hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma

AIM To screen clinically relevant micro RNAs (mi RNAs) silenced by DNA methylation in human hepatocellular carcinoma(HCC).METHODS Knockdown of DNA methyltransferases (DNMTs) using si RNAs and mi RNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated mi RNA downregulation. Confirmation using individual quantitative real-time PCR (qR T-PCR) assays was thenperformed followed by DNA methylation quantification at the promoter of the mi RNA genes. Quantification of DNA methylation and mi RNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.RESULTS mi RNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of mi R-23, mi R-25 and mi R-183. After q RT-PCR confirmation and Cp G island methylation quantification of these miR NAs in cell lines, further analysis in primary HCC specimens showed that hsa-mi R-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2’-deoxycytidine treatment reduced methylation and stimulated expression of mi R-183. In HCC patients, hypermethylation at hsami R-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes.CONCLUSION Our data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.