Expression of zTOR-associated microRNAs in zebrafish embryo treated with rapamycin

详细信息    查看全文

• 作者：Eng-Soon Khor^a ; ^{darren_khor@hotmail.my" class="auth_mail" title="E-mail the corresponding author} ; Suzita Mohd Noor^b ; ^{suzita@um.edu.my" class="auth_mail" title="E-mail the corresponding author} ; Pooi-Fong Wong^a ; ^{wongpf@um.edu.my" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Aging ; Embryonic development ; Mammalian target of rapamycin ; Zebrafish
• 刊名：Life Sciences
• 出版年：2016
• 出版时间：1 April 2016
• 年：2016
• 卷：150
• 期：Complete
• 页码：67-75
• 全文大小：1103 K

文摘

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.

2">Main 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.

Key findings

Rapamycin (400nM) suppressed zTOR pathway by down-regulating the phosphorylation of zTOR-associated proteins such as P70S6K and S6K at both 4 h post-fertilisation (hpf) and 8 hpf while 4E-BP1 was only down-regulated at 8 hpf 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 4 hpf.

Significance

Our findings suggest the involvement of zTOR in embryonic senescence and angiogenesis which could be potentially mediated by selected miRNAs.