Mechanism Of LncRNA NONRATT029757.2 Involved In Cerebral Ischemia Reperfusion Injury Through Regulating Neuronal Autophagy

Stroke is by far the leading cause of death and disability,with ischemic stroke,which accounts for about three-fifths of all strokes,being particularly serious.In China,the incidence and mortality rate of stroke is second to none,and it has become a major burden on the country’s health care system,yet no significant progress has been made in the treatment of stroke.Therefore,it is particularly important to search for mechanisms related to neuronal damage after stroke.Autophagy is the process by which the autophagic lysosomal system degrades damaged organelles and abnormal proteins in cells.There is increasing evidence that autophagy can be activated in a variety of cells in the brain,including neurons,glial cells,and brain microvascular cells,after cerebral ischemia reperfusion.Non-coding RNA includes long non-coding RNA and miRNA,etc.To date,a large number of studies have found that ncRNA,although not capable of coding for proteins,can play regulatory roles in various diseases.In recent years,research on lncRNA has been hot,and a large number of studies have found significant changes in lncRNA expression after ischemic stroke,and a considerable number of lncRNA have been shown to play an important role in the ischemic stroke disease process.However,a large number of lncRNA have not been studied so far,and the mechanisms of the vast majority of lncRNA in ischemic stroke need to be elucidated.Both lncRNA and autophagy play important roles in cerebral ischemic injury.Although some studies have found a close relationship between lncRNA and cerebral ischemic autophagy,the regulatory mechanism of lncRNA involvement in neuronal autophagy after cerebral ischemia is still far from being elucidated,and the study of lncRNA regulation of autophagy and its mechanism still needs to be further explored.Thus,the aim of this paper is to investigate the mechanism of lncRNA involvement in cerebral ischemia-reperfusion injury through autophagy.Part Ⅰ Changes in lncRNA NONRATT029757.2 and autophagy protein expression after cerebral ischemia reperfusionObjective:To explore the change of lncRNA NONRATT029757.2 and autophagy protein in cerebral ischemia reperfusion injury.Methods:1.25 SD rats were randomly divided into Sham group,middle cerebral artery occlusion(MCAO)reperfusion group for 6 h,12 h,24 h and 48 h(n=5),and neurological impairment was scored by Longa score;TTC staining was used to determine Cerebral infarct volume;Western blot detected the expression levels of autophagy proteins LC3 and Beclin 1 in brain tissue on the side of cerebral ischemia reperfusion injury in each group;qRT-PCR quantified the expression of lncRNA NONRATT029757.2 in brain tissue on the side of cerebral ischemia reperfusion injury in each group.2.SD rat cortical primary neurons and PC 12 cell lines were randomly divided into Control,oxygen glucose deprivation(OGD)reperfusion 6 h,12 h,24 h and 48 h groups(n=5),and cell activity was measured by CCK method;Hoechst-PI staining was used to detect cell necrosis;Western blot detected the expression levels of autophagy proteins LC3 and Beclin 1 after OGD reperfusion in each group;qRT-PCR was used to quantify the expression of lncRNA NONRATT029757.2 after OGD reperfusion in each group.Results:1.The results of longa score showed that the ischemia-reperfusion 6 h group,12 h group,24 h group and 48 h group showed different degrees of nerve injury symptoms compared with the Sham group.2.The TTC staining results showed that infarction occurred in the ischemia-reperfusion 24 h group compared with the Sham group,and the difference was statistically significant(P<0.05).3.CCK results and Hoechst-PI staining showed that cell activity was significantly reduced and cell necrosis was significantly increased in the OGD reperfusion 24 h group compared with the Control group(P<0.05).4.Western blot results showed that there were dynamic changes in the expression of autophagy proteins LC3 and Beclin 1 after cerebral ischemia reperfusion and oxygen glucose deprivation reperfusion.Animal experiments:compared with the Sham group,the expression of LC3 and Beclin 1 protein increased at 6 h of reperfusion in MCAO,but the difference was not significant,LC3 and Beclin 1 protein expression increased at 12 h of reperfusion,and continued to increase at 24 h of reperfusion,all differences were statistically significant(P<0.05),LC3 protein remained at a high level at 48 h of reperfusion,and the difference was significant(P<0.05),but Beclin 1 protein expression decreased,and the difference was not statistically significant(P>0.05).Cell experiment:compared with the Control group,LC3 and Beclin 1 protein expression was elevated at 12 h of reperfusion in the PC-12 cell line OGD and continued to increase at 24 h of reperfusion,both differences were statistically significant(P<0.05),LC3 protein remained at a higher level at 48 h of reperfusion,the difference was significant(P<0.05),while Beclin 1 protein expression decreased significantly,and the difference was not statistically significant(P>0.05).5.qRT-PCR results showed that there were dynamic changes in the expression of lncRNA NONRATT029757.2 after cerebral ischemia reperfusion and oxygen glucose deprivation reperfusion.Animal experiments:compared with the Sham group,MCAO reperfusion 6 h lncRNANONRATT029757.2 expression increased,but the difference was not significant,reperfusion 12 h lncRNA NONRATT029757.2 expression significantly increased,the difference was significant(P<0.05),reperfusion 24 h lncRNA NONRATT029757.2 expression was maintained at a high level,the difference was statistically significant(P<0.05),and the expression decreased at 48 h of reperfusion,the difference was not statistically significant(P>0.05).Cell experiment:compared with the Control group,the expression of lncRNA NONRATT029757.2 in primary neuronal OGD increased at 6h of reperfusion,continued to increase at 12 h of reperfusion,and decreased at 24 h and 48 h of reperfusion,but still maintained at a high level,all differences were statistically significant(P<0.05);the expression of lncRNA NONRATT029757.2 in PC12 cell line OGD increased at 6 h of reperfusion,but the difference was not significant(P>0.05),lncRNA NONRATT029757.2 expression increased at 12 h of reperfusion,continued to increase at 24 h of reperfusion,and decreased at 48 h of reperfusion,but remained at a high level,all differences were statistically significant(P<0.05).Part Ⅱ LncRNA NONRATT029757.2 is involved in cerebral ischemia reperfusion injury through upregulation of autophagyObjective:To verify at the molecular level that lncRNANONRATT029757.2 regulates the expression levels of autophagy related proteins and affects OGD tolerance.Methods:1.Experiments were performed at the optimal time intervention point for experiments after OGD reperfusion injury,and PC12 cell lines were randomly divided into Control group,OGD reperfusion group,OGD reperfusion+DMSO group and OGD reperfusion+3-MA group(n=3).Among them,the OGD reperfusion+DMSO group and the OGD reperfusion+3-MA group added DMSO or 3-MA reagent to the culture medium one day in advance.The expression levels of autophagy proteins in each group were detected by Western blot;cell activity was determined by CCK method;and cell necrosis was detected by Hoechst-Pl staining.2.The PC 12 cell lines were randomly divided into Control group,OGD reperfusion group,OGD reperfusion+siRNA NC group and OGD reperfusion+lncRNA NONRATT029757.2 siRNA group(n=3).Among them,OGD reperfusion+siRNA NC group and OGD reperfusion+lncRNA NONRATT029757.2 siRNA group were transfected with siRNA two days in advance.qRT-PCR was used to detect the transfection efficiency of siRNA,and OGD cell models were constructed after successful transfection;Western blot was used to detect the expression level of autophagy proteins in each group;CCK method was used to determine cell activity;Hoechst-PI staining was used to detect cell necrosis.Results:1.Western blot results showed that compared with the Control group,the expression levels of autophagy proteins were increased in the OGD reperfusion group,OGD reperfusion+DMSO group and OGD reperfusion+siRNA NC group,and the differences were statistically significant(P<0.05);compared with OGD reperfusion+DMSO group,the expression levels of autophagy proteins were decreased in the OGD reperfusion+3MA group.The differences were statistically significant(P<0.05);compared with the OGD reperfusion+siRNA NC group,the expression of autophagy proteins was reduced in the OGD reperfusion+lncRNA NONRATT029757.2 siRNA group(P<0.05).2.CCK and Hoechst-PI staining results showed that compared with the Control group,cell activity was significantly lower in the OGD reperfusion group,OGD reperfusion+DMSO group,and OGD reperfusion+siRNA NC group,and cell necrosis was significantly increased;Compared with the OGD reperfusion+DMSO group,cell activity was significantly higher and cell necrosis was significantly reduced in the OGD reperfusion+3-MA group;compared with the OGD reperfusion+siRNA NC group,cell activity was significantly higher and cell necrosis was significantly reduced in the OGD reperfusion+lncRNA NONRATT029757.2 siRNA group,and the differences were statistically significant(P<0.05).Conclusion:LncRNA NONRATT029757.2 can be involved in cerebral ischemia reperfusion injury by elevating the level of autophagy.