hsa-mir-183is frequently methylated and related to poor survival in human hepatocellular carcinoma.
OBJECTIVE
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 thathsa-miR-183is hypermethylated in 30% of HCC (n= 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells,DNMTknockdown and 5-aza-2'-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation athsa-miR-183promoter significantly correlates with poor survival (log-rank testP= 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 athsa-miR-183is specific for the malignant transformation of hepatocytes.


CONCLUSIONS
Our data indicate that hypermethylation ofhsa-miR-183is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.
Sumadi Lukman
SL
Anwar
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.
Till
T
Krech
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.
Britta
B
Hasemeier
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.
Elisa
E
Schipper
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.
Nora
N
Schweitzer
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.
Arndt
A
Vogel
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.
Hans
H
Kreipe
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.
Reena
R
Buurman
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.
Britta
B
Skawran
Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.
Ulrich
U
Lehmann
D000368:Aged; D014408:Biomarkers, Tumor; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis / Q000235:genetics* / Q000401:mortality; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000401:mortality; D008297:Male; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D008875:Middle Aged; D011379:Prognosis; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013447:Sulfites / Q000737:chemistry; D016896:Treatment Outcome
D014408:Biomarkers, Tumor; C533449:MIRN183 microRNA, human; D035683:MicroRNAs; D034741:RNA, Small Interfering; D013447:Sulfites; C042345:hydrogen sulfite
DNA methylation; DNMT knockdown; hsa-miR-183; microRNA microarray