The Role And Mechanism Of Circular RNA CCDC6 In Cerebral Ischemiareperfusion

Purpose:Ischemic stroke is a common neurological disease and the main cause of permanent disability.Clinical treatment,such as thrombolytic therapy,is often limited by the narrow treatment time window.And the long-term curative effect is not good.The degree of neuronal injury after cerebral ischemia is the main factor determining the prognosis.Therefore,it is necessary to choose the treatment to save the injured neurons.However,it is reported that so far only a few therapeutic drugs can alleviate the neurological deficit after stroke,so it is urgent to find new treatments that can reduce neuronal damage.Circular RNA(circ RNA)is a newly discovered endogenous non coding RNA,which is characterized by covalently closed continuous loops produced by reverse splicing.Many circ RNAs are highly expressed in the brain.Although their physiological functions have not been fully determined,studies have found that circ RNAs are involved in neural development and a variety of nervous system diseases.Previous studies found that circccdc6 was up-regulated in the brain of the rat model of middle cerebral artery occlusion(MCAO)in the expression profile of circ RNA after transient focal ischemia.However,the potential role of circccdc6 in ischemic stroke related neuronal injury remains unknown,especially neuronal injury,which has not been determined.Therefore,this study first established the middle cerebral artery occlusion / reperfusion(MCAO/R)rat model and the SH-SY5 Y cell model treated with oxygen glucose deprivation and reoxygenation(OGD/R).We detected the expression of circccdc6 in the two models,and elucidated the mechanism of circccdc6 participating in neuronal thermos ptosis and inflammation through loss of function and function acquisition experiments.To explore the potential of circ CCDC6 as a biomarker of cerebral ischemic nerve injury.It provides new theoretical support for clarifying the function of circ RNA in cerebral ischemia injury,helps to deeply understand the pathophysiological regulation mechanism of ischemic stroke,and lays a foundation for finding biomarkers and therapeutic targets of ischemic stroke based on circ RNA in the future.Methods: 1.Mcao/r rat model was established in vivo.Knockdown of circccdc6 improved the neurological deficit after focal cerebral ischemia in mice: the experimental groups included sham operation control injection group,mcao/r group,mcao/r-control injection group and mcao/r-knockdown of circccdc6 group.QPCR test knockdown efficiency;The neurological deficits of rats in each group were evaluated by improving the neurological deficit score;Nissl staining was used to show the basic neural structure of the brain or spinal cord and detect the injury of nerve cells.During cell death,gsdmd mediates the formation of membrane pores.Therefore,we performed immunofluorescence staining for gsdmd;TUNEL staining was used to determine the number of TUNEL positive cells in rat brain tissue as neuron apoptosis;Detection of inflammatory factor IL-1 by ELISA β、 Expression of IL-18;WB was used to detect the expression of IL-1,IL-18 inflammatory factors and caspase-1,ASC and NLRP3 proteins.Ogd/r model was induced by oxygen glucose deprivation / reperfusion in vitro.The experimental groups included control group,ogd/r group,ogd/r-control group and ogd/roverexpression circccdc6 group.After pc DNA 3.1-circccdc6 was transfected,the transfection efficiency of each group was detected by q PCR.The activity and toxicity of neurons were detected by MTT assay and colorimetry respectively.In addition,the percentage of Caspase-1 positive cells in each group treated with ogd/r was further analyzed by flow cytometry;IL-1 in each group was determined by ELISA β And IL-18;WB detection of IL-1 in each group β、 Protein expression of IL-18,cleaved caspase-1,ASC and NLRP3.(1)in order to study the downstream effectors of circccdc6,through the bioinformatics website https://starbase.sysu.edu.cn/ MiRNA and binding site binding to circccdc6.QPCR was used to detect the expression of mir-128-3p in control group or maco/r rats and ogd/r neurons.Wt-circccdc6 was co transfected with mir-128-3p simulant,and the luciferase activity was detected by double luciferase assay.Rip experiment analyzed the combination of circccdc6 and mir-128-3p.Then,q PCR was used to detect the expression of mir-128-3p after circcdc6 was knocked down or after it passed through the table.(2)Then,we investigated the role of mir-128-3p in ogd/r induced neuronal cell death and inflammation.Mir-128-3p simulant and inhibitor were transfected into SH-SY5 Y cells.The experimental groups included simulant control group,mir-128-3p simulant group,inhibitor control group and mir-128-3p inhibitor group.The expression of mir-128-3p in SH-SY5 Y cells was detected by q PCR to determine the transfection efficiency.LDH release was detected by kit,the percentage of Caspase-1 positive cells was detected by flow cytometry,and IL-1 was detected by ELISA β And IL-18 content and WB determination of IL-1 β 、 Protein expression of IL-18,cleaved caspase-1,ASC and NLRP3.