RUNX3 directly interacts with intracellular domain of Notch1 and suppresses Notch signaling in hepatocellular carcinoma cells.
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.
Gao
Juan
J
Department of Digestive Diseases, Wuhan General Hospital of Guangzhou Command PLA, Wuhan 430070, Hubei Province, PR China.
Chen
Yu
Y
Wu
Kai-Chun
KC
Liu
Jie
J
Zhao
Yan-Qiu
YQ
Pan
Yang-Lin
YL
Du
Rui
R
Zheng
Guo-Rong
GR
Xiong
Yi-Min
YM
Xu
Hua-Lin
HL
Fan
Dai-Ming
DM
D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000235:genetics / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D000072056:Transcription Factor HES-1; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology; D014162:Transfection
D051792:Basic Helix-Loop-Helix Proteins; D050677:Core Binding Factor Alpha 3 Subunit; D004247:DNA; D004151:Dipeptides; D018398:Homeodomain Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D010446:Peptide Fragments; D011480:Protease Inhibitors; D051881:Receptor, Notch1; D011993:Recombinant Fusion Proteins; D000072056:Transcription Factor HES-1; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; C497761:NOTCH1 protein, human; C497570:RBPJ protein, human; C493729:Runx3 protein, human; C078493:HES1 protein, human