Effect of iNOS and NF-kappaB gene silencing on beta-cell survival and function.
OBJECTIVE
Type I diabetes results from beta-cell death and dysfunction, induced by infiltration of immune cells and local production of inflammatory cytokines. Therefore, we investigated the effect of iNOS and NF-kappaB gene silencing on beta-cell survival and function.


METHODS
Rat insulinoma INS-1E cells were transfected with chemically synthesized siRNA after complex formation with Lipofectamine 2000. Cells were then treated with a cocktail of inflammatory cytokines (IL-1beta+ TNF-alpha+ IFN-alpha), and glucose stimulated-insulin response and viability were determined. iNOS and NF-kappaB gene expression was assessed at mRNA level by real time RT-PCR. The effect of gene silencing was also correlated with cytokine-induced NO production and apoptosis.


RESULTS
Transfection of INS-1E cells with siRNAs silenced iNOS and NF-kappaB gene expression and reduced NO production in a sequence-specific manner without causing significant loss of cell viability and function. However, the abrogation of NO production did not prevent INS-1E cells from cytokine-induced apoptosis, suggesting that this event may not be totally dependent on NO production.


CONCLUSIONS
The gene silencing approach presented here is capable of attenuating the effects of inflammatory cytokines, such as iNOS expression and NO production and it will help to identify new target genes to improve islet transplantation.
De Paula
Daniel
D
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil.
Bentley
M Vitória L B
MV
Mahato
Ram I
RI
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D015870:Gene Expression; D020868:Gene Silencing; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D007340:Insulinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D007371:Interferon-gamma / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology; D008954:Models, Biological; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D053583:Interleukin-1beta; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II