BACKGROUND: Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of diseases. Keloid is an intricate lesion that is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skin with the aid of a cDNA microarray to explore the molecular mechanism underlying keloid formation. MATERIALS AND METHODS: The polymerase chain reaction products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs were isolated from freshly excised human keloids and normal skins and then were purified to mRNAs by Oligotex. Both the mRNAs from keloids and normal skins were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. RESULTS: Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250 genes, including TGF-beta1 and NGF, were upregulated (2.98%) and 152 downregulated (1.81%). Analyses of collagen, fibronectin, proteoglycan, growth factors, and apoptosis-related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with the published biochemical and clinical observations of keloids. Higher expression of TGF-beta(1) and NGF in keloids versus normal skins was also testified with reverse transcription polymerase chain reaction method. CONCLUSIONS: DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help to understand the molecular mechanism of keloid formation.
BACKGROUND: Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of diseases. Keloid is an intricate lesion that is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skin with the aid of a cDNA microarray to explore the molecular mechanism underlying keloid formation. MATERIALS AND METHODS: The polymerase chain reaction products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs were isolated from freshly excised human keloids and normal skins and then were purified to mRNAs by Oligotex. Both the mRNAs from keloids and normal skins were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. RESULTS: Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250 genes, including TGF-beta1 and NGF, were upregulated (2.98%) and 152 downregulated (1.81%). Analyses of collagen, fibronectin, proteoglycan, growth factors, and apoptosis-related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with the published biochemical and clinical observations of keloids. Higher expression of TGF-beta(1) and NGF in keloids versus normal skins was also testified with reverse transcription polymerase chain reaction method. CONCLUSIONS: DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help to understand the molecular mechanism of keloid formation.
Authors: Fernando Vázquez-Villa; Marcos García-Ocaña; José A Galván; Jorge García-Martínez; Carmen García-Pravia; Primitiva Menéndez-Rodríguez; Carmen González-del Rey; Luis Barneo-Serra; Juan R de Los Toyos Journal: Tumour Biol Date: 2015-03-12