LncRNA BIRF Participates In Brain Ischemic Tolerance Induced By Cerebral Ischemic Preconditioning Through Upregulating GLT-1 Via Sponging MiR-330-5p

Cerebral ischemic disease is a serious hazard to modern human health with high morbidity,high mortality and high disability rate.It brings heavy burden to patients and their families.Therefore,it is an important task to study the pathogenesis of cerebral ischemic diseases and improve the resistance of neurons to ischemic injury.Studies have shown that excitatory toxicity of glutamate is an important mechanism of neuron injury during cerebral ischemia.The homeostasis of glutamate in brain mainly depends on glial glutamate transporter-1(GLT-1).Regulating GLT-1 and enhancing glutamate uptake rate in extracellular fluid is an effective method to prevent neurotoxic effects of glutamate during cerebral ischemia.Preconditioning with mild,short-term ischemia that does not cause neuron injury can protect neurons from subsequent,more severe ischemia that usually causes neuron injury.This phenomenon is known as brain ischemic tolerance(BIT).Preconditioning of animals with mild,short-term ischemia is called cerebral ischemic preconditioning(CIPC).Previous studies of our group found that cerebral ischemic preconditioning can up-regulate GLT-1expression in astrocytes and enhance brain ischemic tolerance.However,more in-depth mechanisms need to be elucidated as to how CIPC upregulates GLT-1and induces brain ischemic tolerance.Non-coding RNA(ncRNA)is a class of RNA that does not have protein-coding function.,which include long non-coding RNA(lncRNA)and micro RNA(miRNA).They play an important regulatory role in various diseases.In the cytoplasm,lncRNAs can competitively bind miRNAs with mRNA as competing endogenou RNA(ceRNA),thereby inhibiting the regulation of miRNAs on mRNA expression.Thus lncRNAs play important roles in the occurrence and development of diseases.Studies have shown that lncRNA-miRNA-mRNA is involved in the regulation of oxidative stress,inflammation,apoptosis,autophagy and other pathophysiological processes induced by ischemia-reperfusion through the mechanism of ceRNA in ischemic stroke.In neurodegenerative diseases,the regulation of GLT-1 by miRNA has been reported.Therefore,this study aims to explore whether lncRNA-miRNA regulates GLT-1 through the ceRNA mechanism and thus participates in the induction of cerebral ischemia tolerance.Part One LncRNA BIRF upregulates GLT-1 and participates in BIT induced by cerebral ischemic preconditioningObjective:1.Construct ceRNA regulatory network through high-throughput sequencing and bioinformatics analysis.And explore the lncRNAs involved in the induction of BIT.2.To verify the relationship between lncRNA BIRF and GLT-1expression and BIT induced by cerebral ischemic preconditioning.Methods:1.High-throughput sequencing and validationThe neurons and astrocytes were co-cultured from cortex of newborn mice within 24 h after birth,and randomly divided into 2 groups(n=2):(1)Control group: After 2 d of co-culture,astrocytes and neurons were cultured in normal medium and normoxic incubator without OGD treatment;(2)IPC group: Astrocytes and neurons were replaced with glucose-free DMEM medium for 45 min OGD,and normal medium was replaced after treatment and placed in normoxic incubator.Astrocytes were collected after 6 h and sent to the Sinotech Genomics Corporation for high-throughput sequencing of lncRNA.Five up-regulated and five down-regulated lncRNAs were selected from the sequencing results,and the credibility of the sequencing results was verified by qRT-PCR in 3 batches of independent cultured astrocytes.2.Prediction analysis of ceRNA networkGLT-1 centered ceRNA interaction network analysis was commissioned by Sinotech Genomics Corporation.3.The effects of IPC on lncRNA 1-7 expressionBased on the fold change and expression abundance of lncRNAs,seven lncRNAs were selected for qRT-PCR validation.Co-cultured astrocytes and neurons were randomly divided into 2 groups(n=3):(1)Control group;(2)IPC Group.Cells were collected at 6 h after IPC and the expression of lncRNA 1-7 was detected by qRT-PCR.4.The effects of lncRNA1-7 knockdown on OGD toleranceSeven lncRNAs siRNA were transfected into co-cultured astrocytes respectively,and the knockdown efficiency was detected by qRT-PCR 48 