Abstract
RUNX3 takes a strong suppressive effect in many tumors including hepatocellular carcinoma (HCC). HES-1, a downstream target of Notch signaling, is shown to be decreased in human HCC cell line SMMC7721 with RUNX3 gene transfection. Since Notch signaling is oncogenic in HCC, RUNX3 might exert its inhibitory effect in HCC partly through the suppression on Notch signaling. To investigate the possible mechanism of the down-regulation of HES-1 by RUNX3, we performed Western blot and reporter assay and found that RUNX3 suppressed intracellular domain of Notch1 (ICN1)-mediated transactivation of Notch signaling while it did not alter the expression of ICN1 and recombination signal binding protein-J kappa (RBP-J) in SMMC7721 cells. Besides, confocal microscopy, co-immunoprecipitation and GST pull-down assays showed that RUNX3 could co-localize with ICN1 and RBP-J, forming a complex with these two molecules in nucleus of SMMC7721 cells by its direct interaction with ICN1. Furthermore, RUNX3 was recruited to RBP-J recognition motif of HES-1 promoter, which was identified by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Taken together, these findings indicate that RUNX3 suppresses Notch signaling in HCC SMMC7721 cells by its interaction with ICN1 and thus recruitment to the RBP-J recognition motif of downstream genes of Notch signaling.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Basic Helix-Loop-Helix Transcription Factors / genetics
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Basic Helix-Loop-Helix Transcription Factors / metabolism
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Carcinoma, Hepatocellular / metabolism*
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Cell Line, Tumor
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Cell Nucleus / metabolism
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Core Binding Factor Alpha 3 Subunit / genetics
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Core Binding Factor Alpha 3 Subunit / metabolism*
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DNA / genetics
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DNA / metabolism
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Dipeptides / pharmacology
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Gene Expression / drug effects
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Gene Expression / genetics
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Gene Expression Regulation, Neoplastic / physiology*
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Homeodomain Proteins / genetics
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Homeodomain Proteins / metabolism
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Humans
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Immunoglobulin J Recombination Signal Sequence-Binding Protein / genetics
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Immunoglobulin J Recombination Signal Sequence-Binding Protein / metabolism
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Liver Neoplasms / metabolism*
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Peptide Fragments / genetics
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Peptide Fragments / metabolism
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Promoter Regions, Genetic / genetics
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Protease Inhibitors / pharmacology
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Protein Binding / physiology
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Protein Interaction Domains and Motifs / physiology*
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Receptor, Notch1 / genetics
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Receptor, Notch1 / metabolism*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Signal Transduction / physiology*
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Transcription Factor HES-1
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Transcriptional Activation / drug effects
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Transcriptional Activation / physiology
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Transfection
Substances
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Basic Helix-Loop-Helix Transcription Factors
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Core Binding Factor Alpha 3 Subunit
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Dipeptides
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Homeodomain Proteins
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Immunoglobulin J Recombination Signal Sequence-Binding Protein
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N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester
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NOTCH1 protein, human
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Peptide Fragments
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Protease Inhibitors
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RBPJ protein, human
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Receptor, Notch1
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Recombinant Fusion Proteins
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Runx3 protein, human
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Transcription Factor HES-1
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HES1 protein, human
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DNA