title: Synthetic small interfering RNA down-regulates caspase-3 and affects apoptosis, IL-1 β, and viability of porcine proximal tubular cells.
abstract: Proximal tubular cells are most vulnerable to ischemia reperfusion injury (IRI) in renal transplantation. Caspase-3 can be up-regulated by IRI due to a variety of pathogenic processes such as oxidative damage. This study utilized synthetic small interfering RNA (siRNA) to posttranscriptionally silence target gene, caspase-3, may represent a feasible approach to produce transient effects, but avoid side actions caused by viral vectors. The porcine proximal tubular cells (LLC-PK1), with or without the stimulation of hydrogen peroxide (H(2) O(2) , an oxidizer), were transfected with synthetic caspase-3 siRNA using a cationic lipid-based transfection regent. The expression of caspase-3 at mRNA and protein level was assessed at different times posttransfection and its downstream biological events were also monitored. The caspase-3 mRNA was posttranscriptionally silenced by its siRNA up to 50% after 24 h. The active caspase-3 protein was increased by transfection reagent alone and H(2) O(2) in a dose- and time-dependent manner. Both the precursor and active protein of caspase-3 were decreased by siRNA after 48 h and maintained up to 96 h at least, with a consistent change in its activity. Consequently, apoptotic cells and active IL-1β protein expression was reduced by caspase-3 siRNA; cell viability, especially with H(2) O(2) treatment, was also improved. Taken together, caspase-3 and apoptosis are sensitive markers for cellular injury; using synthetic siRNA silencing caspase-3 may provide not only a valid approach for underlying mechanisms of diseases, but also a potential therapeutic intervention for a wide range of acute clinical disorders including IRI in renal transplantation.
authors.affiliation: Transplant Group, Department of Infection, Immunity and Inflammation, University of Leicester, Leicester General Hospital, University Hospitals of Leicester, Leicester, United Kingdom. by5@le.ac.uk
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.forename: Joshua E
authors.initials: JE
authors.lastname: Elias
authors.forename: Maureen
authors.initials: M
authors.lastname: Bloxham
authors.forename: Michael L
authors.initials: ML
authors.lastname: Nicholson
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D053148:Caspase 3 / Q000235:genetics; D061945:Caspase Inhibitors; D002470:Cell Survival / Q000235:genetics; D015536:Down-Regulation; D053583:Interleukin-1beta / Q000235:genetics; D016030:Kidney Transplantation; D007687:Kidney Tubules, Proximal / Q000201:enzymology* / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000235:genetics*; D015427:Reperfusion Injury / Q000201:enzymology* / Q000473:pathology / Q000517:prevention & control*; D034421:Sus scrofa; D014162:Transfection
chemical_list: D061945:Caspase Inhibitors; D053583:Interleukin-1beta; D034741:RNA, Small Interfering; D053148:Caspase 3