hours later.A total of 5 siRNA with high efficiency were screened for the following experiments.Co-cultured astrocytes and neurons were randomly divided into 3 groups(n=3):(1)IPC + OGD group: 45 min OGD was regarded as IPC,and after 24 h interval,4 h OGD was performed as injury OGD;(2)IPC + OGD + lncRNA 1-5 siRNA group: lncRNA 1-5 siRNA was added to the medium before IPC respectively,and other steps were the same as IPC + OGD group;(3)IPC + OGD + lncRNA siRNA NC group: lncRNA siRNA NC was added to the medium before IPC,and other steps were the same as IPC + OGD group.One day after reoxygenation,CCK8 assay was used to evaluate the neuronal activity of each group to explor whether lncRNA1-5 siRNA could inhibit OGD tolerance induced by IPC.5.The effect of lncRNA 1-5 silencing on GLT-1 protein expressionCo-cultured astrocytes and neurons were randomly divided into 3 groups(n=3):(1)IPC Group;(2)IPC + lncRNA 1-5 siRNA group: lncRNA 1-5siRNA were respectively transfected to the astrocytes before IPC,and other steps were the same as IPC group;(3)IPC + lncRNA siRNA NC group:lncRNA siRNA NC was transfected to the astrocytes before IPC,and other steps were the same as IPC group.Astrocytes were collected at 6 h after reoxygenation,and the effect on GLT-1 protein expression was studied by western blot.6.The subcellular localization of lncRNACo-cultured astrocytes(n=3)were used to detect the subcellular localization of lncRNA BIRF by fluorescence in situ hybridization(FISH).7.The effects of cerebral ischemic preconditioning on lncRNA BIRF expressionThe global brain ischemic model was established by four-vessel occlusion.Ten adult male Wistar rats were randomly separated into two groups(n=5):(1)Sham group: Sham operation of global brain ischemia;(2)CIPC group: The rats were subjected to a global brain ischemia for 3 min,and then the reperfusion was recovered.The expression of lncRNA BIRF in hippocampal CA1 region of rats was detected by qRT-PCR 2 d after the last ischemia.8.The effect of lncRNA BIRF knockdown on brain ischemia toleranceThe global brain ischemic model was established by four-vessel occlusion.The rats were randomly divided into the following groups(n=5):(1)Sham group;(2)IS group: 8 min global cerebral ischemia,and then the reperfusion was recovered;(3)CIPC + IS group: 3 min of global brain ischemia was regarded as CIPC,and after 2 days interval,perform 8 min of global brain ischemia as injury ischemia;(4)CIPC + IS + sh-BIRF group:sh-BIRF was injected into the lateral ventricle of rats before CIPC,and the other procedures were the same as CIPC + IS group;(5)CIPC + IS + sh-NC group: sh-NC was injected into the lateral ventricle of rats before CIPC,and the other procedures were the same as CIPC + IS group.The survival of neurons was observed by thionine staining at 7th day after the last ischemia.9.The effect of lncRNA BIRF knockdown on GLT-1 protein expressionThe global brain ischemic model was established by four-vessel occlusion.The rats were randomly divided into the following groups(n=5):(1)Sham group;(2)CIPC group;(3)CIPC + sh-BIRF group: sh-BIRF was injected into the lateral ventricle of rats before CIPC,and the other procedures were the same as the CIPC group;(4)CIPC + sh-NC group: sh-NC was injected into the lateral ventricle of rats before CIPC,and the other procedures were the same as the CIPC group.The expression of GLT-1 in hippocampal CA1 region of rats was observed by western blot at 2 d after last ischemia.Results:1.LncRNA expression profile of astrocytes in IPC group was significantly differentHigh-throughput sequencing results showed that 323 lncRNAs were significantly down-regulated and 329 lncRNAs were significantly up-regulated in the IPC group compared with the control group.The results of lncRNA sequencing were verified by qRT-PCR in three independent cultured astrocytes.2.Construct ceRNA interaction network with GLT-1 centeredAccording to the sequencing results of lncRNAs and miRNAs,45 lncRNAs and 60 miRNAs participated in the ceRNA interaction network with GLT-1 centered,and these ncRNAs may participate in the regulation of GLT-1through the ceRNA mechanism.3.The expression changes of target lncRNAs in IPC groupAccording to the lncRNA expression level and change fold in lncRNA expression profile,7 lncRNAs were selected as target lncRNAs for