Synthetic small interfering RNA down‐regulates caspase‐3 and affects apoptosis, IL‐1β, 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.