title: Expression of zTOR-associated microRNAs in zebrafish embryo treated with rapamycin.
abstract: OBJECTIVE
  MicroRNAs (miRNAs) are vital in modulating lifespan and various biological processes including vascular function. The pivotal roles of mammalian target of rapamycin (mTOR) in regulating senescence and angiogenesis have been extensively described. However, the roles of its orthologue, zebrafish target of rapamycin (zTOR) in senescence and angiogenesis remain to be unravelled. In the present study, we aimed to investigate the role of zTOR and identify miRNAs associated with senescence and angiogenesis.
  
  
  METHODS
  Zebrafish embryos were treated with rapamycin and the inhibition of zTOR and its downstream proteins were validated by immunoblotting. Following the treatment, melanocyte density was quantitated, and senescence and angiogenic responses were determined by senescence-associated beta-galactosidase (SA-β-gal) and endogenous alkaline phosphatase (ALP) staining, respectively. Relative expression of microRNAs were determined by quantitative RT-PCR.
  
  
  RESULTS
  Rapamycin (400 nM) suppressed zTOR pathway by down-regulating the phosphorylation of zTOR-associated proteins such as P70S6K and S6K at both 4h post-fertilisation (hpf) and 8hpf while 4E-BP1 was only down-regulated at 8hpf when compared to their respective vehicle controls. Treatment with rapamycin also resulted in significant suppression of melanocyte development and senescence-associated beta-galactosidase (SA-β-gal) activity, and perturbed the development of intersegmental vessels (ISVs) of zebrafish embryos. In addition, the expressions of dre-miR-9-5p and -3p, dre-miR-25-3p and dre-miR-124-3p were significantly up-regulated in embryos treated with rapamycin from 4hpf.
  
  
  CONCLUSIONS
  Our findings suggest the involvement of zTOR in embryonic senescence and angiogenesis which could be potentially mediated by selected miRNAs.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. Electronic address: darren_khor@hotmail.my.
authors.forename: Eng-Soon
authors.initials: ES
authors.lastname: Khor
authors.affiliation: Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. Electronic address: suzita@um.edu.my.
authors.forename: Suzita Mohd
authors.initials: SM
authors.lastname: Noor
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. Electronic address: wongpf@um.edu.my.
authors.forename: Pooi-Fong
authors.initials: PF
authors.lastname: Wong
mesh_terms: D000375:Aging / Q000235:genetics; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015536:Down-Regulation / Q000187:drug effects; D004625:Embryo, Nonmammalian; D008544:Melanocytes / Q000187:drug effects; D035683:MicroRNAs / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D018919:Neovascularization, Physiologic / Q000235:genetics; D020123:Sirolimus / Q000494:pharmacology*; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000096:biosynthesis* / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
chemical_list: D035683:MicroRNAs; C571017:Torc1 protein complex, zebrafish; D014157:Transcription Factors; D029961:Zebrafish Proteins; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D020123:Sirolimus
keywords: Aging; Embryonic development; Mammalian target of rapamycin; Zebrafish