Bin1 gene promoter region occurred in the EC109 cell after treatment with 5-Aza-dc. In the EC109 cell treated with 5-Aza-dc, expressions of Bin1 mRNA and its protein obviously up-regulated, proliferation ability of the EC109 cell remarkably decreased, the EC109 cell was blocked at G0/G1 stage, proportion of the EC109 cell at S stage evidently reduced, as well as expressions of Cyclin D1 and CDK4 proteins were markedly down-regulated (all P<0.05). After the EC109 cell in demethylation status was transfected with Bin1 siRNA, expressions of Bin1 mRNA and its protein significantly decreased and proliferation ability of the EC109 cell evidently enhanced (all P<0.05).Conclusion: Bin1 gene promoter region in the EC109 cell appeared in complete demethylation status. Demethylation of 5-Aza-dc could enhance expression of Bin1 in the EC109 cell of ESCC, which could induce cell cycle arrest and inhibit proliferation of the EC109 cell through decrease of cell cycle- related protein expression. It was confirmed that epigenetic changes could be related to malignant proliferation of the ESCC cell, which could provide novel idea for therapy of the esophageal carcinoma.

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）；河北省高层次人才培养项目资助（No.A201401040）