subsequent experiments,and it was found that 6 lncRNAs up-regulated and 1 lncRNA down-regulated after IPC treatment.qRT-PCR results showed that the expression levels of NONRATT010451.2,NONRATT021426.2,NONRATT029757.2,NONRATT 001933.2,NONRATT010720.2 and NONRATT009133.2 in IPC group were significantly higher than those in the Control group(FC>2)(P < 0.05),and only NONRATT026094.2 expression was down-regulated(P < 0.05).4.The effect of target lncRNAs on neuron activitySiRNA of NONRATT010720.2,NONRATT021426.2,NONRATT010451.2,NONRATT009133.2 and NONRATT001933.2 lncRNA were transfected respectively into co-cultured astrocytes.Then the co-cultured cells were treated with IPC+OGD.CCK8 results showed that compared with IPC +OGD + siRNA NC group,the neurons activity in IPC+OGD+siRNA group were significantly decreased(P < 0.05).5.NONRATT009133.2 had the greatest effect on GLT-1 protein expressionWestern blot results showed that NONRATT021426.2,NONRATT009133.2 and NONRATT001933.2 inhibited GLT-1 expression(P < 0.05).And NONRATT009133.2 had the most significant inhibitory effect.Therefore,NONRATT009133.2 was selected for follow-up study.According to its function,it was named “Brain Ischemia Related Factor”(lncRNA BIRF).6.LncRNA BIRF subcellular localization in astrocytes.FISH results showed that lncRNA BIRF was highly expressed in both cytoplasm and nucleus of astrocytes,suggesting that lncRNA BIRF may play an important regulatory role both in cytoplasm and nucleus.7.The lncRNA BIRF expression was increased by cerebral ischemic preconditioningqRT-PCR result showed that lncRNA BIRF expression in CIPC group was 5.2128 times higher than that in Sham group(P < 0.05).8.Down-regulation of lncRNA BIRF inhibits the induction of brain ischemia toleranceThionine staining results showed that the neurons in hippocampal CA1 subfield of IS group were severely damaged,and the damage of the neurons in CIPC+IS group was significantly reduced.However,in sh-BIRF+CIPC+IS group,the sh-BIRF was prior injected through lateral ventricles to knockdown lncRNA BIRF expression,which inhibited ischemic tolerance and aggravated neuronal injury.These results suggest that lncRNA BIRF can promote the induction of cerebral ischemia tolerance.9.Down-regulation of lncRNA BIRF can inhibit GLT-1 expression during brain ischemia toleranceWestern blot results showed that lncRNA BIRF expression was inhibited by lateral ventricular injection of sh-BIRF before CIPC,and GLT-1 elevation induced by CIPC was inhibited.Therefore,lncRNA BIRF can up-regulate GLT-1 expression.Summary: lncRNA expression profiles were significantly different after IPC treatment,and lncRNA BIRF expression increased,which may promote ischemic tolerance induced by cerebral ischemic preconditioning through up-regulating GLT-1.Part Two miR-330-5p participates in brain ischemic tolerance induced by cerebral ischemic preconditioning through targeting GLT-1Objective: To demonstrate that miR-330-5p regulates the induction of brain ischemia tolerance by directly targeting GLT-1 in molecular,cellular and in vivo levels.Methods:1.High-throughput sequencing and validationmiRNA high-throughput sequencing was performed by Sinotech Genomics Corporation,and the expression differences between the Control group and the IPC group were compared.Five up-regulated and five down-regulated miRNAs were selected from the sequencing results to verify the reliability of the sequencing results by qRT-PCR in 3 batches of independent cultured astrocytes.2.Screening miRNAs that can interact with lncRNA BIRF and GLT-1 to form ceRNA regulatory networkAccording to miRNA sequencing results and ceRNA network prediction,miRNAs with down-regulated expression and binding sites to lncRNA BIRF and GLT-1 mRNA in IPC group were selected.miR-22-3p and miR-330-5p were selected for the following studies.The binding sites of lncRNA,miRNA and GLT-1 were analyzed and predicted.3.To demonstrate the effects of miR-22-3p or miR-330-5p overexpression on GLT-1 protein expressionCo-cultured astrocytes and neurons were randomly divided into 4 groups(n=3):(1)IPC Group;(2)IPC + miRNA mimics NC group: miRNA mimics negative control was pre-transfected before IPC,and the other steps were the same as IPC group;(3)IPC + miR-22-3p mimics group: miR-22-3p mimics was pre-transfected before IPC,and the other steps