摘要目的 观察DNA甲基化及缺氧与沉默缺氧诱导因子-1α(HIF-1α)对食管癌细胞中B细胞淋巴瘤/白血病-2(bcl-2)/腺病毒E1B 19 000相互作用蛋白3(BNIP3)基因表达的影响.方法 收集食管癌细胞株KE4、Kyse-140、Caes-17和TE-7,采用逆转录-聚合酶链反应(RT-PCR)和Western blot分别检测BNIP3 mRNA和蛋白表达水平,甲基化特异性聚合酶链反应(MSP)检测BNIP3启动子区甲基化状态.MIC-101乏氧培养系统模拟肿瘤缺氧微环境,实时荧光定量聚合酶链反应(FQ-PCR)和Western blot检测缺氧(1％O2)、常氧及小干扰RNA(siRNA)沉默HIF-1α后Caes-17细胞中BNIP3mRNA和蛋白表达水平.结果 BNIP3在KE4和Caes-17细胞中表达水平均较高,而在TE-7和Kyse-140细胞中表达较低;同时MSP结果显示,在TE-7和Kyse-140细胞中BNIP3启动子区发生了明显的甲基化,经5-氮杂-2'-脱氧胞苷(5-Aza-CdR)处理2个细胞株后,BNIP3启动子区甲基化状态得到了逆转,同时其表达水平显著升高.缺氧条件下,Caes-17细胞中BNIP3 mRNA (4.35±0.20)和蛋白(1.23±0.04)的表达水平较常氧时(BNIP3 mRNA:1.07 ±0.12,BNIP3蛋白:0.28 ±0.03)显著升高(P＜0.05),siRNA转染Caes-17细胞后能显著下调HIF-1α的表达(抑制率为90％),并导致BNIP3基因的表达(BNIP3 mRNA:1.12 ±0.14,BNIP3蛋白:0.34±0.03)受到明显的抑制.结论 BNIP3基因启动子区甲基化是导致其在食管癌细胞中表达较弱的重要原因之一,甲基化酶抑制剂可逆转BNIP3的甲基化状态重新恢复其表达;缺氧能使HIF-1α蛋白的表达升高,同时上调BNIP3的表达水平.

abstractsObjective To clarify the significance of DNA methylation on the expression of B-cell lymphoma/Leukemia-2 (bcl-2)/adenovirus E1B 19 000-interacting protein 3 (BNIP3) gene in esophageal cancer ceils,and to investigate the effects of hypoxia and silencing hypoxia inducible factor-1α (HIF-1α) gene by small interfering RNA (siRNA) on the expression of BNIP3 in Caes-17 cells.Methods KE4,Kyse-140,Caes-17 and TE-7 cells were cultured in vitro,reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression lever of BNIP3 mRNA and protein.Furthermore,the promoter methylation status of BNIP3 gene was detected by methylation specific PCR (MSP).Modular incubator chamber (MIC-101) was used to mimic tumor hypoxic microenvironment.The expression of BNIP3 mRNA and protein was detected by using fluorescence real-time quantitative polymerase chain reaction (FQ-PCR) and Western blotting under nonnoxia,hypoxia (1％ O2) or transfected with HIF-1α-siRNA in Caes-17 cells.Results The expression of BNIP3 was high in KE4 and Caes-17 cells,however,low expression was found in TE-7 and Kyse-140 cells.Hypermethylation status of BNIP3 gene promoter region was observed in TE-7 and Kyse-140 cells by MSP.After treated with 5-Aza-CdR,it was found to effectively reverse BNIP3 gene methylation in these two cell lines and strongly up-regulate the expression lever of BNIP3.The expression of BNIP3 mRNA (4.35 ±0.20) and protein (1.23 ±0.04) were increased under hypoxia than those under normoxia [BNIP3 mRNA (1.07 ±0.12),BNIP3 protein (0.28 ± 0.03)] (P ＜ 0.05).After siRNA transfection,the HIF-1 α was down-regulated efficiently in Caes-17 cells (inhibition ratio:90％),and BNIP3 gene [BNIP3 mRNA (1.12 ± 0.14)、BNIP3 protein (0.34 ± 0.03)] was down-regulated as well.Conclusion The promoter hypermethylation may be one of the predominant inactivation mechanisms of the BNIP3 gene in human esophageal cancer cell lines.Methytransferase inhibitor can restore the expression of BNIP3 by reversing methylation status.Hypoxia can increase protein level of HIF-la and up-regulate the gene expression of BNIP3.