title: Epigenetic inactivation of transforming growth factor-β1 target gene HEYL, a novel tumor suppressor, is involved in the P53-induced apoptotic pathway in hepatocellular carcinoma.
abstract: OBJECTIVE
  Hairy/enhancer-of-split related with YRPW motif-like (HEYL) protein was first identified as a transcriptional repressor. It is a downstream gene of the Notch and transforming growth factor-β pathways. Little is known about its role in the pathogenesis of hepatocellular carcinoma (HCC).
  
  
  METHODS
  Eighty surgically resected paired HCC and adjacent non-cancerous tissues were analyzed for HEYL expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). HCC cells were transfected with pHEYL-EGFP vector to overexpress the HEYL gene or infected with specific shHEYL lentiviral vector to silence HEYL gene expression. HEYL expressional analysis and functional characterization were assessed by 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide assays, flow cytometry, RT-qPCR, western blotting and methylation-specific PCR.
  
  
  RESULTS
  We determined that HEYL expression was inactivated in more than 75% of HCC. In addition, overexpression of HEYL in SK-Hep 1 cells caused apoptosis by the cleavage of caspase 3 and poly (ADP-ribose) polymerase. We discovered that HEYL apoptosis was preceded by serine 15 phosphorylation and accumulation of P53. Molecular analysis revealed that HEYL overexpression led to increased p16, p19, p21, p27 and Bad protein expression, and reduced c-Myc, Bcl-2 and Cyclin B1 expression. Epigenetic silencing of HEYL expression by DNA hypermethylation in HCC directly correlated with loss of HEYL expression in HCC.
  
  
  CONCLUSIONS
  HEYL is frequently downregulated by promoter methylation in HCC. HEYL may be a tumor suppressor of liver carcinogenesis through upregulation of P53 gene expression and activation of P53-mediated apoptosis.
authors.affiliation: Department of Surgery, Division of Hepatobiliary Pancreatic Surgery, Kaohsiung Medical University, Kaohsiung, Taiwan.|Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Kung-Kai
authors.initials: KK
authors.lastname: Kuo
authors.affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
authors.forename: Shu-Fang
authors.initials: SF
authors.lastname: Jian
authors.affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
authors.forename: Yi-Jin
authors.initials: YJ
authors.lastname: Li
authors.affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
authors.forename: Shi-Wei
authors.initials: SW
authors.lastname: Wan
authors.affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
authors.forename: Ching-Chieh
authors.initials: CC
authors.lastname: Weng
authors.affiliation: Department of Research and Development, Eternal Chemical, Kaohsiung, Taiwan.
authors.forename: KuanTe
authors.initials: K
authors.lastname: Fang
authors.affiliation: Division of Internal Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.|Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan.|Department of Internal Medicine, Division of Gastroenterology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
authors.forename: Deng-Chyang
authors.initials: DC
authors.lastname: Wu
authors.affiliation: Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan.|Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
authors.forename: Kuang-Hung
authors.initials: KH
authors.lastname: Cheng
keywords: HEYL; P53; apoptosis; hepatocellular carcinoma; promoter hypermethylation