were the same as IPC group;(4)IPC + miR-330-5p mimics group: miR-330-5p mimics was pre-transfected before IPC,and the other steps were the same as IPC group.Astrocytes were collected in each group 6 h after the reoxygenation,and the influence on GLT-1 protein expression was studied by western blot.miRNA with the most obvious regulation effect was selected for subsequent experiments.4.Dual-luciferase reporter assays to verify whether miR-330-5p specifically regulates GLT-1To verify whether miR-330-5p can directly bind to the predicted target of GLT-1.The sequences of GLT-1 gene targets(wt and mut)were constructed into dual-luciferase reporter gene vectors,and miR-330-5p mimics was synthesized and co-transfected into HEK293-T cells.The groups are as follows(n=3):(1)miRNA mimics NC + GLT-1-wt;(2)miR-330-5p mimics +GLT-1-wt;(3)miRNA mimics NC + GLT-1-mut;(4)miR-330-5P mimics +GLT-1-mut.At the 24 h after transfection,the fluorescence intensity of luciferase in each group was detected using the dual-luciferase(?) reporter assay system,thus confirming that GLT-1 was the target gene of miR-330-5p.5.The effects of IPC on miR-330-5p expressionCo-cultured astrocytes and neurons were randomly divided into 2 groups(n=3):(1)Control group;(2)IPC group.Cells were collected at 6 h after reoxygenation,and the expression of miR-330-5p was detected by qRT-PCR.6.The effect of miR-330-5p overexpression on OGD toleranceCo-cultured astrocytes and neurons were randomly divided into 5 groups(n=3):(1)Control group;(2)OGD group;(3)IPC + OGD Group;(4)IPC +OGD + miR-330-5p mimics group: miR-330-5p mimics was transfected to the astrocytes before IPC,and the other steps were the same as IPC + OGD group;(5)IPC + OGD + miRNA mimics NC group: miRNA mimics NC was transfected to the astrocytes before IPC,and the other steps were the same as IPC + OGD group.CCK8 assay was used to evaluate the activity of neurons 1d after the reoxygenation,and to investigate whether miR-330-5p mimics could inhibit OGD tolerance induced by IPC.7.The effects of cerebral ischemic preconditioning on miR-330-5p expressionThe global brain ischemic model was established by four-vessel occlusion.The rats were randomly divided into the following groups(n=5):(1)Sham group;(2)CIPC group.The expression of miR-330-5p was detected by qRT-PCR in the hippocampal CA1 region of rats 2 d after the last ischemia.8.The effect of miR-330-5p overexpression on brain ischemia toleranceThe global brain ischemic model was established by four-vessel occlusion.The rats were randomly divided into the following groups(n=5):(1)Sham group;(2)IS group;(3)CIPC + IS group;(4)CIPC + IS + miR-330-5p group: miR-330-5p was injected into the lateral ventricle before CIPC,and the other procedures were the same as CIPC + IS group;(5)CIPC + IS + miR-NC group: miR-NC was injected into the lateral ventricle before CIPC,and the other procedures were the same as CIPC + IS group.The samples were collected at 7th day after the last ischemia,and thionine staining was used to observe the survival of neurons,and to observe whether miR-330-5p mimics could block the induction of brain ischemia tolerance.9.The effects of miR-330-5p overexpression on GLT-1 expressionThe global brain ischemic model was established by four-vessel occlusion.The rats were randomly divided into the following groups(n=5):(1)Sham group;(2)CIPC group;(3)CIPC + miR-330-5p group: miR-330-5p was injected into the lateral ventricle before CIPC,and the other procedures were the same as CIPC group;(4)CIPC + miR-NC group: miR-NC was injected into the lateral ventricle before CIPC,and the other procedures were the same as CIPC group.The expression of GLT-1 in hippocampal CA1 region of rats was observed by Western blot 2 days after the last ischemia.Results:1.miRNA expression profile of astrocytes in IPC group was significantly differentHigh-throughput sequencing results showed that 91 miRNAs were significantly down-regulated and 134 miRNAs were significantly up-regulated in the IPC group compared with the control group.The results of miRNA sequencing were verified by qRT-PCR in 3 batches of independent cultured astrocytes.2.The binding sites of miRNAs to lncRNA BIRF and GLT-1 mRNA were predictedThrough the RNAhybride database,one binding site was predicted between miR-330-5p and lncRNA BIRF,miR-330-5p and