MiR-199a And Its Target Gene Expression In The 3-nitro-acid-induced Rat Cerebral Ischemic Tolerance Process

Background and Objectives:Preconditioning of brain tissues with sublethal stresses can result in resistance to subsequent lethal ischemic events in a response called cerebral ischemic tolerance. Elucidation of its mechanism is of paramount importance for future clinical application. The miRNAs are currently considered as important posttranscriptional gene regulators. Recently it is shown that ischemic preconditioning induces a temporal change in the cerebral miRNA profile. In our study, we induced cerebral ischemic tolerance by intraperitoneal injection of3-NPA to rats, and detected the expression of miR-199a and its predicted target genes during the process.Methods:1. Construction of3-NPA induced cerebral ischemic tolerance model:The rats were administrated with3-NPA or vehicle (saline)3days before permanent occlusion of the middle cerebral artery (MCAO). Neurological Severity Scale (NSS) scores and infarction volumes were measured the next day after operation.2. Measurement of the expression of miR-199a and its predicted target genes during3-NPA preconditioning:The rats fell in7groups randomly. While the control group was treated with vehicle (saline), the other6groups were treated with3-NPA and sacrificed2,3,4,5,6and7days later respectively. Bilateral cortical, hippocampal and striatal tissues were rapidly removed and homogenated for total RNA and protein extraction. MiR-199a and its predicted target genes were detected by quantitative real-time PCR and western blotting.Results:1. The rats treated with3-NPA had improved NSS scores after MCAO than the rats treated with vehicle. The infarction volume of rats of3-NPA group (245.43±25.24mm3) was also reduced by21.76%compared to that of control group (313.67±26.29mm3)(p=0.003).2. MiR-199a was downregulated in all tested brain regions during3-NPA preconditioning. MiR-199a in the rat cortex and striatum was down regulated in an early phase (2days after3-NPA injection) and a late phase (4days after3-NPA injection) separately, while in the hippocampus it was only down regulated in the early phase (2days after3-NPA injection). MiR-199a reduced more significantly in the striatum (66.3%) and hippocampus (54.9%) than in the cortex (27.6%). The expression of predicted targets of miR-199a-HIF-1α,Sirtl and VEGFA-showed no change at mRNA level but increased at protein level during3-NPA induced brain ischemic tolerance. Conclusion:The induction of brain ischemic tolerance by3-NPA is a reliable model. MiR-199a was down regulated in an early and late phase during3-NPA preconditioning, which suggested that miR-199a might play a role in both the early and late phase of brain ischemic tolerance. The different response of miR-199a in different brain regions offered a new possible explanation of the regional variation in ischemic tolerance. HIF-1α, Sirt1and VEGFA were regulated in a posttranscriptional manner during3-NPA preconditioning, which indicated that they might partly be regulated by miR-199a.