title: MicroRNA-345 induces apoptosis in pancreatic cancer cells through potentiation of caspase-dependent and -independent pathways.
abstract: BACKGROUND
  Previously, miR-345 was identified as one of the most significantly downregulated microRNAs in pancreatic cancer (PC); however, its functional significance remained unexplored.
  
  
  METHODS
  miR-345 was overexpressed in PC cells by stable transfection, and its effect on growth, apoptosis and mitochondrial-membrane potential was examined by WST-1, Hoechst-33342/Annexin-V, and JC-1 staining, respectively. Gene expression was examined by quantitative reverse-transcription-PCR and/or immunoblotting, and subcellular fractions prepared and caspase-3/7 activity determined by commercially available kits. miR-345 target validation was performed by mutational analysis and luciferase-reporter assay.
  
  
  RESULTS
  miR-345 is significantly downregulated in PC tissues and cell lines relative to normal pancreatic cells, and its expression decreases gradually in PC progression model cell lines. Forced expression of miR-345 results in reduced growth of PC cells because of the induction of apoptosis, accompanied by a loss in mitochondrial membrane potential, cytochrome-c release, caspases-3/7 activation, and PARP-1 cleavage, as well as mitochondrial-to-nuclear translocation of apoptosis-inducing factor. These effects could be reversed by the treatment of miR-345-overexpressing PC cells with anti-miR-345 oligonucleotides. BCL2 was characterised as a novel target of miR-345 and its forced-expression abrogated the effects of miR-345 in PC cells.
  
  
  CONCLUSIONS
  miR-345 downregulation confers apoptosis resistance to PC cells, and its restoration could be exploited for therapeutic benefit.
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Srivastava
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA.
authors.forename: Arun
authors.initials: A
authors.lastname: Bhardwaj
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA.
authors.forename: Sumit
authors.initials: S
authors.lastname: Arora
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA.
authors.forename: Nikhil
authors.initials: N
authors.lastname: Tyagi
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA.
authors.forename: Seema
authors.initials: S
authors.lastname: Singh
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Andrews
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA.
authors.forename: Steve
authors.initials: S
authors.lastname: McClellan
authors.affiliation: Department of Mathematics and Statistics, University of South Alabama, Mobile, AL, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.affiliation: 1] Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA [2] Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL, USA.
authors.forename: Ajay P
authors.initials: AP
authors.lastname: Singh
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D053148:Caspase 3 / Q000235:genetics*; D053179:Caspase 7 / Q000235:genetics*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics; D045304:Cytochromes c / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D008928:Mitochondria / Q000235:genetics; D010190:Pancreatic Neoplasms / Q000235:genetics*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D015398:Signal Transduction / Q000235:genetics*; D051028:bcl-2-Associated X Protein / Q000235:genetics
chemical_list: C561417:MIRN345 microRNA, human; D035683:MicroRNAs; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D053179:Caspase 7