GLT-1 mRNA,and one binding site was predicted between miR-22-3p and lncRNA BIRF,miR-22-3p and GLT-1 mRNA.3.Overexpression of miR-330-5p can significantly reduce GLT-1 protein expressionWestern blot results showed that GLT-1 protein expression was significantly decreased by transfection of miR-330-5p mimics before IPC,while it was not significantly affected by transfection of miR-22-3p mimics.Therefore,miR-330-5p was selected for subsequent experiments.4.miR-330-5p can specifically bind to GLT-1 mRNADual-luciferase reporter gene analysis was performed to confirm GLT-1-CDS as a direct target of miR-330-5p.Cells transfected with both GLT-1-wt and miR-330-5p mimics showed significantly reduced relative luciferase activity compared with other groups.5.IPC down-regulated miR-330-5p expressionqRT-PCR results showed that miR-330-5p expression was significantly decreased in IPC group compared with control group(P < 0.05).6.Overexpression of miR-330-5p inhibits the induction of OGD toleranceCCK8 showed that the activity of neurons decreased significantly after OGD.IPC can protect neurons and improve cell activity;However,pre-transfection miR-330-5p mimics before IPC + OGD,the cell activity decreased again.It was suggested that overexpression of miR-330-5p could inhibit OGD tolerance induced by IPC.7.Cerebral ischemic preconditioning significantly reduced miR-330-5p expressionqRT-PCR result showed that miR-330-5p expression was significantly decreased in CIPC group compared with Sham group(P < 0.05).8.Overexpression of miR-330-5p inhibits brain ischemia toleranceThionine staining showed that CIPC induced brain ischemic tolerance and alleviated neuronal death,but the effect was inhibited by lateral ventricular injection of miR-330-5p before CIPC.9.Overexpression of miR-330-5p reduces GLT-1 protein expressionWestern blot result showed that overexpression of miR-330-5p inhibited the elevation of GLT-1 expression induced by CIPC.Summary: miRNA expression profile was significantly different after IPC.and miR-330-5p expression decreased.Overexpression of miR-330-5p down-regulated GLT-1 expression by directly targeting GLT-1 mRNA,thus inhibiting brain ischemia tolerance induced by CIPC.Part Three lncRNA BIRF/miR-330-5p/GLT-1 axis participating in the induction of brain ischemic tolerance by cerebral ischemic preconditioningObjective:1.Demonstrate the interaction between lncRNA BIRF,miR-330-5p and GLT-1 in molecular level.2.Verify that the ceRNA interaction network constituted by lncRNA BIRF/miR-330-5p/GLT-1 could regulate GLT-1 expression and OGD tolerance in cellular level.Methods:1.Demonstrated the interaction between miR-330-5p and lncRNA BIRFThe binding site between miR-330-5p and lncRNA BIRF were demonstrated by dual-luciferase reporter assays.The dual-luciferase reporter gene(wild type and mutant type)containing the binding site between lncRNA BIRF and miR-330-5p was constructed and co-transfected into HEK293-T cells with miR-330-5p mimics.The groups are as follows(n=3):(1)miRNA mimics NC + BIRF-wt;(2)miR-330-5p mimics + BIRF-wt;(3)miRNA mimics NC + BIRF-mut;(4)miR-330-5p mimics + BIRF-mut.At the 24 h after transfection,the fluorescence intensity of luciferase in each group was detected using the dual-luciferase(?) reporter assay system,thus confirming that lncRNA BIRF can bind with miR-330-5p.2.Demonstrated that lncRNA BIRF could competitively bind miR-330-5p with GLT-1Dual-luciferase reporter assays was performed using HEK293-T cells.Which demonstrated that lncRNA BIRF can competitively bind miR-330-5p to relieve the inhibitory effect of miR-330-5p on GLT-1.The sequence of lncRNA BIRF was constructed into the vector pc DNA3.1.The vector,dual-luciferase reporter gene containing GLT-1 binding site and miR-330-5p mimics were co-transfected into HEK293-T cells using Lipofectamine3000.The control group was only transfected with miR-330-5p mimics and GLT-1dual-luciferase reporter gene.The groups are as follows(n=3):(1)miR-330-5p mimics + GLT-1-wt;(2)miR-330-5p mimics + GLT-1-wt+pc DNA3.1 vector;(3)miR-330-5p mimics+GLT-1-wt+pc DNA3.1-BIRF.At the 24 h after transfection,the fluorescence intensity of luciferase in each group was detected using the dual-luciferase(?) reporter assay system,which could demonstrate that lncRNA BIRF could relieve the inhibitory effect of miR-330-5p on