TNFα诱导激活NF-κB上调抑癌基因CDKN2B表达

Transcriptional Upregulation of CDKN2B by TNFα-activated NF-κB

NF-κB通过转录调控其靶基因,在肿瘤发生发展和精准治疗中起关键作用。过表达抑癌基因细胞周期蛋白依赖性激酶抑制剂2B(CDKN2B)可抑制肿瘤细胞增殖并诱导凋亡,但是否受NF-κB调节尚无报道。本文发现,NF-κB直接结合并上调CDKN2B基因:在TNFα处理的He La细胞内,CDKN2B的基因区覆盖大量NF-κB结合峰,其中富集倍数大于20的结合峰有14个。TRANSFAC软件分析发现,NF-κB结合峰内包含大量经典的κB位点。Ch IP-q PCR证明,TNFα诱导NF-κB结合CDKN2B基因。将NF-κB结合峰中心区DNA片段插入荧光素酶报告基因载体中,发现该DNA片段的插入使荧光素酶相对活性上调至7.88倍,TNFα处理又使其相对活性提高到2.37倍,且NF-κB/p65 siRNA显著干扰其相对活性的升高。免疫荧光检测显示,TNFα诱导激活NF-κB进入细胞核,而NF-κB/p65 siRNA阻止它入核。此结果提示,我们成功构建了具有NF-κB转录活性差异的细胞模型,q PCR检测两个已知的NF-κB靶基因NFKB2和STAT5A的表达,进一步验证该细胞模型构建成功。利用此细胞模型,发现受TNFα诱导激活的NF-κB,能够上调CDKN2B基因。总之,本文发现抑癌基因CDKN2B是NF-κB新的靶基因,NF-κB直接结合并上调CDKN2B基因在转录水平的表达。本研究为抑癌基因CDKN2B的抗肿瘤应用奠定了基础。

NF-κB plays a key role in the development and precise treatment of tumor by transcriptionally regulating its target gene. Overexpression of the tumor suppressor gene CDKN2B inhibits the proliferation of tumor cells and induces apoptosis. The present study found that NF-κB bound and transcriptionally upregulated CDKN2B. Initially,we found that the CDKN2B gene covered a large number of NF-κB-bound peaks in TNFα-treated He La cells. Among these peaks,there were 14 peaks with fold enrichment over20. The TRANSFAC software showed that there were many classical κB sites in the NF-κB-bound peaks.Besides,Ch IP-q PCR showed that TNFα induced the binding of NF-κB to the CDKN2B gene. Moreover,the DNA fragment of NF-κB-bound peak summit region was inserted into the reporter gene plasmid vector. The insertion increased relative luciferase activity to 7. 88-fold. The treatment of TNFα also increased the relative luciferase activity to 2. 37-fold,and NF-κB/p65 siRNA significantly interfered withthe increase. Immunofluorescence assays showed that TNFα induced activation of NF-κB into the nucleus,and NF-κB/p65 siRNA prevented it from entering the nucleus. This finding indicated that we successfully constructed the cell model with different transcriptional activity of NF-κB. We also detected the expression of two known NF-κB target genes,NFKB2 and STAT5 A,thus confirming the cell model.Using this cell model,we found that NF-κB upregulated the CDKN2B gene in TNFα-treated cells. In conclusion,the tumor suppressor gene CDKN2B was bound and upregulated by NF-κB. This study provides insights into the anti-tumor application of CDKN2B.