Histone deacetylases activate hepatocyte growth factor signaling by repressing microRNA-449 in hepatocellular carcinoma cells.
OBJECTIVE
Histone deacetylation regulates chromatin remodeling and transcriptional down-regulation of specific genomic regions; it is altered in many types of cancer cells. We searched for microRNAs (miRs) that are affected by histone deacetylation and investigated the effects in hepatocellular carcinoma (HCC) cells.


METHODS
HCC cell lines (HepG2, HLE, HLF, and Huh7) and immortalized liver cell lines (THLE-2 and THLE-3) were incubated with the histone deacetylase inhibitor trichostatin A. Differentially expressed messenger RNAs (mRNAs) and miRs were identified by expression profiling. Small interfering RNAs were used to reduce levels of histone deacetylases (HDAC)1-3, and HCC cell lines were transfected with miR-449. We evaluated growth of xenograft tumors from modified cells in nude mice. Cells were analyzed by immunoblot and luciferase reporter assays. We analyzed HCC samples from 23 patients.


RESULTS
HDAC1-3 were up-regulated in HCC samples from patients. In cell lines, inhibition of HDAC significantly increased levels of hsa-miR-449a. c-MET mRNA, which encodes the receptor tyrosine kinase for hepatocyte growth factor, is a target of miR-449. Incubation of HCC cells with trichostatin A or transfection with miR-449 reduced expression of c-MET and phosphorylation of extracellular signal-regulated kinases 1 and 2 (downstream effectors of c-MET), increased apoptosis, and reduced proliferation. Huh-7 cells transfected with miR-449 formed tumors more slowly in mice than cells expressing control miRs. HCC samples from patients had lower levels of miR-449 and higher levels of c-MET than human reference.


CONCLUSIONS
In HCC cells, up-regulation of HDAC1-3 reduces expression of miR-449. miR-449 binds c-MET mRNA to reduce its levels, promoting apoptosis and reducing proliferation of liver cells. Expression of miR-449 slows growth of HCC xenograft tumors in mice; this miR might function as a tumor suppressor.
Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany.
Reena
R
Buurman
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
Engin
E
Gürlevik
Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany.
Vera
V
Schäffer
Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany.
Marlies
M
Eilers
Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany.
Maria
M
Sandbothe
Institute of Pathology, Hannover Medical School, Hannover, Germany.
Hans
H
Kreipe
Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany; Institute of Pathology, Hannover Medical School, Hannover, Germany.
Ludwig
L
Wilkens
Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany.
Brigitte
B
Schlegelberger
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
Florian
F
Kühnel
Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany. Electronic address: skawran.britta@mh-hannover.de.
Britta
B
Skawran
D020413:3' Untranslated Regions; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D049109:Cell Proliferation; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D057809:HEK293 Cells; D056945:Hep G2 Cells; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D056284:Histone Deacetylase 1 / Q000378:metabolism; D056464:Histone Deacetylase 2 / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008113:Liver Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D035683:MicroRNAs / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D047368:Tumor Burden; D000099186:Histone Deacetylase 3
D020413:3' Untranslated Regions; D017228:Hepatocyte Growth Factor; D056284:Histone Deacetylase 1; D056464:Histone Deacetylase 2; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases; D006877:Hydroxamic Acids; D035683:MicroRNAs; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019859:Proto-Oncogene Proteins c-met; D012333:RNA, Messenger; D000099186:Histone Deacetylase 3; C066855:HGF protein, human; C544530:MIRN449 microRNA, human; C012589:trichostatin A; C535150:MAPK1 protein, human; C110550:HDAC1 protein, human; C533423:HDAC2 protein, human