Hagos Engda G, Ghaleb Amr M, Kumar Amrita, Neish Andrew S, Yang Vincent W
Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
Am J Cancer Res. 2011 Jan 1;1(1):85-97.
Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. METHODS: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. RESULTS: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. CONCLUSIONS: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions.
Krüppel样因子4（KLF4）是一种锌指转录因子，在细胞增殖、分化和发育过程中具有多种调节功能。KLF4在炎症、肿瘤发生以及体细胞重编程为诱导多能干细胞（iPS细胞）的过程中也发挥作用。为深入了解KLF4调节这些过程的机制，我们进行了DNA微阵列分析，以鉴定野生型和Klf4基因缺失的小鼠胚胎成纤维细胞（MEF）中差异表达的基因。
通过DNA微阵列检测从野生型或Klf4等位基因缺失的小鼠胚胎中分离出的成纤维细胞的表达谱。差异表达的基因提交至注释、可视化与整合发现数据库（DAVID）。还使用 Ingenuity 通路分析（IPA）和基因集富集分析（GSEA）对微阵列数据进行分析以确定通路。通过蛋白质免疫印迹法对具有生物学相关性的选定基因进行分析，以确认微阵列分析结果，从而确定mRNA水平与蛋白质水平之间的相关性。
与野生型MEF相比，在Klf4基因缺失的MEF中鉴定出163个上调基因和88个下调基因，其变化倍数至少为1.5且P值<0.05。Klf4基因缺失的MEF中许多上调基因编码原癌基因、生长因子、细胞外基质和细胞周期激活剂。相比之下，编码肿瘤抑制因子的基因以及参与JAK-STAT信号通路的基因在Klf4基因缺失的MEF中下调。IPA和GSEA还鉴定出了受KLF4调节的各种通路。最后，对选定靶基因的蛋白质免疫印迹法证实了微阵列数据所揭示的变化。
这些数据不仅与先前关于KLF4在肿瘤抑制和体细胞重编程中作用的功能研究一致，还揭示了介导KLF4功能的新靶基因。
