title: miR-375 inhibits autophagy and reduces viability of hepatocellular carcinoma cells under hypoxic conditions.
abstract: OBJECTIVE
  Tumor cells survive hypoxic conditions by inducing autophagy. We investigated the roles of microRNAs (miRNAs) in regulating autophagy of hepatocellular carcinoma (HCC) cells under hypoxic conditions.
  
  
  METHODS
  We used gain- and loss-of-function methods to evaluate the effect of miRNAs on autophagy in human HCC cell lines (Huh7 and Hep3B) under hypoxic conditions. Autophagy was quantified by immunoblot, immunofluoresence, and transmission electron microscopy analyses, and after incubation of cells with bafilomycin A1. We used a luciferase reporter assay to confirm associations between miRNAs and their targets. We analyzed growth of HCC xenograft tumors in nude mice.
  
  
  RESULTS
  miR-375 was down-regulated in HCC cells and tissues; it inhibited autophagy under hypoxic conditions by suppressing the conversion of LC3I to LC3II and thereby autophagic flux. The ability of miR-375 to inhibit autophagy was independent of its ability to regulate 3'-phosphoinositide-dependent protein kinase-1-AKT-mammalian target of rapamycin signaling, but instead involved suppression of ATG7, an autophagy-associated gene. miR-375 bound directly to a predicted site in the 3' untranslated region of ATG7. Up-regulating miR-375 or down-regulating ATG7 inhibited mitochondrial autophagy of HCC cells, reduced the elimination of damaged mitochondria under hypoxia, increased release of mitochondrial apoptotic proteins, and reduced viability of HCC cells. In mice, xenograft tumors that expressed miR-375 had fewer autophagic cells, larger areas of necrosis, and grew more slowly than tumors from HCC cells that expressed lower levels of miR-375.
  
  
  CONCLUSIONS
  miR-375 inhibits autophagy by reducing expression of ATG7 and impairs viability of HCC cells under hypoxic conditions in culture and in mice. miRNAs that inhibit autophagy of cancer cells might be developed as therapeutics.
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Chang
authors.forename: Wei
authors.initials: W
authors.lastname: Yan
authors.forename: Xingxing
authors.initials: X
authors.lastname: He
authors.forename: Lemeng
authors.initials: L
authors.lastname: Zhang
authors.forename: Chuanjiang
authors.initials: C
authors.lastname: Li
authors.forename: Hai
authors.initials: H
authors.lastname: Huang
authors.forename: Gary
authors.initials: G
authors.lastname: Nace
authors.forename: David A
authors.initials: DA
authors.lastname: Geller
authors.forename: Jusheng
authors.initials: J
authors.lastname: Lin
authors.forename: Allan
authors.initials: A
authors.lastname: Tsung
mesh_terms: D000818:Animals; D001343:Autophagy; D000071193:Autophagy-Related Protein 7; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000503:physiopathology*; D045744:Cell Line, Tumor; D002470:Cell Survival; D006801:Humans; D000860:Hypoxia; D008113:Liver Neoplasms / Q000235:genetics / Q000503:physiopathology*; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics
chemical_list: C540933:MIRN375 microRNA, human; D035683:MicroRNAs; C509349:ATG7 protein, human; D000071193:Autophagy-Related Protein 7; D044764:Ubiquitin-Activating Enzymes