title: Ghrelin inhibition of ethanol-induced gastric epithelial cell apoptosis is mediated by miR-21.
abstract: OBJECTIVE
  To investigate the underlying mechanism of ghrelin-induced gastro-protection in a cell culture model of ethanol-induced gastric epithelial cell injury.
  
  
  METHODS
  The human gastric epithelial cell line GES-1 was incubated with ghrelin (0.01-1 µM), 1 µM ghrelin and 1 µM D-Lys3-growth hormone releasing peptide-6 (GHRP-6), or 1 µM ghrelin and 400 nM antagomiR-21 for 24 h, followed by treatment with 8% ethanol for 3 h to induce apoptosis. Cell viability was determined by MTT assays and flow cytometry was used for detection of apoptosis rates. miR-21 transcription was analyzed by qRT-PCR and Akt, Bcl-2, Bax and caspase 3 expressions were measured by Western blot.
  
  
  RESULTS
  Flow cytometry and a quantitative RT-PCR analysis of the expression of miR-21 showed that ghrelin inhibited apoptosis in a dose dependent manner through a signaling pathway that was both growth hormone secretagogue receptor (GHS-R) and miR-21 dependent, as the antiapoptotic effect of ghrelin was blocked by both D-Lys3-GHRP-6 and antagomiR-21, respectively. Western blotting of Akt, Bcl-2, Bax, and caspase 3 showed that the levels of the antiapoptotic proteins, Akt and Bcl-2, in the cells pretreated with ghrelin alone were higher than those in the cells pretreated with D-Lys3-GHRP-6 or antagomiR-21. By contrast, the levels of the proapoptotic proteins, Bax and caspase 3, in the cells pretreated with ghrelin alone were lower than those in the cells pretreated with D-Lys3-GHRP-6 or antagomiR-21.
  
  
  CONCLUSIONS
  Ghrelin inhibits GES-1 cell apoptosis through GHS-R-dependent signaling in which miR-21 activates the PI3K/Akt pathway, which upregulates Bcl-2 and downregulates Bax and caspase 3 expression.
authors.affiliation: Department of Gastroenterology, Jinshan Hospital of Fudan University Shanghai, China.
authors.forename: Miao
authors.initials: M
authors.lastname: Jiang
authors.affiliation: Department of TCM, Jinshan Hospital of Fudan University Shanghai, China.
authors.forename: Peng-Fei
authors.initials: PF
authors.lastname: Gao
authors.affiliation: Department of Gastroenterology, Jinshan Hospital of Fudan University Shanghai, China.
authors.forename: Huan-Qing
authors.initials: HQ
authors.lastname: Li
authors.affiliation: Department of Gastroenterology, Jinshan Hospital of Fudan University Shanghai, China.
authors.forename: Pei-Ying
authors.initials: PY
authors.lastname: Tian
authors.affiliation: Department of Gastroenterology, Jinshan Hospital of Fudan University Shanghai, China.
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Fan
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D000431:Ethanol / Q000633:toxicity*; D005753:Gastric Mucosa / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D054439:Ghrelin / Q000494:pharmacology*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D054440:Receptors, Ghrelin / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
chemical_list: D051017:Apoptosis Regulatory Proteins; C520836:GHRP-6, Lys(3)-; D054439:Ghrelin; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D009841:Oligonucleotides; D009842:Oligopeptides; D054440:Receptors, Ghrelin; D000431:Ethanol; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt
keywords: Ghrelin; PI3K/Akt; apoptosis; gastric epithelial cells; miR-21