LncRNA SOX2-OT Regulates Mir-27a-3p CDIP1 In Cerebral Ischemia Reperfusion Injury

Purposes:CIRI is considered to be one of the most common causes of disability in the world and one of the diseases that cause the most deaths.In addition,it is worth noting that the sequelae of CIRI,including disability and paralysis,are still a big problem.Although there are some CIRI treatments,few patients receive the appropriate treatment at the correct time window.Therefore,there is an urgent need to find a target gene to explore new strategies for I/R.Studies have shown that LncRNA SOX2-OT directly binds to miR-27a-3p,and LncRNA SOX2-OT as a sponge can negatively regulate the expression of miR-27a-3p.Currently,miR-27a-3p has been proven to inhibit cerebral ischemia-reperfusion injury.Therefore,we assume that LncRNA SOX2-OT may play a role in cerebral ischemia-reperfusion injury by regulating the expression of miR-27a-3p.This article aims to explore the role of LncRNA SOX2-OT in cerebral ischemia-reperfusion injury and analyze its potential molecular mechanism.Methods:CIRI rat model construction:SD rats undergo right middle cerebral artery occlusion(MCAO)to construct a CIRI rat model.In order to study the effect of LncRNA SOX2-OT on CIRI rats,stereotactic injection was used to inject LncRNA SOX2-OT-siRNA,control-siRNA,LncRNA SOX2-OT-siRNA+inhibitor control or LncRNA SOX2 into the cerebral cortex before I/R.LncRNA SOX2-OT-siRNA+miR-27a-3p inhibitor.Subsequently,the neurological deficit of the CIRI rat model was analyzed and evaluated,and the cerebral infarct volume after I/R in the rat was quantitatively analyzed.At the same time,the apoptosis of the rat brain tissue,cleaved-Caspase3,and GAPDH protein were analyzed.Analysis;Detect LDH,SOD,MDA,IL-6,IL-1β,TNF-α through the kit;qRT-PCR experiment is used to detect the expression level of LncRNA SOX2-OT and miR-27a-3p.OGD/R cell model construction:Replace the PC 12 cell culture medium with glucose-free DMEM,and incubate at 37℃ for 3 hours in an anaerobic incubator with 95%N2 and 5%CO2 to simulate OGD damage.After OGD exposure,the cells were reoxygenated by maintaining the cells in 10%FBS-containing DMEM with glucose under normoxic conditions for 48 hours.In order to study the effect of LncRNA SOX2-OT on OGD/R PC 12 cells,PC 12 cells were transfected with LncRNA SOX2-OT-siRNA,control-siRNA,inhibitor control,miR-27a-3p inhibitor,LncRNA SOX2-OT-siRNA.+inhibitor control or LncRNA SOX2-OT-siRNA+miR-27a-3p inhibitor 24 hours later,perform OGD/R treatment.Subsequently,MTT was used to detect cell viability;FCM to detect cell apoptosis;Western blot to detect cleaved-Caspase3 protein expression;kit to detect LDH,SOD,MDA;ELISA to detect IL-6,IL-1β,and TNF-α.In addition,TargetScan predicted the binding site between miR-27a-3p and CDIP1,and the dual luciferase reporter gene experiment verified the binding site between miR-27a-3p and CDIP1.In order to study the effect of miR-27a-3p on OGD/R PC 12 cells and the molecular mechanism,PC 12 cells were transfected with mimic control,miR-27a-3p mimic,miR-27a-3p mimic+control-plasmid,miR-27a-3p mimic+CDIP 1-plasmid 24 hours later,OGD/R treatment was performed.Subsequently,MTT detects cell viability;FCM detects cell apoptosis;Western blot detects cleaved-Caspase3 protein expression;kit detects LDH,SOD,MDA;ELISA detects IL-6,IL-1β and TNF-α.Results:In the CIRI rat model experiment,MCAO significantly induced brain damage in rats;at the same time,brain tissue cell apoptosis was also significantly increased compared with the control group,and the expression of cleaved-Caspase3 was significantly increased;IL-6,IL-1β,and TNF-α The secretion in the brain tissue is significantly increased;the release of LDH activity and MDA in the brain tissue is significantly increased,while the SOD activity is significantly reduced in the brain tissue.Compared with the Sham group,the expression level of LncRNA SOX2-OT in the brain tissue of the