TNFα and LTβ stimulated UM-SCC 46 gene expression [LTB]

To investigate the regulatory mechanisms and targets of the activation of NF-kappaB and inflammatory pathways, we treated HPV(-) head and neck cancer line UM-SCC 46 cells with TNFα and LTβ at different time points, and compared the gene expression by microarray. TNFα uniquely induced 172 genes, LTβ specifically induced 202 genes, while 155 genes were induced by both ligands. Total RNA samples were isolated from UM-SCC 46 cells after TNFα or LTβ treatment at different time points, and the gene expression were compared with untreated cell controls. UM-SCC 46 cells were treated with TNFα (20ng/ml) or LTβ (100ng/ml) for 1, 3, 6, 12, and 24 hours. RNA was isolated and global gene expression was examined by beads based array using Illumina HumanWG-6-v3-BeadChip (Illumina, CA). Raw data were imported to the GenomeStudio software, and normalized data were imported to Parte to detect differentially expressed genes by using an ANOVA test. Hierarchical clustering of altered gene expression when treated with LTβ was compared to untreated cells using MeV software. The heat map represents two-fold increased gene expression when compared to untreated samples.

为探究核因子κB（NF-kappaB）与炎症通路激活的调控机制及作用靶点，我们以人乳头瘤病毒阴性（HPV(-)）头颈癌细胞系UM-SCC 46为研究对象，采用肿瘤坏死因子α（TNFα）与淋巴毒素β（LTβ）进行不同时长的处理，并通过基因芯片技术比较各组的基因表达谱。其中，TNFα可特异性诱导172个基因的表达，LTβ可特异性诱导202个基因的表达，另有155个基因可被两种配体共同诱导。我们从经TNFα或LTβ处理不同时长的UM-SCC 46细胞中提取总RNA，并以未处理的细胞作为对照开展基因表达比较；具体处理方案为：以20ng/ml的TNFα或100ng/ml的LTβ分别处理细胞1、3、6、12及24小时。随后提取总RNA，采用基于微球的芯片阵列技术，使用Illumina HumanWG-6-v3-BeadChip芯片（美国加利福尼亚州Illumina公司）检测全基因组基因表达水平。原始数据导入GenomeStudio软件进行预处理，将标准化后的数据集导入Parte软件，通过方差分析（ANOVA）筛选差异表达基因；采用MeV软件对LTβ处理组与未处理对照组的差异表达基因进行分层聚类分析。本研究的热图展示了相较于未处理样本，表达量上调两倍的基因。