Identification and investigation of methylated genes in hepatoma.
Gene silencing due to aberrant DNA methylation plays an important role in carcinogenesis. Previous microarray analysis demonstrated that 14 genes, including hepatocyte growth factor activator inhibitor 2/placental bikunin (HAI2/PB) gene, showed particularly high inductions after 5-aza-2'deoxycytidine (5Aza-dC) treatment in multiple hepatoma cell lines. In the present study, we studied all of these genes except for the HAI2/PB gene and examined DNA methylation status and levels of acetylated histones using bisulphite genomic sequencing and the chromatin immunoprecipitation (ChIP) assay, respectively. Aberrant methylation in primary hepatoma tissues was also examined using methylation-specific polymerase chain reaction (MSP). Genes for E-cadherin, collagen type I alpha 2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), connective tissue growth factor (CTGF) and fibronectin 1 exhibited aberrant methylation in several hepatoma cell lines. The ChIP assay showed that DNA methylation and deacetylation of histones generally coexist except for fibronectin 1. In further studies of 24 primary hepatoma tissues, methylation signals for COL1A2, IGFBP2, CTGF and fibronectin 1 were detected in 13, 18, 4 and 10 patients, respectively. In conclusion, aberrant methylation of COL1A2, IGFBP2, CTGF and fibronectin 1 genes were detected in hepatoma cell lines. We also demonstrated that the methylation of 5'CpG islands and histone deacetylation generally coexisted in the regulation of gene expression except for fibronectin 1. The results of MSP in hepatoma tissues suggested that some of these genes might be involved in the development or progression of hepatoma.
Chiba
Tetsuhiro
T
Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo Ward, Chiba 260-8670, Japan.
Yokosuka
Osamu
O
Fukai
Kenichi
K
Hirasawa
Yuichi
Y
Tada
Motohisa
M
Mikata
Rintaro
R
Imazeki
Fumio
F
Taniguchi
Hideki
H
Iwama
Atsushi
A
Miyazaki
Masaru
M
Ochiai
Takenori
T
Saisho
Hiromitsu
H
D000328:Adult; D000368:Aged; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D055513:Connective Tissue Growth Factor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D018076:DNA, Complementary / Q000235:genetics*; D005260:Female; D005353:Fibronectins / Q000235:genetics; D006657:Histones / Q000235:genetics; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D008113:Liver Neoplasms / Q000235:genetics*; D008297:Male; D046228:Microarray Analysis / Q000379:methods; D008875:Middle Aged; D009857:Oncogenes / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
C526317:CCN2 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D018076:DNA, Complementary; D005353:Fibronectins; D006657:Histones; D017874:Immediate-Early Proteins; D018971:Insulin-Like Growth Factor Binding Protein 2; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D055513:Connective Tissue Growth Factor