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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 m RNA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 investigate the effect of demethylation of Binomial integrin-1 (Bin1) on the expression of Bin1 gene and the proliferation of esophageal squamous cell carcinoma EC109 cells, and to explore its possible mechanism. Methods: Methylation-specific polymerase chain reaction (MSP) was used to detect the 5-Aza-2’-deoxycytidine (5-Aza-dc) The methylation status of the Bin1 promoter region of EC109 cells was detected by qPCR, Western blotting and MTT assay. The expression of the Bin1 siRNA transfection with 5-Aza-dc and 5-Aza-dc alone Cell Bin1 mRNA and protein expression and cell proliferation were detected by flow cytometry and Western blotting. The changes of cell cycle and expression of Cyclin D1 and CDK4 in EC109 cells treated with 5-Aza-dc were detected by flow cytometry. Results: Demethylation of the Bin1 gene promoter region of EC109 cells occurred after 5-Aza-dc demethylation treatment. The mRNA and protein levels of Bin1 in Bin1 demethylated EC109 cells treated with 5-Aza-dc were significantly increased (all P <0.05), and the cell proliferation was significantly decreased (P <0.05). The cells arrested in G0 / G1 phase. The proportion of cells in S phase was significantly decreased, and the expressions of cyclin D and CDK4 were significantly down-regulated (all P <0.05). Bin1 demethylated EC109 cells transfected Bin1 siRNA, Bin1 mRNA and protein expression was significantly downregulated, cell proliferation increased (P <0.05). CONCLUSION: Bin1 promoter region is completely methylated in EC109 cells. Demethylation of Bin-1 gene can increase the expression of Bin1 in EC109 cells and decrease the expression of cell cycle-associated proteins Blocking inhibits EC109 cell proliferation. Confirmed that epigenetic changes may be associated with malignant proliferation of esophageal cancer cells may provide a new way for the treatment of esophageal cancer.