(3)TXNIP has been proved to be a proinflammatory factor in i/r injury.The activation of txnip/nlrp3 inflammatory pathway is a key factor in blepharoptosis.WB was used to detect the expression of TXNIP in vivo and in vitro.With regard to the change of TXNIP expression mediated by mir-128-3p,quantitative PCR was performed to detect the protein level of TXNIP in neurons transfected with mir-128-3p mimics or mir-128-3p inhibitors.The complementary sequences of mir-128-3p and TXNIP 3’UTR were searched on bioinformatics websites,and then the targeting relationship was determined by double Luciferase Report Analysis and rip experiment.(4)Finally,rescue experiments were conducted to determine the effect of circccdc6/mir-128-3p/txnip/nlrp3 axis on ogd/r induced neuronal cell death and inflammation.The experimental groups were Si NC group,Si circccdc6+pcdna3.1 group and Si circccdc6+pcdna3.1-txnip group.The expression of mir-128-3p in mir-groups was detected by q PCR;WB was used to detect the expression of TXNIP in each group.In function,CCK8 was used to measure the cell viability of each group;LDH release of each group was detected by the kit;The percentage of Caspase-1 positive cells was detected by flow cytometry;WB determination of IL-1 β、 Protein expression of IL-18,cleaved caspase-1,ASC and NLRP3.Results: 1.(1)after shrna-circccdc6 injection,the enhanced expression of circccdc6 induced by mcao/r treatment was inhibited;(2)Neurological function evaluation showed that mcao/r rats had severe nerve injury,which could be alleviated by knocking down circccdc6;(3)Nissl staining further showed that the neurons of mcao/r rats were swollen,the cytoplasm was vacuolized,and the number of Nissl bodies decreased.Knocking down circccdc6 could alleviate these symptoms;(4)Mcao/r treatment increased the fluorescence intensity of gsdmd,but this trend was limited by circcdc6 gene knockout;(5)The number of TUNEL positive cells in brain tissue of rats undergoing mcao/r surgery increased,and IL-1 β And IL-18,IL-1 β、 The protein expression of IL-18,cleaved caspase-1,ASC and NLRP3 increased;When circccdc6 was knocked down,all the changes caused by mcao/r were reversed.(1)After pc DNA 3.1-circccdc6 was transfected,the expression of circccdc6 in neurons induced by ogd/r treatment increased further;(2),under ogd/r treatment,neuronal viability was inhibited and LDH release was increased,while the overexpression of circccdc6 had an enhanced effect;(3)Ogd/r treatment could induce the percentage of Caspase-1 positive cells and IL-1 β And IL-18 and IL-1 β、 The protein expression of IL-18,cleaved caspase-1,ASC and NLRP3 increased;In addition,the upregulation of CIRC ccdc6 further promoted all these changes.(1)it was predicted that 20 miRNAs might bind to circccdc6,especially mir-128-3p;Mir-128-3p and circccdc6 have binding sites at chr10:61564232-61564251[-].It is reported that mir-128-3p is down regulated in spinal cord i/r injury,and its overexpression can improve neuroinflammation and apoptosis.Similar results were obtained in this study.Mir-128-3p was down regulated in both maco/r rats and ogd/r neurons.Double Luciferase Report experiment showed that CO transfection of wt-circccdc6 with mir-128-3p simulant could reduce luciferase activity.Rip experiment further supported the increase of circccdc6 and mir-128-3p in ago2 group.Knockdown or overexpression of circcdc6 up-regulated and down regulated the expression of mir-128-3p,respectively.In conclusion,circccdc6 competitively binds mir-128-3p.(2)Transfection of mir-128-3p mimics and inhibitors up-regulated and down regulated the expression of mir-128-3p in SH-SY5 Y cells,respectively.After the up regulation of mir-128-3p,the activity of neurons increased,the release of LDH decreased,the percentage of Caspase-1 positive cells and IL-1 β And IL-18 content and IL-1 β、 The protein expression of IL-18,cleavage caspase-1,ASC and NLRP3 decreased;The knockout of mir-128-3p had the opposite effect(Fig.4b-f).In conclusion,mir-128-3p is involved in the regulation of neuronal cell death and inflammation.(3)In vivo and in vitro,TXNIP expression was increased.Transfection of mir-128-3p mimics or mir-128-3p inhibitors decreased and increased TXNIP protein levels in neurons,respectively.The complementary sequences of mir-128-3p and TXNIP 3’UTR were searched on bioinformatics websites,and then the targeting relationship was determined by double luciferase reporter analysis and rip experiment.In short,the binding of mir-128-3p TXNIP 3 ’UTR has a targeting relationship.(4)Knockout of circccdc6 could promote the expression of mir-128-3p,while overexpression of TXNIP had no effect on the expression of mir-128-3p;Overexpression of TXNIP could reverse the inhibition of TXNIP protein expression induced by circcdc6 knockout.Functionally,when TXNIP was up-regulated,circccdc6 silencing mediated protection against ptosis and inflammation was blocked.Obviously,circcdc6 affects neuronal cell death and inflammation by regulating the mir-128-3p/txnip/nlrp3 pathway.Conclusion:(1)knockdown of circccdc6 can improve the cell death and inflammation induced by mcao/r.(2)In vitro,overexpression of circccdc6 aggravated ogd/r induced neuronal cell death and inflammation..(3)in the mechanism,circccdc6 mediates mir-128-3p and activates txnip/nlrp3,thereby promoting ogd/r induced neuronal cell death and inflammation.Circccdc6 may provide a new strategy for the treatment of mcao/r.