title: Reprogramming using microRNA-302 improves drug sensitivity in hepatocellular carcinoma cells.
abstract: BACKGROUND
  Although studies have shown that Oct4, Sox2, Klf4, and c-Myc (OKSM)-mediated induced pluripotent stem cell (iPSC) technology sensitizes cancer cells to drugs, the potential risk of inserting c-Myc and random insertions of exogenous sequences into the genome persists. Several authors, including us, have presented microRNA (miRNA)-mediated reprogramming as an alternative approach. Herein, we evaluated the efficacy of miRNA-mediated reprogramming on hepatocellular carcinoma (HCC) cells.
  
  
  METHODS
  Among three miRNAs (miR-200c, miR-302s, and miR-369s) that were previously presented for miRNA-mediated reprogramming, miR-302 was expressed at low levels in HCC cells. After transfecting three times with miR-302, the cells were incubated in ES medium for 3 weeks and then characterized.
  
  
  RESULTS
  iPSC-like spheres were obtained after the 3-week incubation. Spheres presented high NANOG and OCT4 expression, low proliferation, high apoptosis, low epithelial-mesenchymal transition marker expression (N-cadherin, TGFBR2), and sensitization to drugs. Several miRNAs were changed (e.g., low oncomiR miR-21, high miR-29b). cMyc was decreased, and methylation was elevated on histone 3 at lysine 4 (H3K4). Differentiated cells expressed markers of each germ layer (GFAP, FABP4, and ALB). AOF2 (also known as LSD1 or KDM1), one of the targets for miR-302, was repressed in iPSC-like-spheres. Silencing of AOF2 resulted in similar features of iPSC-like-spheres, including cMyc down-regulation and H3K4 methylation. In drug-resistant cells, sensitization was achieved through miR-302-mediated reprogramming.
  
  
  CONCLUSIONS
  miR-302-mediated iPSC technology reprogrammed HCC cells and improved drug sensitivity through AOF2 down-regulation, which caused H3K4 methylation and c-Myc repression.
authors.affiliation: Department of Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan.
authors.forename: Chikato
authors.initials: C
authors.lastname: Koga
authors.forename: Shogo
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nagano
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Tomimaru
authors.forename: Naoki
authors.initials: N
authors.lastname: Hama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Wada
authors.forename: Koichi
authors.initials: K
authors.lastname: Kawamoto
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Eguchi
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Konno
authors.forename: Hideshi
authors.initials: H
authors.lastname: Ishii
authors.forename: Koji
authors.initials: K
authors.lastname: Umeshita
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Doki
authors.forename: Masaki
authors.initials: M
authors.lastname: Mori
mesh_terms: D017209:Apoptosis; D015820:Cadherins / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics*; D002454:Cell Differentiation; D049109:Cell Proliferation; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D058750:Epithelial-Mesenchymal Transition; D020868:Gene Silencing; D056945:Hep G2 Cells; D056466:Histone Demethylases / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics*; D008745:Methylation; D035683:MicroRNAs / Q000235:genetics*; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D014162:Transfection
chemical_list: D015820:Cadherins; D018398:Homeodomain Proteins; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; C532169:MIRN302A microRNA, human; C489427:MYC protein, human; D035683:MicroRNAs; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; D018125:Receptors, Transforming Growth Factor beta; D056466:Histone Demethylases; C495195:KDM1A protein, human; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II