The Effect Of Knocking-down DJ-1Expression On The Delayed Protection Of Anoxic Preconditioning In H9c2Cells

Objective:To clarify whether DJ-1as an endogenous protective protein involvedin the delayed protection of anoxic preconditioning (APC) in H9c2cells, viaconstructing DJ-1gene-specific small interfering RNA (siRNA) expression vectorsand investigating effects of DJ-1gene silence on the delayed cardioprotection of APCagainst anoxia/reoxygenation injury.Methods:1. Three candidate RNA interference (RNAi) sequences targetingDJ-1were designed and inserted into pGPU6/GFP/Neo expression vector toconstruct the recombinant plasmids. Subsequently, these recombinant plasmids weretransiently transfected into H9c2cells according to the instructions of themanufacturer. Their efficacies in extinguishing DJ-1expression were evaluated byRT-PCR and Western Blot. The RNAi recombinant plasmid testified being mosteffective was used for subsequent experiments.2. The H9c2cells were randomly divided into following five groups:①controlgroup;②pGPU6/GFP/Neo-shDJ-1+Control group;③anoxia/reoxygenation (A/R)group;④anoxia preconditioning (APC) group;⑤pGPU6/GFP/Neo-shDJ-1+APCgroup. After experimental treatment, expression of DJ-1was detected by RT-PCRand Western blot. Cell viability was detected by Methyl thiazolyl tetrazolium (MTT)method. Lactate dehydrogenase (LDH), antioxidant enzymes (superoxide dismutase,catalase and glutathione peroxidase), and malondialdehyde (MDA) were measured bya colorimetric method. Meanwhile,intracellular ROS level was measured by flowcytometry.Results:1. The enzyme digestion analysis and DNA sequencing confirmed thatRNAi expression vector was constructed successfully, including three RNAirecombinant plasmids of targeting DJ-1(pGPU6/GFP/Neo-shDJ-1-A, B, C) andnegative control recombinant plasmid (pGPU6/GFP/Neo-sh-NC). Furthermore,pGPU6/GFP/Neo-shDJ-B was shown to induced most effectively DJ-1genesilencing and therefore was used in subsequent experiments.2. H9c2cells subjected to A/R had significant increases of LDH activity and reduction of cell viability as compared with untreated cells (P<0.01). At the sametime, H9c2cells subjected to A/R had a rapid and significant increase in intracellularROS level and concomitantly increase of MDA content and reduction of antioxidantenzymes activity (SOD, CAT, and GSH-Px) compared with untreated cells (all,p<0.01). This data suggests that H9c2cells treated with A/R appear significantoxidative stress injury. However, APC efficiently attenuated A/R-induced viabilityloss and LDH leakage, inhibited sI/R-induced the elevation of ROS and MDAcontents followed by the increase of antioxidant enzymes (SOD, CAT, and GSH-Px)activities. This shows that APC can induce the delayed cardioprotective effectsagainst A/R injury by inhibiting A/R-induced oxidative stress. Moreover, the resultsshowed that APC can significant up-regulate the expression of DJ-1mRNA andprotein. However, when DJ-1expression was specifically knocked down by siRNA,the delayed cardioprotection induced by APC was abolished, and the inhibitoryeffect of APC on A/R-induced oxidant stress was also reversed.Conclusion:1. RNAi expression vector targeting DJ-1is successfullyconstructed; it provides a foundation for further exploration of the role of DJ-1inmyocardial I/R injury and protection.2. DJ-1is as an endogenous protective protein involved in the delayed protectionof anoxic preconditioning. Moreover, DJ-1is required for APC to inhibitA/R-induced oxidant stress.