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目的探讨甲基化酶抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-dC)及组蛋白去乙酰化酶抑制剂曲古菌素A(TSA)对胰腺癌Panc-1细胞系中TFPI-2基因甲基化水平及基因表达的影响。方法 Panc-1细胞经5-Aza-dC和TSA单独或联合处理后,应用甲基化特异性聚合酶链反应(MSP),逆转录聚合酶链反应(RT-PCR),蛋白印迹法(Western Blot)检测细胞TFPI-2基因启动子区甲基化状态和mRNA及蛋白表达情况。结果 Panc-1细胞经5-Aza-dC单独处理或与TSA联合处理后,TFPI-2基因启动子区高甲基化状态产生逆转,表现为非甲基化,原本不表达TFPI-2 mRNA及蛋白的Panc-1细胞重新表达,TFPI-2mRNA相对表达量为0.821±0.050和0.887±0.042,蛋白表达量为0.613±0.092和0.712±0.059,两者作用效果相似。经TSA单独处理的Panc-1细胞TFPI-2基因启动子区仍为异常高甲基化状态,原本不表达的TFPI-2基因未见重新表达。结论 Panc-1细胞中TFPI-2基因启动子区高甲基化可能是导致该基因失活的主要原因;5-Aza-dC单独作用或与TSA联合作用均能逆转TFPI-2基因的高甲基化状态,使该基因重新表达,但TSA对受抑制的TFPI-2基因无逆转作用。
Objective To investigate the effects of 5-Aza-dC and TSA, a methylase inhibitor, on pancreatic cancer Panc-1 cells Department of TFPI-2 gene methylation levels and gene expression. METHODS: Panc-1 cells were treated with 5-Aza-dC and TSA alone or in combination. The methylation-specific polymerase chain reaction (MSP), reverse transcription polymerase chain reaction (RT- Blot) were used to detect the methylation status of TFPI-2 gene promoter region and mRNA and protein expression. Results After treated with 5-Aza-dC alone or in combination with TSA, the hypermethylation status of TFPI-2 gene promoter region was reversed in Panc-1 cells, showing nonmethylation and no expression of TFPI-2 mRNA and protein Panc-1 cells were re-expressed. The relative expression levels of TFPI-2 mRNA were 0.821 ± 0.050 and 0.887 ± 0.042, and the protein expression levels were 0.613 ± 0.092 and 0.712 ± 0.059, respectively. The TFPI-2 gene promoter region of Panc-1 cells treated with TSA was still abnormally hypermethylated, and no expression of TFPI-2 gene was observed. Conclusion The hypermethylation of TFPI-2 promoter region in Panc-1 cells may be the main reason of inactivation of this gene. 5-Aza-dC alone or in combination with TSA can reverse the hypermethylation of TFPI-2 gene, The gene was re-expressed, but TSA did not reverse the inhibitory TFPI-2 gene.