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目的:检测食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)细胞株TE13中(bridge integration factor1,Bin1)甲基化状态,分析去甲基化药物5-Aza-d C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-d C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-d C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 the methylation status of bridge integration factor 1 (Bin1) in esophageal squamous cell carcinoma (ESCC) cell line, and to analyze the effect of bin1 on demethylation of demethylated drug 5-Aza-d C Expression level, methylation status and cell biological activity changes to explore the possible mechanism of ESCC and treatment strategies. Methods: The expression of Bin1 mRNA in TE13 cells was detected by qRT-PCR before and after demethylation. The methylation status of Bin1 promoter in ESCC cell line TE13 was detected by MSP method. The methylation status of Bin1 promoter was detected by scratch assay and Transwell assay TE13 cells migration and invasion ability, Western blotting assay Bin1 and matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) protein expression changes. Results: The expression of Bin1 in the TE13 cell line was completely methylated and the expression of Bin1 mRNA was low. The expression of Bin1 mRNA was significantly increased after 5-Aza-d C treatment (P <0.01). The scratch test and Transwell test showed that Methylation could significantly decrease the migration and invasion ability of TE13 cells (P <0.01), and the expression of Bin1 protein was significantly increased after treatment with 5-Aza-d C (P <0.01). The expressions of MMP-2 and MMP- Decreased (all P <0.01). Conclusion: DNA methylation is one of the important mechanisms of Bin1 expression or deletion, and may affect the migration and invasion ability of esophageal squamous cell carcinoma TE13 cells by regulating the expression of MMP-2 and MMP-9.