title: Restored expression of the tumor suppressor gene RUNX3 reduces cancer stem cells in hepatocellular carcinoma by suppressing Jagged1-Notch signaling.
abstract: Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene that is downregulated in various cancers. In the present study, we analyzed the regulatory function of RUNX3 on Jagged-1 (JAG1) expression and cancer stem cell (CSC) signaling in hepatocellular carcinoma (HCC). Eleven HCC cell lines and 30 human HCC tissues were used. RUNX3 and JAG1 expression levels were analyzed by immunoblotting and immunohistochemistry. Ectopic RUNX3 expression was induced by introducing RUNX3 cDNA into the RUNX3-negative HCC cell line Hep3B and Huh7 cells. Furthermore endogenous RUNX3 expression was knocked down by RUNX3 siRNA in SK-Hep-1 cells. In order to analyze JAG1 transcriptional regulation, we conducted reporter assays, chromatin immunoprecipitation (ChIP) assays and electrophoretic mobility shift assays (EMSAs). Tumorigenicity was analyzed using a SCID mouse liver injection model. An inverse correlation was observed between RUNX3 expression and JAG1 expression in most HCC cell lines and tissues. Restoring RUNX3 expression decreased the expression of JAG1 in Hep3B and Huh7 cells, whereas JAG1 expression was upregulated in RUNX3 siRNA-treated SK-Hep-1 cells. Reporter assays, ChIP assays and EMSAs revealed that RUNX3 directly bound to the transcriptional regulatory region of JAG1 and suppressed JAG1 transcription. Moreover, RUNX3 restoration downregulated CSCs by suppressing JAG1-mediated Notch signaling. The tumorigenic capacity of RUNX3-expressing Hep3B cells was lower compared to that of control Hep3B cells. RUNX3 expression suppressed JAG1 expression and resulted in downregulation of tumorigenesis by suppression of JAG1-mediated CSCs.
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. shin12470123@yahoo.co.jp
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Nishina
authors.forename: Hidenori
authors.initials: H
authors.lastname: Shiraha
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Nakanishi
authors.forename: Shigetomi
authors.initials: S
authors.lastname: Tanaka
authors.forename: Minoru
authors.initials: M
authors.lastname: Matsubara
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takaoka
authors.forename: Masayuki
authors.initials: M
authors.lastname: Uemura
authors.forename: Shigeru
authors.initials: S
authors.lastname: Horiguchi
authors.forename: Junro
authors.initials: J
authors.lastname: Kataoka
authors.forename: Masaya
authors.initials: M
authors.lastname: Iwamuro
authors.forename: Takahito
authors.initials: T
authors.lastname: Yagi
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D000072100:Jagged-1 Protein; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D008875:Middle Aged; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D051880:Receptors, Notch / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction; D014158:Transcription, Genetic; D014183:Transplantation, Heterologous; D047368:Tumor Burden; D055815:Young Adult
chemical_list: D002135:Calcium-Binding Proteins; D050677:Core Binding Factor Alpha 3 Subunit; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; C000606274:Jag1 protein, mouse; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; D051880:Receptors, Notch; D011994:Recombinant Proteins; C493729:Runx3 protein, human; D000072099:Serrate-Jagged Proteins; D049409:Luciferases, Firefly