Metastatic suppressor genes inactivated by aberrant methylation in gastric cancer.
OBJECTIVE
To screen out the differentially methylated DNA sequences between gastric primary tumor and metastatic lymph nodes, test the methylation difference of gene PTPRG between primary gastric tumor and metastatic lymph nodes, and test the regulatory function of 5-aza-2'-deoxycytidine which is an agent with suppression on methylation and the level of methylation in gastric cancer cell line.


METHODS
Methylated DNA sequences in genome were enriched with methylated CpG islands amplification (MCA) to undergo representational difference analysis (RDA), with MCA production of metastatic lymph nodes as tester and that of primary tumor as driver. The obtained differentially methylated fragments were cloned and sequenced to acquire the base sequence, which was analyzed with bioinformatics. With methylation-specific PCR (MSP) and RT-PCR, methylation difference of gene PTPRG was detected between primary tumor and metastatic lymph nodes in 36 cases of gastric cancer. Methylation of gene PTPRG and its regulated expression were observed in gastric cancer cell line before and after being treated with methylation-suppressive agent.


RESULTS
Nineteen differentially methylated sequences were obtained and located at 5' end, exons, introns and 3' end, in which KL59 was observed to be located at 9p21 as the first exon of gene p16 and KL22 to be located at promoter region of PRPRG. KL22, as the probes, was hybridized with driver, tester and 3-round RDA products respectively with all positive signals except with the driver. Significant difference was observed in both methylation rate of gene PTPRG and PTPRG mRNA expression rate between primary tumor and metastatic lymph nodes. Demethylation of gene PTPRG, with recovered expression of PTPRG mRNA, was observed after gastric cancer cell line being treated with methylation-suppressive agent.


CONCLUSIONS
Difference exists in DNA methylation between primary tumor and metastatic lymph nodes of gastric cancer, with MCA-RDA as one of the good analytical methods. Significant difference exists in methylation of gene PTPRG between primary tumor and metastatic lymph nodes of gastric cancer. Methylation level in gastric cancer cell line can be decreased by 5-aza-2'-deoxycytidine, which is the methylation-suppressive agent, with PTPRG expression being recovered.
Wang
Jian-Feng
JF
Department of Surgical Oncology, the First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China.
Dai
Dong-Qiu
DQ
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D019175:DNA Methylation / Q000187:drug effects; D004273:DNA, Neoplasm / Q000378:metabolism*; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016147:Genes, Tumor Suppressor / Q000502:physiology*; D005820:Genetic Testing; D006801:Humans; D008198:Lymph Nodes / Q000378:metabolism / Q000473:pathology; D008207:Lymphatic Metastasis / Q000235:genetics / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5 / Q000235:genetics / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology
D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D000077209:Decitabine; C516850:PTPRG protein, human; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5; D001374:Azacitidine