丝裂霉素C通过核因子-κB上调SK-HEP-1细胞中肿瘤坏死因子受体相关蛋白1基因表达

Mitomycin-C up-regulated the expression level of the TNF-α receptor associate factor-1 gene through NF-κB in SK-HEP-1 cell

目的 研究丝裂霉素C(MMC)对肿瘤坏死因子受体相关蛋白 1(TRAF1)基因表达的影响 ,检测核因子 κB(NF κB)在TRAF1基因表达的转录调控中的作用。方法 免疫组织化学检测NF κB细胞定位 ,半定量逆转录 聚合酶链反应 (RT PCR)检测TRAF1基因的mRNA水平 ,染色质免疫沉淀检测NF κB同TRAF1基因启动子序列的结合。结果 MMC作用后SK HEP 1细胞浆中的NF κB进入细胞核 ,MMC作用 8h后TRAF1基因的mRNA水平升高 ,MMC作用后NF κB同TRAF1启动子序列结合。结论 MMC能够通过NF κB入核启动TRAF1基因的转录从而上调TRAF1基因的表达 ,染色质免疫沉淀是体内研究转录调控的优秀方法。

Objective To study the effect of Mitomycin-C (MMC) on the expression level of TNFα receptor associate factor 1 (TRAF1) gene in SK-HEP-1 cell and to further investigate the transcription regulation mechanism of the NF-κB on TRAF1 gene expression in the response of SK-HEP-1 cell to the chemical drug.Methods The cellular distribution of NF-κB was detected by immunohistochemistry method in SK-HEP-1 cells treated with MMC.The mRNA expression of TRAF1 gene was determined by RT-PCR and the binding of NF-κB to the promoter of TRAF1 gene was detected by chromosome immunoprecipitation (ChIP).Results MMC stimulated the NF-κB influx into the nucleus from plasma.After treatment with 5 mg/L concentration MMC for 8 h,the expression level of TRAF1 gene was elevated.NF-κB could bind the promoter sequence of TRAF1 gene after MMC treatment.Conclusion MMC could up-regulate the expression of TRAF1 gene in theSK-HEP-1 cells through the NF-κB binding the promoter of TRAF1 gene in transcription level.ChIP is the excellent tool to investigate the transcription regulation in vivo.