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©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Nora Schweitzer, Arndt Vogel, Hans Kreipe, Reena Buurman, Britta Skawran, Ulrich Lehmann
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany
Sumadi Lukman Anwar, Department of Surgery, Faculty of Medicine Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
Nora Schweitzer, Arndt Vogel, Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule Hannover, Hannover D30625, Germany
Reena Buurman, Britta Skawran, Institute of Human Genetics, Medizinische Hochschule Hannover, D30625 Hannover, Germany
Author contributions: Anwar SL and Lehmann U conceived and designed the experiments; Anwar SL, Hasemeier B and Shipper E performed the experiments; Anwar SL, Krech T, Buurman R, Vogel A, Kreipe H, Skawran B and Lehmann U contributed to reagents/materials/analysis tools and analysed the data; Anwar SL and Lehmann U contributed to the writing of the manuscript; all authors made critical revisions related to the intellectual content of the manuscript, and approved the final version of the article to be published.
Supported by the Deutsche Forschungsgemeinschaft, No. (DFG) SFB-TRR77 "Liver cancer" (Project B1).
Institutional review board statement: Primary tumor specimens were collected at the time of surgery as leftover from diagnostics at the Hannover Medical School Germany following protocol approved by the local ethics committee ("Ethik-Kommission der Medizinischen Hochschule Hannover", head: Prof. Dr. Tröger).
Conflict-of-interest statement: all authors declared no conflict of interest.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Ulrich Lehmann, professor, Institute of Pathology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
lehmann.ulrich@mh-hannover.de
Telephone: +49-511-5324501 Fax: +49-511-5325799
Received: October 4, 2016
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
AIM
To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC).
METHODS
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.
RESULTS
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.
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.
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.
