A New Strategy to Find Targets for Anticancer Therapy: Chemokine CXCL14/BRAK Is a Multifunctional Tumor Suppressor for Head and Neck Squamous Cell Carcinoma

Table 2

Regulation of gene expression of tumor-related genes by epidermal growth factor (EGF).

Name	Control	+EGF	Ratio-1	+EGF	Control	Ratio-2	1/Ratio-2
	Cy3	Cy5		Cy3	Cy5
MMP1	2240	10470	4.67	2673	579	0.22	4.61
Vimentin	4596	16877	3.67	5170	1896	0.37	2.73
TIMP3	2755	2113	0.77	631	1404	2.22	0.45
IGFBP3	6081.20	1361	0.22	401	1643	4.09	0.24
BRAK	7174	2089	0.29	654	4637	7.09	0.14

Total RNA was extracted with TRIzol reagent (Invitrogen) from cells treated with EGF for 24 h and untreated control cells and fluorescent cDNAs were prepared separately by a single round of reverse transcription in the presence of fluorescent Cy3-deoxy UTP for EGF-treated cells and Cy5-deoxy UTP for untreated ones. Cy3-deoxy UTP- and Cy5-deoxy UTP-labeled cDNAs were mixed and hybridized with IntelliGene Human Cytokine CHIP Ver. 3.0 (TAKARA). cDNA microarray analysis was performed according to the manufacturer’s protocol. The same experiment was repeated by reversing the labels, that is, using Cy5-deoxy UTP for EGF-treated cells and Cy3-deoxy UTP for the untreated ones. The hybridized chips were analyzed by ScanArray 4000 (Perkin-Elmer, Wellesley, MA, USA). MMP1: matrix metalloproteinase-1, TIMP3: tissue inhibitor of matrix metalloproteinase 3, IGFBP3: insulin-like growth factor-binding protein 3.