Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo.
BACKGROUND
Many miR genes are located within or around CpG islands. It is unclear whether methylation of these CpG islands represses miR transcription regularly. The aims of this study are to characterize gastric carcinoma (GC)-related methylation of miR CpG islands and its relationship with miRNA expression.


METHODS
Methylation status of 9 representative miR CpG islands in a panel of cell lines and human gastric samples (including 13 normal biopsies, 38 gastritis biopsies, 112 pairs of GCs and their surgical margin samples) was analyzed by bisulfite-DHPLC and sequencing. Mature miRNA levels were determined with quantitative RT-PCR. Relationships between miR methylation, transcription, GC development, and clinicopathological characteristics were statistically analyzed.


RESULTS
Methylation frequency of 5 miR CpG islands (miR-9-1, miR-9-3, miR-137, miR-34b, and miR-210) gradually increased while the proportion of methylated miR-200b gradually decreased during gastric carcinogenesis (Ps < 0.01). More miR-9-1 methylation was detected in 62%-64% of the GC samples and 4% of the normal or gastritis samples (18/28 versus 2/48; Odds ratio, 41.4; P < 0.01). miR-210 methylation showed high correlation with H. pylori infection. miR-375, miR-203, and miR-193b methylation might be host adaptation to the development of GCs. Methylation of these miR CpG islands was consistently shown to significantly decrease the corresponding miRNA levels presented in human cell lines. The inverse relationship was also observed for miR-9-1, miR-9-3, miR-137, and miR-200b in gastric samples. Among 112 GC patients, miR-9-1 methylation was an independent favourable predictor of overall survival of GC patients in both univariate and multivariate analysis (P < 0.02).


CONCLUSIONS
In conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 methylation may be a GC-specific event. Methylation of miR CpG islands may significantly down-regulate their transcription regularly.
Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Division of Cancer Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, No,52 Haidian District, Beijing 100142, China. dengdajun@bjmu.edu.cn
Yantao
Y
Du
Zhaojun
Z
Liu
Liankun
L
Gu
Jing
J
Zhou
Bu-dong
BD
Zhu
Jiafu
J
Ji
Dajun
D
Deng
D000328:Adult; D000368:Aged; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D057809:HEK293 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D053208:Kaplan-Meier Estimate; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D015999:Multivariate Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
C545567:MIRN137 microRNA, human; C533896:MIRN200 microRNA, human; C543800:MIRN210 microRNA, human; C522425:MIRN34 microRNA, human; C541874:MIRN92 microRNA, human; D035683:MicroRNAs