GLT-1.3.Explore whether miR-330-5p inhibitor could block the effect of lncRNA BIRF down-regulation on OGD toleranceCo-cultured astrocytes and neurons were randomly divided into 7 groups(n=3):(1)Control group;(2)OGD group;(3)IPC + OGD group;(4)IPC +OGD + siRNA NC group;(5)IPC + OGD + lncRNA BIRF siRNA group;(6)IPC + OGD + lncRNA BIRF siRNA + miR-330-5p inhibitor group: lncRNA BIRF siRNA + miR-330-5p inhibitor were pre-transfected before IPC,and the other steps were the same as IPC + OGD group;(7)IPC + OGD + lncRNA BIRF siRNA + miRNA inhibitor NC group: lncRNA BIRF siRNA + miRNA inhibitor NC were pre-transfected before IPC,and the other steps were the same as IPC + OGD group.CCK8 assay was used to evaluate the cellular activity of neurons 1 day after reoxygenation.4.To observe whether miR-330-5p inhibitor could block the effect of lncRNA BIRF down-regulation on GLT-1 protein expressionCo-cultured astrocytes and neurons were randomly divided into 5 groups(n=3):(1)Control group;(2)IPC Group;(3)IPC + lncRNA BIRF siRNA group;(4)IPC + lncRNA BIRF siRNA + miR-330-5p inhibitor group:lncRNA BIRF siRNA + miR-330-5p inhibitor were pre-transfected before IPC,and the other steps were the same as IPC group;(5)IPC + lncRNA BIRF siRNA + miRNA inhibitor NC group: lncRNA BIRF siRNA + miRNA inhibitor NC were pre-transfected before IPC,and the other steps were the same as IPC group.Astrocytes were collected 6 h after reoxygenation,and the effect on GLT-1 protein expression was studied by western blot.Results:1.miR-330-5p can directly bind to lncRNA BIRFmiR-330-5p mimics significantly reduced relative luciferase activity of BIRF-wt compared with the miRNA mimics NC + BIRF-wt group.However,if the lncRNA BIRF binding site was mutated,this effect disappeared.2.lncRNA BIRF can relieve the inhibitory effect of miR-330-5p on GLT-1According to the result 4 of the second part,miR-330-5p could bind with GLT-1 and reduce relative luciferase activity of GLT-1-wt.On this basis,when lncRNA BIRF overexpression plasmid pc DNA3.1-lncRNA BIRF was transfected into HEK293-T cells,the luciferase activity was found to be increased again.The results showed that lncRNA BIRF could target miR-330-5p and reverse the inhibitory effect of miR-330-5p on GLT-1.3.miR-330-5p inhibitor blocked the inhibitory effect of lncRNA BIRF down-regulation on OGD tolerance inductionThe results of the first part 4 proved that lncRNA BIRf siRNA could inhibit the induction of OGD tolerance and significantly decrease cell activity.On this basis,the effect of lncRNA BIRF siRNA could be blocked by transfection of miR-330-5p inhibitor into astrocytes and cell activity was increased.Therefore,it can be proved that the inhibitory effect of lncRNA BIRF down-regulation on OGD tolerance can be blocked by miR-330-5p.4.miR-330-5p inhibitor blocked the effect of lncRNA BIRF down-regulation on GLT-1 protein expressionThe first part 5 proved that lncRNA BIRF siRNA could inhibit GLT-1expression.On this basis,the effect of lncRNA BIRF siRNA could be blocked by transfection of miR-330-5p inhibitor into astrocytes simultaneously,and the expression of GLT-1 protein was increased.Therefore,it can be proved that down-regulated lncRNA BIRF can inhibit GLT-1 expression by miR-330-5p.Summary: lncRNA BIRF/miR-330-5p/GLT-1 can participate in brain ischemia tolerance induced by cerebral ischemic preconditioning through ceRNA mechanism.Conclusion:1.lncRNA expression profiles were significantly different after IPC treatment,and lncRNA BIRF expression increased,which may promote ischemic tolerance induced by cerebral ischemic preconditioning through up-regulating GLT-1.2.miRNA expression profile was significantly different after IPC.and miR-330-5p expression decreased.Overexpression of miR-330-5p down-regulated GLT-1 expression by directly targeting GLT-1 mRNA,thus inhibiting brain ischemia tolerance induced by CIPC.3.lncRNA BIRF/miR-330-5p/GLT-1 can participate in brain ischemia tolerance induced by cerebral ischemic preconditioning through ceRNA mechanism.These results suggest that cerebral ischemic preconditioning can induce significant differently changes in ncRNA expression profile,and the regulatory mechanism of lncRNA BIRF/miR-330-5p/GLT-1 axis plays an important role in the process of brain ischemic tolerance induced by cerebral ischemic preconditioning.