Identification of genes up-regulated by histone deacetylase inhibition with cDNA microarray and exploration of epigenetic alterations on hepatoma cells.
OBJECTIVE
Epigenetics is the key factor in the regulation of gene expression. We conducted cDNA microarray analysis to screen for genes induced by histone deacetylase (HDAC) inhibition and examined epigenetic alterations.


METHODS
Microarray analysis was performed in six hepatoma cell lines and primary hepatocytes treated with trichostatin A (TSA). mRNA expression of several genes was examined by reverse transcription-polymerase chain reaction in TSA-treated cells and hepatoma samples. Acetylated histones and methylation status in 5'CpG islands was assessed by chromatin immunoprecipitation (ChIP) assay and bisulfite genomic sequencing, respectively.


RESULTS
Fifty-seven genes showed greater than 2-fold change after TSA treatment in multiple cell lines. Among them, four genes including p21(WAF1) exhibited substantial induction (greater than 5-fold changes). Decreased mRNA levels of these genes in hepatoma tissues were observed in more than half of patients. ChIP assay, in general, demonstrated a good correlation between mRNA expression and histone acetylation, but only a limited correlation with the methylated DNA in the promoter region.


CONCLUSIONS
We identified 57 up-regulated genes by TSA treatment in hepatoma cells and some of them appeared to be cancer-related genes in hepatomas. The alterations in acetylated histones are likely closely associated with gene expression.
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
Arai
Makoto
M
Tada
Motohisa
M
Fukai
Kenichi
K
Imazeki
Fumio
F
Kato
Masaki
M
Seki
Naohiko
N
Saisho
Hiromitsu
H
D000107:Acetylation; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics; D045744:Cell Line, Tumor; D055513:Connective Tissue Growth Factor; D018899:CpG Islands; D050759:Cyclin-Dependent Kinase Inhibitor p21; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D017874:Immediate-Early Proteins / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
C526317:CCN2 protein, human; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017931:DNA Primers; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D055513:Connective Tissue Growth Factor; C012589:trichostatin A