MCAO model group was significantly increased,while the expression level of miR-27a-3p was significantly reduced.The expression level of LncRNA SOX2-OT in the brain tissue of the MCAO model group rats was significantly increased,while miR-27a-3p was significantly reduced;compared with the MCAO+control-siRNA group,the MCAO+LncRNA SOX2-OT-siRNA group The brain tissue LncRNA SOX2-OT decreased significantly,while miR-27a-3p increased significantly.Compared with the MCAO+LncRNA SOX2-OT-siRNA+inhibitor control group,the brain tissue miR-27a-3p of MCAO+LncRNA SOX2-OT-siRNA+miR-27a-3p inhibitor rats was significantly reduced;at the same time,Compared with the sham group,the neurological deficit score of the MCAO model group was significantly increased,and the cerebral infarction volume was significantly increased;the neurological deficit score of the MCAO+LncRNA SOX2-OT-siRNA group was significantly reduced,and the cerebral infarction volume was significantly reduced;The brain tissue of rats in the MCAO+LncRNA SOX2-OT-siRNA group significantly reduced apoptosis and the expression of cleaved-Caspase3 in the brain tissue;IL-6,IL-in the brain tissue of the MCAO+LncRNA SOX2-OT-siRNA group The secretion of 1β,and TNF-α in brain tissue is significantly reduced;the release of LDH activity and MDA content in brain tissue is significantly reduced,and SOD activity is significantly increased in brain tissue,but these reduction effects are significantly reduced by miR-27a-3p inhibitor reverse.In the experiment of OGD/R cell model,LncRNA SOX2-OT was significantly increased in OGD/R model group cells,and miR-27a-3p was significantly decreased;LncRNA SOX2-OT was significantly increased.in O GD/R+LncRNA SOX2-OT-siRNA group cells LncRNA SOX2-OT was significantly Decreased,miR-27a-3p significantly increased;SOX2-OT-siRNA+miR-27a-3p inhibitor cells significantly decreased miR-27a-3p.The cell apoptosis and cleaved-Caspase3 expression in the OGD/R model group were significantly increased;the cell apoptosis and cleaved-Caspase3 expression in the OGD/R+LncRNA SOX2-OT-siRNA group were significantly decreased,and the OGD/R model group cells were IL-6,The secretion level of IL-1β and TNF-α is significantly increased;LDH activity and MDA release in cells are significantly increased,and SOD activity is significantly reduced in cells;In OGD/R+LncRNA SOX2-OT-siRNA group cells,The secretion levels of-1β and TNF-α were significantly reduced;LDH activity and MDA release in cells were significantly reduced,and SOD activity was significantly increased in cells.These reductions were significantly reversed by miR-27a-3p inhibitor.CDIP1 and miR-27a-3p were significantly increased in OGD/R model group cells;miR-27a-3p was significantly increased in OGD/R+miR-27a-3p mimic group cells,CDIP1 was significantly decreased,and CDIP1 increased significantly in 27a-3p mimic+CDIP1-plasmid cells.Apoptosis and cleaved-Caspase3 expression of cells in the OGD/R model group were significantly increased;in the OGD/R+miR-27a-3p mimic group,cell apoptosis and cleaved-Caspase3 expression were significantly decreased.The secretion levels of IL-6,IL-1β and TNF-α in OGD/R model group cells were significantly increased;LDH activity and MDA release in cells were significantly increased,SOD activity was significantly reduced in cells;In OGD/R+miR-27a-3p mimic group,The secretion of IL-6,IL-1β,and TNF-αin the cell supernatant of the mimic group was significantly reduced;the LDH activity and the release of MDA in the cells were significantly reduced,and the SOD activity was significantly increased in the cells.These reduction effects were significantly reversed by CDIP1-plasmid.Conclusion:In summary,LncRNA SOX2-OT gene silencing reduces cerebral ischemia-reperfusion injury by regulating miR-27a-3p/CDIP1.The above research results provide a new idea for LncRNA SOX2-OT as a new biomarker and potential therapeutic target for cerebral ischemia-reperfusion injury.