title: Hypoxia induces the overexpression of microRNA-21 in pancreatic cancer cells.
abstract: BACKGROUND
  Pancreatic cancer cells exist in a hypoxic microenvironment containing numerous factors that impact tumor survival, proliferation, and metastasis. MicroRNAs (miRs) are differentially expressed in cancer but also altered by hypoxia. We hypothesized that hypoxia could induce expression of miR-21, an oncomir in pancreatic cancer cells.
  
  
  METHODS
  We examined how hypoxia regulates miR-21 expression in pancreatic cancer cell lines (BxPC-3, AsPC-1) by stem-loop RT-PCR. Chromatin immunoprecipitation assays were used to study how hypoxia alters hypoxia-inducible factor (HIF)-1α binding to the hypoxia response element of miR-21. BxPC-3 and AsPC-1 cells were transfected with a constitutively stable HIF-1α subunit or vector control (pcDNA3.1) to determine the influence of miR-21 in normoxia. The effect of mature miR-21 sense and antisense oligonucleotides on proliferation and apoptosis in hypoxic and normoxic conditions was assessed via WST-1 assay and flow cytometry.
  
  
  RESULTS
  MiR-21 levels increased in all cell lines grown in hypoxic conditions versus normoxia, whereas siRNA targeting HIF-1α reduced miR-21 expression. Hypoxic conditions resulted in direct binding of HIF-1α to the predicted binding site in miR-21. Transfection with a constitutively stable HIF-1α expression plasmid in normoxia resulted in upregulated miR-21, similar to that seen in hypoxia. Cells transfected with antisense constructs targeting miR-21 had reduced proliferation and increased apoptosis in normoxia, whereas miR-21 overexpression abrogated hypoxia-associated reductions in proliferation.
  
  
  CONCLUSIONS
  MiR-21 is induced by hypoxia in pancreatic cancer cells via HIF-1α upregulation. MiR-21 overexpression allows cells to avoid apoptosis in a hypoxic microenvironment. Inhibition of miR-21 expression may increase cellular susceptibility to hypoxia in pancreatic cancer.
authors.affiliation: Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Mace
authors.forename: Amy L
authors.initials: AL
authors.lastname: Collins
authors.forename: Sylwia E
authors.initials: SE
authors.lastname: Wojcik
authors.forename: Carlo M
authors.initials: CM
authors.lastname: Croce
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Lesinski
authors.forename: Mark
authors.initials: M
authors.lastname: Bloomston
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology / Q000503:physiopathology*; D017209:Apoptosis / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival / Q000502:physiology; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000502:physiology; D035683:MicroRNAs / Q000502:physiology*; D010190:Pancreatic Neoplasms / Q000473:pathology / Q000503:physiopathology*; D015854:Up-Regulation / Q000502:physiology*
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C524940:MIRN21 microRNA, human; D035683:MicroRNAs
keywords: HIF-1alpha; Hypoxia; Micro-RNA; Pancreatic cancer