Hypermethylation downregulates Runx3 gene expression and its restoration suppresses gastric epithelial cell growth by inducing p27 and caspase3 in human gastric cancer.
OBJECTIVE
Runx family transcription factors are integral components of transforming growth factor-beta signaling pathways and have been implicated in cell cycle regulation, differentiation, apoptosis, and malignant transformation. The silencing of tumor suppressor genes by aberrant hypermethylation occurs frequently in human cancer. It has been noted previously that Runx3 is regarded as an important tumor suppressor gene.


METHODS
Reverse transcription polymerase chain reaction was used to measure Runx3 and the DNA methyltransferase 1 (Dnmt1) messenger RNA (mRNA) expression level of paired samples of primary gastric cancer and corresponding non-cancerous gastric mucosae, which were obtained from surgically resected specimens of 70 patients. Western blot was used to detect the expression of Runx3 at protein levels. The promoter methylation status was measured by using methylation-specific polymerase chain reaction. We used Annexin V-FITC/PI assay to detect cell apoptosis, and the cell cycle was also analyzed. In order to examine the cell cycle and/or apoptosis, we determined p27 and caspase 3 expression by immunohistological analysis.


RESULTS
Our results demonstrate a loss or substantial decrease of Runx3 expression in 70 cases of gastric tumors as compared with that in normal gastric mucosa (0.5749 +/- 0.3580 vs 1.7252 +/- 0.4085, P < 0.05). The protein levels of the Runx3 gene were significantly lower in gastric cancers than those in adjacent normal tissues. The hypermethylation of Runx3 was involved in 50% (28/56) of gastric cancer tissues, which had reduced Runx3 mRNA expression. The differences of the Dnmt1 mRNA level were significant between the methylated and unmethylated Runx3 cancerous groups. Runx3 methylation was significantly correlated with increased Dnmt1 (r = 0.64, P < 0.01). Enforced restoration of Runx3 expression led to the induction of cell apoptosis and upregulation of p27 and caspase3 expression in vitro.


CONCLUSIONS
Our results suggest that a decrease of Runx3 expression by DNA hypermethylation is frequently associated with the evolution of gastric cancer. Runx3 was an independent prognostic factor and a potential therapeutic target for gastric cancer.
Chen
Weichang
W
Department of Gastroenterology, First Affiliated Hospital of Soochow University, China. weichangchen@126.com
Gao
Nan
N
Shen
Yuling
Y
Cen
Jian-Nong
JN
D000328:Adult; D000368:Aged; D017209:Apoptosis; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics / Q000378:metabolism*; D018899:CpG Islands; D050760:Cyclin-Dependent Kinase Inhibitor p27; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005260:Female; D005753:Gastric Mucosa / Q000378:metabolism* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D013997:Time Factors; D015854:Up-Regulation; D055815:Young Adult
C436291:CDKN1B protein, human; D050677:Core Binding Factor Alpha 3 Subunit; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; C493729:Runx3 protein, human; D050760:Cyclin-Dependent Kinase Inhibitor p27; D000077209:Decitabine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; C505407:CASP3 protein, human; D053148:Caspase 3; D001374:Azacitidine