Efficient delivery of siRNA into cytokine-stimulated insulinoma cells silences Fas expression and inhibits Fas-mediated apoptosis.
Fas/FasL interactions have been proposed as a potentially important mechanism mediating beta-cell death in type 1 diabetes. Recent investigations suggest RNA interference, afforded by small interfering RNAs (siRNA), can provide specific and robust gene silencing in mammalian cells. The current study attempted to investigate the effects of silencing Fas expression with siRNA on Fas-mediated apoptosis in mouse insulinoma cells following cytokine incubation. Our results indicate that siRNA is capable of rapid inhibition of cytokine-induced Fas mRNA production and cell surface Fas protein. A complete suppression of the total Fas protein was only observed after prolonged incubation with siRNA, suggesting a slow turn-over of Fas protein. Moreover, siRNA significantly inhibited Fas-mediated beta-cell apoptosis assessed by Caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assays, the extent of which positively correlated with the level of cell surface Fas. These observations provide additional evidence supporting a role for the Fas-mediated pathway in beta-cell destruction, and suggest that siRNA targeting Fas may be of therapeutic value in preventing type 1 diabetes and improving islet cell viability in transplantation.
Burkhardt
Brant R
BR
University of Pennsylvania, Children's Hospital of Philadelphia, Department of Pathology, PA, USA.
Lyle
Rachel
R
Qian
Keping
K
Arnold
Anne-Sophie
AS
Cheng
Henrique
H
Atkinson
Mark A
MA
Zhang
Y Clare
YC
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D020287:In Situ Nick-End Labeling; D050417:Insulin-Secreting Cells / Q000276:immunology / Q000502:physiology; D007340:Insulinoma / Q000378:metabolism*; D051379:Mice; D034741:RNA, Small Interfering / Q000378:metabolism*; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D034741:RNA, Small Interfering; D019014:fas Receptor; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases