| ObjectiveTo explore the effects and molecular mechanisms of lncRNA DLX6-AS1 on cerebral ischemia/reperfusion-induced neuronal apoptosis in vivo and in vitro.MethodsPart 1.The effect of lncRNA DLX6-AS1 on neurological function and neuronal apoptosis after cerebral ischemia-reperfusion injury in vivo and in vitro.The models of middle cerebral artery occlusion/reperfusion(MCAO/R)in C57BL/6 mice and glucose and oxygen deprivation/reperfusion(OGD/R)in N2 a and SH-SY5 Y cells were established to explore the role of lncRNA DLX6-AS1 in the process of cerebral ischemia-reperfusion injury.The mice were intracerebroventricularly injected with DLX6-AS1-sh RNA lentivirus.After 7 days of injection,the mice were exposed to 1hour of ischemia and 24 hours of reperfusion,and then the neuronal apoptosis and acute neurological injury were evaluated by behavioral score,TTC and TUNEL staining;meanwhile,the cleaved-caspase-3 protein level was detected by western blot;Further,water maze test,pole test and cresol violet staining were performed to evaluate the effect of lncRNA DLX6-AS1 on long-term neurological injury in the mice after 1 hour of ischemia and 28 days of reperfusion.In addition,N2 a and SH-SY5 Y cells were transfected with DLX6-AS1-sh RNA,and then exposed to OGD 3h/R 24 h.The neuronal apoptosis and cleaved-caspase-3 protein level of N2 a cells and SH-SY5 Y cells were evaluated by TUNEL staining and western blot.Part 2.The regulating mechanisms of lncRNA DLX6-AS1 on cerebral ischemia/reperfusion or OGD/R-induced neuronal apoptosis in vivo and in vitro.Bioinformatics methods were used to predict the target mi RNA(mi R-149-3p)for lncRNA DLX6-AS1 as a ce RNA to compete and its downstream target genes.The level of mi R-149-3p in MCAO/R mouse brain tissue and in OGD/R-treated cells were detected by RT-q PCR.To detect lncRNA DLX6-AS1 sponging ability of mi R-149-3p,we constructed DLX6-AS1 WT or MUT luciferase reporter vectors and mi R-149-3p overexpression vector or negative vector.Then the luciferase activity was evaluated after co-transfection of lncRNA DLX6-AS1 luciferase reporter vectors and mi R-149-3p overexpression vector or negative vector.In order to verify the effect of lncRNA DLX6-AS1 targeting mi R-149-3p on OGD/R-induced apoptosis of N2 a cells,the N2 a cells were transfected with DLX6-AS1-sh RNA combined with or without mi R-149-3p mimic or mi R-149-3p inhibitor before OGD/R.The apoptosis rate was detected by TUNEL staining,and the level of cleaved-caspase-3 protein was detected by western blot after OGD/R.For the purpose of verifying the regulatory mechanism of DLX6-AS1 with mi R-149-3p on BOK,BOK protein level was detected after transfection of DLX6-AS1-sh RNA,mi R-149-3p mimic or mi R-149-3p inhibitor and then treated with cerebral ischemia/R and OGD/R.Further the effect of BOK on neuronal apoptosis was investigated.BOK level was downregulated in N2 a cells via si RNA transfection and the effects of BOK on the apoptosis of N2 a cells was detected by TUNEL staining,and the level of cleaved-caspase-3 protein was also detected.ResultsPart 1.After cerebral ischemia 1 h/ reperfusion 24 hours,the level of lncRNA DLX6-AS1 was significantly up-regulated in mouse brain tissue,the same results were found in OGD/R-treated N2 a cells and SH-SY5 Y cells.Knockdown of lncRNA DLX6-AS1 significantly improved the neurological deficit of acute injury mice,reduced the volume of cerebral infarction and the rate of neuronal apoptosis,and inhibited the level of cleaved-caspase-3 protein.The results of water maze,pole test and cresol violet staining showed that knockdown of lncRNA DLX6-AS1 significantly improved the cognitive and motor function,reduced brain atrophy and alleviated the long-term neurological injury after 28 days of ischemia-reperfusion in mice.In N2 a cells and SH-SY5 Y cells treated with OGD/R,downregulation of lncRNA DLX6-AS1 level significantly inhibited neuronal apoptosis induced by OGD/R and reduce the level of cleaved-caspase-3 protein which indicates that lncRNA DLX6-AS1 plays an important role in promoting neuronal apoptosis in cerebral ischemia-reperfusion injury.Part 2.Bioinformatics technology predicts that lncRNA DLX6-AS1 participated in the process of neuronal apoptosis induced by cerebral ischemia/reperfusion injury as a competitive endogenous RNA of mi R-149-3p.The results of RT-q PCR showed that the expression of mi R-149-3p was significantly down-regulated in mouse MCAO/R and in vitro OGD/R injury model,which was contrary to the change trend of lncRNA DLX6-AS1.Dual luciferase reporter gene assay confirmed that lncRNA DLX6-AS1 and mi R-149-3p could combine with each other.Overexpression of mi R-149-3p significantly reduced the number of apoptosis-positive cells and the level of cleaved-caspase-3 protein,and inhibited OGD/R-induced cell apoptosis.However,when mi R-149-3p inhibitor was used to interfere its expression,the rate of cell apoptosis and the level of cleaved-caspase-3 protein were significantly increased.Co-transfection of DLX6-AS1-sh RNA and mi R-149-3p inhibitor significantly reversed the protective effect of DLX6-AS1-sh RNA on ischemia/reperfusion injury.Bioinformatics analysis predicted that BOK may be the target gene of mi R-149-3p,and the level of BOK protein was significantly up-regulated in the model of in vivo cerebral ischemia-reperfusion and in vitro OGD/R.Knockdown of lncRNA DLX6-AS1 inhibited the level of BOK protein in vivo and in vitro,indicating that there is a positive correlation between BOK and lncRNA DLX6-AS1 expression.Mi R-149-3p inhibitor could reverse the inhibitory effect of DLX6-AS1-sh RNA on the level of BOK protein,while the level of BOK was down-regulated after transfection with mi R-149-3p mimic,suggesting that there was a negative correlation between BOK and the expression of mi R-149-3p.Downregulation of BOK expression could significantly inhibit the level of cleaved-caspase-3 protein,thus to the inhibitory effect in apoptosis of N2 a cells induced by OGD/R ConclusionLncRNA DLX6-AS1,as a competitive endogenous RNA,can competitively bind to mi R-149-3p,indirectly regulate the expression of target protein BOK,which plays an important role in the process of cerebral ischemia-reperfusion injury.Knockdown of lncRNA DLX6-AS1 expression can alleviate the neuronal apoptosis induced by cerebral ischemia-reperfusion. |
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