Expression Change Of MicroRNA-155 And Rheb/mTOR Signaling Pathways After Cerebral Ischemia/reperfusion Injury In Rats

The incidence of stroke,the disability rate and the mortality are high in China,and the proportion of ischemic stroke is more than 85%,which is widely concerned by scholars.It is recognized that ischemia/reperfusion injury is one of pathophysiological processes for ischemic stroke and confirmed by many international studies,which become one of the research focus.Recent studies show that mTOR signaling pathways is involved in the process of cerebral ischemia/reperfusion injury,but the specific molecular mechanisms and action targets are still not very clear.mTOR signaling molecule regulate protein translation,cell growth and proliferation by targeting downstream molecules,such as 4Ebp1,S6k1.Nowadays,small RNA,known as microRNA(miRNA),is a type of long-chain non-coding RNA,whose length is about 20-22 bp.miRNA and its corresponding target molecules mRNA pair complementary at non-coding regions,so that the target mRNA molecule is inhibited at translation level or directly led to the degradation,thus participate in the regulation of cell growth and differentiation,energy metabolism,apoptosis,and other important and basic cellular physiological processes.miR-155 is a member of miRNA family,with characteristics of miRNA,involved in diseases such as cancer,inflammation,myocardial infarction occurrence and development.Studies indicated that miR-155 possibly played a role in cerebral ischemia,but its exact changes in brain ischemia/reperfusion injury was not clear.Under the anoxic condition,miR-155 regulated mRNA molecules of mTOR signaling pathways,such as Rheb,RICTOR,RPS6KB2,thus induced autophagy,delay cell cycle progression,and promoted cell apoptosis,which demonstrated whether miR-155 regulate mTOR signaling pathways in the process of cerebral ischemia/reperfusion injury by targeting Rheb,which is still unknown.We designed and carried out this study to observe expression change of miR-155 and Rheb/mTOR signaling pathways in ischemia /reperfusion rats models,in order to research the role of miR-155 in cerebral ischemia/reperfusion injury.Methods:Focal transient cerebral ischemia-reperfusion injury was induced in the right middle cerebral artery of male S-D rats using Longa modified suture occlusion.The rats were randomly divided into four groups according to re-perfusion time,which is 6h,12 h,24h and 72 h after re-perfusion,and the relatively control group received sham surgery(n=8).TTC staining was used to calculate ischemic infarction volume.Quantitative real time PCR was applied to detect the expression of miR-155 and the mRNA levels of Rheb,mTOR,4Ebp1 and S6k1 in ischemic core,boundary zone and contralateral hemisphere after cerebral ischemia-reperfusion injury respectively.Results:(1)The largest volume of ischemic infarction appeared in 24 h after cerebral ischemia-reperfusion,decreased at 48 h and continued to decrease at 72 h.Compared with the Sham group,ischemic infarction volume was higher(P < 0.05).(2)Compared with the Sham group,miR-155 expression increased within 72 h after cerebral ischemia-reperfusion in both ischemic core and boundary zone(P﹤0.05).It was the highest at 24 h,then began to decline,and almost reached to normal level at 72 h in boundary zone.It was the highest level at 24 h and 48 h,then began to decline,and was still higher than nornal level at 72 h in ischemic core.(3)Compared with the Sham group,Rheb mRNA expression increased within 72 h after cerebral ischemia-reperfusion in both ischemic core and boundary zone(P﹤0.05).It was maintained at a higher level within 72 h in boundary zone.It began to increase at 24 h,reached to the highest level at 48 h,then began to decline,and was still higher than normal level at 72 h in ischemic core.(4)Compared with the Sham group,mTOR mRNA expression increased within 72 h after cerebral ischemia-reperfusion in both ischemic core and boundary zone(P﹤0.05).It began to rise within 24 h,and reached to the highest level at 48 h and 72 h in boundary zone.It didn,t rise within 24 h,then began to rise,reached to the highest level at 48 h,afterwards fell rapidly,and almost reached to normal levels at 72 h in ischemic core.(5)Compared with the Sham group,4Ebp1 mRNA expression increased within 72 h after cerebral ischemia-reperfusion in both ischemic core and boundary zone(P﹤0.05).It increased a little within 24 h,then began to rise,reached to the highest level at 48 h,fell rapidly,and closed to normal level at 72 h in boundary zone.It began to rise within 24 h,rised rapidly,reached to the highest at 48 h,then began to decline,and closed to normal level at 72 h in ischemic core.(6)Compared with the Sham group,S6k1 mRNA expression increased within 72 h after cerebral ischemia-reperfusion in both ischemic core and boundary zone(P﹤0.05).It began to increase within 24 h,rised a little at 48 h,and slowly increased at 72 h in boundary zone.It increased significantly within 24 h,reached to the highest at 48 h,then began to decline,and was still higher normal level at 72 h in ischemic core.Conclusion:(1)The ischemic infarction volume decreased gradually after cerebral ischemia-reperfusion in rats;(2)The expression of miR-155 was increased in cerebral ischemia-reperfusion injury,and the higher level associated with reperfusion time,indicating that miR-155 may participate in the process of brain injury after cerebral ischemia-reperfusion;(3)The expression of Rheb,mTOR,4Ebp1 and S6k1 was increased in cerebral ischemia-reperfusion injury,indicating that Rheb/mTOR signaling pathways may participate in brain injury after cerebral ischemia-reperfusion.