Published online Mar 7, 2017. doi: 10.3748/wjg.v23.i9.1568
Peer-review started: October 10, 2016
First decision: October 20, 2016
Revised: December 13, 2016
Accepted: February 7, 2017
Article in press: February 8, 2017
Published online: March 7, 2017
Processing time: 152 Days and 14.5 Hours
To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC).
Knockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.
miRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that hsa-miR-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 miR-183. In HCC patients, hypermethylation at hsa-miR-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.
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.
Core tip: A comprehensive screening using microRNA microarray in hepatocellular carcinoma (HCC) cells after DNMT1-, DNMT3A-, and/or DNMT3B-knockdown revealed upregulation of miR-23, miR-25, and miR-183. Using primary HCC tumor tissues, we confirmed frequent DNA hypermethylation at the hsa-mir-183 promoter. Hypermethylation of hsa-miR-183 was not found in benign liver tumors, adjacent tumor tissues as well as healthy livers and significantly correlated with poor prognosis. Therefore it represents a potential novel diagnostic and prognostic marker in HCC.