MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous 
silencers of target genes. Some tumor-suppressive miRNAs are known to be 
epigenetically silenced by promoter DNA methylation in cancer. In the present 
study, we aimed to identify miRNA genes that are silenced by DNA 
hypermethylation in hepatocellular carcinoma (HCC). We screened for miRNA genes 
with promoter DNA hypermethylation using a genome-wide methylation microarray 
analysis in HCC cells. It was found that miR-335, which is harbored within an 
intron of its protein-coding host gene, MEST, was downregulated by aberrant 
promoter hypermethylation via further methylation assays, including 
methylation-specific PCR, combined bisulfite and restriction analysis, bisulfite 
sequencing analysis and 5-aza-2'-deoxycytidine treatment. The expression levels 
of miR-335 significantly correlated with those of MEST, supporting the notion 
that the intronic miR-335 is co-expressed with its host gene. The levels of 
miR-335/MEST methylation were significantly higher in 18 (90%) out of 20 primary 
HCC tumors, compared to their non-tumor tissue counterparts (P<0.001). The 
expression levels of miR-335 were significantly lower in 25 (78%) out of 32 
primary HCC tumors, compared to their non-tumor tissue counterparts (P=0.001). 
Furthermore, the expression levels of miR-335 were significantly lower in HCC 
tumors with distant metastasis compared to those without distant metastasis 
(P=0.02). In conclusion, our results indicate that expression of miR-335 is 
reduced by aberrant DNA methylation in HCC.