title: MIR494 reduces renal cancer cell survival coinciding with increased lipid droplets and mitochondrial changes.
abstract: BACKGROUND
  miRNAs can regulate cellular survival in various cancer cell types. Recent evidence implicates the formation of lipid droplets as a hallmark event during apoptotic cell death response. It is presently unknown whether MIR494, located at 14q32 which is deleted in renal cancers, reduces cell survival in renal cancer cells and if this process is accompanied by changes in the number of lipid droplets.
  
  
  METHODS
  769-P renal carcinoma cells were utilized for this study. Control or MIR494 mimic was expressed in these cells following which cell viability (via crystal violet) and apoptotic cell numbers (via Annexin V/PI staining) were assessed. By western blotting, MIR494 cellular responses were validated using MIR494 antagomir and Argonaute 2 siRNA. Transmission electron microscopy (TEM) was performed in MIR494-transfected 769-P cells to identify ultrastructural changes. LipidTOX green neutral lipid staining and cholesterol measurements were conducted to assess accumulation of lipids droplets and total cholesterol levels, respectively, in MIR494 expressing 769-P cells. Indirect immunofluorescence and western analyses were also performed to examine changes in mitochondria organization. Co-transfection of MIR494 mimic with siRNA targeting LC3B and ATG7 was conducted to assess their contribution to formation of lipid droplets in MIR494-expressing cells.
  
  
  RESULTS
  MIR494 expression reduces viability of 769-P renal cancer cells; this was accompanied by increased cleaved PARP (an apoptotic marker) and LC3B protein. Further, expression of MIR494 increased LC3B mRNA levels and LC3B promoter activity (2.01-fold; 50% increase). Interestingly, expression of MIR494 markedly increased multilamellar bodies and lipid droplets (by TEM and validated by LipidTOX immunostaining) while reducing total cholesterol levels. Via immunocytochemistry, we observed increased LC3B-associated endogenous punctae upon MIR494 expression. In contrast to ATG7 siRNA, knockdown of LC3B reduced the numbers of lipid droplets in MIR494-expressing cells. Our results also identified that MIR494 expression altered the organization of mitochondria which was accompanied by co-localization with LC3B punctae, decreased PINK1 protein, and altered Drp1 intracellular distribution.
  
  
  CONCLUSIONS
  Collectively, our findings indicate that MIR494 reduces cell survival in 769-P renal cancer cells which is accompanied by increased lipid droplet formation (which occurs in a LC3B-dependent manner) and mitochondrial changes.
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL, 33620, USA. punashidutta@mail.usf.edu.
authors.forename: Punashi
authors.initials: P
authors.lastname: Dutta
authors.affiliation: Department of Integrative Biology, University of South Florida, Tampa, FL, 33620, USA. ehaller@usf.edu.
authors.forename: Edward
authors.initials: E
authors.lastname: Haller
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL, 33620, USA. aesharp@usf.edu.
authors.forename: Arielle
authors.initials: A
authors.lastname: Sharp
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL, 33620, USA. mnanjund@usf.edu.
authors.forename: Meera
authors.initials: M
authors.lastname: Nanjundan
mesh_terms: D017209:Apoptosis / Q000235:genetics; D001343:Autophagy / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D066292:Lipid Droplets / Q000378:metabolism* / Q000473:pathology; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology
chemical_list: C502070:MAP1LC3B protein, human; C547168:MIRN494 microRNA, human; D035683:MicroRNAs; D008869:Microtubule-Associated Proteins