Promoter hypermethylation and histone hypoacetylation contribute to pancreatic-duodenal homeobox 1 silencing in gastric cancer.
OBJECTIVE
The expression of pancreatic-duodenal homeobox 1 (PDX1) in gastric cancer is aberrantly reduced. The aim of this study was to elucidate the regulation of DNA methylation and histone acetylation at the promoter for PDX1 silencing in gastric cancer.


METHODS
PDX1 expression in response to demethylation and acetylation was detected in human gastric cancer cell lines by reverse transcription-polymerase chain reaction (PCR) and western blot. Four CpG islands within the 5'-flanking region of PDX1 gene were analyzed with their transcription activities being detected by dual luciferase assay. Promoter hypermethylation was identified in gastric cancer cell lines and cancer tissues by methylation-specific PCR or bisulfite DNA sequencing PCR analysis. Histone acetylation was determined by chromatin immunoprecipitation (ChIP) assay.


RESULTS
Demethylation by 5'-aza-2'-deoxycytidine (5'-aza-dC) and/or acetylation by trichostatin A (TSA) restored PDX1 expression in gastric cancer cells. Hypermethylation was found in four CpG islands in six of seven cancer cell lines. However, only the distal CpG island located in the promoter fragment of PDX1, F383 (c.-2063 to -1681 nt upstream of the ATG start codon) displayed significant transcriptional activity that could be suppressed by SssI methylase and increased by 5'-aza-dC and TSA. More than 70% of the single CpG sites in F383 were methylated with hypermethylation of F383 fragment more common in gastric cancerous tissues compared with the paired normal tissues (P < 0.05). ChIP assay showed F383 was also associated with low hypoacetylation level of the histones.


CONCLUSIONS
Promoter hypermethylation and histone hypoacetylation contribute to PDX1 silencing in gastric cancer.
Ma
Juan
J
Department of Gastroenterology and Hepatology, Guangdong General Hospital, Guangzhou 510080, China.
Wang
Ji-De
JD
Zhang
Wen-Jing
WJ
Zou
Bing
B
Chen
Wen-Ji
WJ
Lam
Colin S C
CS
Chen
Min-Hu
MH
Pang
Roberta
R
Tan
V P Y
VP
Hung
I F
IF
Lan
Hui-Yao
HY
Wang
Qi-Yi
QY
Wong
Benjamin C Y
BC
D000107:Acetylation; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D056572:Histone Deacetylase Inhibitors; D018398:Homeodomain Proteins; D006877:Hydroxamic Acids; D012333:RNA, Messenger; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; C012589:trichostatin A; D001374:Azacitidine