5-Aza-dC对胰腺癌细胞系Panc-1中TFPI-2基因甲基化水平及表达的影响
Effect of 5-Aza-dC on Expression and Methylation of TFPI-2 Gene in Panc-1 Pancreatic Cancer
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摘要: 目的 探讨甲基化酶抑制剂5-氮杂-2′-脱氧胞苷(5-A2a-dc)对胰腺癌细胞系Panc-1中抑癌基因组织因子途径抑制物-2（TFPI-2）甲基化水平及基因表达的影响。方法用不同浓度5-Aza-dC处理胰腺癌细胞系Panc-1。用甲基化特异性PCR（MS-PCR），反转录聚合酶链（RT-PCR）及蛋白印迹实验（Western blot）检测药物处理前后Panc-1细胞TFPI-2基因的甲基化状态，mRNA及蛋白表达的改变。结果MSP检测Panc-1细胞TFPI-2基因药物作用后异常甲基化得到逆转，RT-PCR检测到不同浓度5-Aza-dC处理后TFPI-2基因mRNA重新表达，相对表达量分别为（0.211±0.087），（0.327±0.068），（0.609±0.017），Western blot检测TFPI-2基因蛋白重新表达，相对表达量分别为（0.429±0.121），（0.675±0.044），（1.132±0.124），以上作用呈时间、剂量依赖性（P<0.05），差异具有统计学意义。结论胰腺癌细胞系Panc-1中抑癌基因TFPI-2启动子高甲基化可能是导致该基因表达下调甚至失活的主要原因。5-Aza-dC能够较成功的逆转胰腺癌细胞Panc-1中TFPI-2基因的高甲基化状态，并能恢复TFPI-2基因的mRNA及蛋白重新表达。Abstract: Objective To investigate the effects of 5-aza-2，-deoxycytidine(5-Aza-dC)，a methylation inhibitor，on the expression and methylation of TFPI-2 gene in Panc-1 cell lines of pancreatic cancer. Methods Panc-1 cell line was treated with different dosages of 5-Aza-dC.TFPI-2 gene DNA，mRNA and protein were determined by MSP，RT-PCR and Western blot respectively. Results MSP detection showed that the TFPI-2 gene hypermethylation has effectively been reversed by 5-Aza-dC.Moreover，the expression levels of TFPI-2 mRNA treated with 5-Aza-dC were increased (0.211 ± 0.087，0.327 ± 0.068 and 0.609 ± 0.017，respectively).Western blot indicated that 5-Aza-dC could recover the TFPI-2 protein expression (0.429 ± 0.121，0.675 ± 0.044 and 1.132 ± 0.124 respectively).These effects within certain extent were dose and time dependent with statistical significance (P<0.05). Conclusion The hypermethylation of promoter region was a main cause for transcriptional inactivation of TFPI-2 gene in Panc-1 cell lines.5-Aza-dC might effectively reactivate the gene transcription through a demethylation role.
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