Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2005) 9 P4

BES2005 Poster Presentations Diabetes and metabolism (35 abstracts)

Silencing of leptin and resistin gene expression by RNA interference in vitro

RE Brown 1 , E Ur 2,3 & M Wilkinson 1,2,3

1Department of Physiology & Biophysics, Dalhousie University, Halifax, Canada; 2Department of Obstetrics & Gynaecology, IWK Health Centre, Halifax, Canada; 3Division of Endocrinology & Metabolism, QEII, Halifax, Canada.

In previous work we demonstrated that various adipokines, including leptin and resistin, were expressed in the hypothalamus and anterior pituitary gland. We hypothesized that a practical approach to investigate the putative central role of such factors would be to selectively silence gene expression by RNA interference (RNAi). We tested this hypothesis using model in vitro systems : C6 glioblastoma cells , which we reported express leptin, and 3T3-L1 adipocytes which are known to produce resistin. Four different small interfering RNA (siRNA) targets were designed for each gene. We determined optimal transfection protocols for each cell line using fluorescein-labelled siRNAs. Effective conditions concentrated the siRNA molecules in the perinuclear region of the cells. In C6 cells siRNA L7 reduced leptin mRNA by more than 50% (RT-PCR; p<0.025), and protein by 55% (Western analysis; p<0.01) relative to a scramble siRNA control. In 3T3-L1 cells, siRNA R12 reduced resistin mRNA by 60% (p<0.0001) and resistin protein by 75% (p<0.03).

More detailed studies in C6 cells showed that : (a) the silencing effect of siRNA L7 on leptin mRNA was time- and dose-dependent (maximal at 100 nM); (b) the attenuation of leptin expression was coincident with a 2-fold increase in C6 cell death (p<0.02) and (c) siRNA L7 also blocked the dbcAMP-induced increase in leptin mRNA normally observed in C6 cells. Further experiments in C6 cells with a stability-enhanced siRNA (siSTABLE L7), revealed that leptin mRNA was significantly reduced for almost twice as long relative to the effect of unmodified siRNA L7. Our data indicate that silencing of brain adipokine genes, such as leptin and resistin, is feasible. Supported by the NSHRF , IWK and the Atlee Endowment.

Volume 9

24th Joint Meeting of the British Endocrine Societies

British Endocrine Societies 

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