KCNQ1OT1 Promotes Autophagy By Regulating MiR-200a/FOXO3/ATG7 Pathway In Cerebral Ischemic Stroke

Objectives: Acute ischemic cerebrovascular disease is one of the major diseases affecting human health and life-threatening in the world.There are about 2.5 million new patients with cerebrovascular disease in China every year.Compared with Europe and the United States,the incidence and mortality of cerebrovascular disease in China is much higher than that of cardiovascular disease.Ischemic stroke(IS)refers to ischemic necrosis or softening of localized brain tissue caused by blood circulation disorder,ischemia and hypoxia.Ischemic stroke is an important classification in cerebrovascular disease,accounting for about 80% of patients with cerebrovascular disease in China.The disease has a high rate of disability and death,which consumes a lot of social and medical resources,and brings patients great physical and mental pain.Currently,recanalization is the core treatment for acute ischemic stroke.Intravenous thrombolysis or mechanical thrombectomy in the effective time window can save the cells in the ischemic penumbra zone,which is at the critical state of perfusion,thereby reducing the neurological deficit of the patient and disability mortality rate,improving the survival rate,and ultimately improving the quality of life and prognosis of patients.However,due to the limitations of early diagnosis conditions and treatment time windows,there are still a few patients who are able to receive recanalization therapy.Besides,ischemia/reperfusion injury leads to unsatisfied results of treatment.Therefore,exploring an effective and specific indicator of early diagnosis,treatment and prognosis of ischemic stroke at the molecular level is an urgent problem to be solved.Neuronal cell death secondary to ischemia is the leading cause of disability and death in stroke.Oxygen and glucose consumption after cerebral ischemia,which reduces the energy supply of normal structures and functions of cells and tissues,leading to a series of side reactions such as ion homeostasis imbalance,reactive oxygen species(ROS)and reactive nitrogen species(RNS)production and mitochondrial damage.After ischemia and hypoxia injury,brain cells may exhibit necrotic manifestations such as swelling,rupture and cell lysis,or apoptotic expression such as chromatin condensation and DNA fragmentation.In this process,cell necrosis and apoptosis may occur simultaneously.The cerebral blood flow in the ischemic penumbra is between electrical failure and energy failure.There are residual blood flow or collateral circulation in such regional brain tissue.There are still a large number of surviving neurons,where the injury of brain tissue is reversible.If the cerebral blood could recover rapidly in a short time,nerve cells can survive and restore function.Autophagy is a process to meet the metabolic needs of the body itself and the renewal of organelles.The cytoplasmic proteins or organelles themselves engulf and envelop form vesicles,which are then combined with lysosomes to degrade and metabolize.Both the physiological and pathological conditions,there is autophagy in the cell.In the pathological situation,autophagy can play a protective role and can play a destructive role.Therefore,it is often regarded as a "double-edged sword".KCNQ1OT1(potassium voltage-gated channel subfamily Q member 1 opposite strand 1)is located at 11p15.5 and is a non-coding antisense RNA that can regulate epigenetic silencing as a blotting gene cluster.KCNQ1OT1 is up-regulated in various diseases such as diabetes,lung cancer,kidney cancer and glioma.Additioncally,KCNQ1OT1 is closely related to the occurrence of left ventricular dysfunction in acute myocardial infarction.However,the expression of KCNQ1OT1 in acute ischemic stroke and its biological effects have not been reported at home and abroad.This study preliminarily explored the expression changes of KCNQ1OT1 after ischemiareperfusion injury and the potential regulation mechanism of autophagy induced by ischemia-reperfusion injury.Methods: 42 cases of acute stroke patients who underwent intravenous thrombolysis in the Department of Neurology in Shengjing Hospital from October 2016 to July 2017,and 40 cases healthy controls were collected in this study.Obtain plasma.A transient middle cerebral artery occlusion(tMCAO)model and a cellular oxygen and glucose deprivation reoxygenation(OGD/R)model were established to simulate ischemiareperfusion(I/R)injury conditions in vivo and in vitro,respectively.Lentivirus was injected into lateral ventricle of mice to silence the expression of KCNQ1OT1 in brain tissue.We constructed KCNQ1OT1 expression silencing plasmid stably transfected mouse neuroblastoma(N2a)cell line,miR-200 a overexpressing or silencing plasmid transfected N2 a cell line,FOXO3 overexpressing or silencing plasmid stably transfected N2 a cell line and ATG7 silenced plasmid stably transfected N2 a cell line.The expression levels of KCNQ1OT1,miR-200 a,FOXO3 and ATG7 in brain tissue of mice and N2 a cells were detected by qRT-PCR.The protein expression levels of FOXO3,ATG7,LC3 B II and SQSTM1 in brain tissue and N2 a were detected by Western blot.The expression and distribution of LC3 B in cells were observed directly by immunofluorescence.Cell viability changes were detected by the CCK-8 method.Apoptosis was detected by flow cytometry and TUNEL staining.The formation and number of autophagosomes were observed by transmitted electron microscopy.The infected adenovirus mRFP-GFP-LC3 B monitors autophagic flow in cells.Dual luciferase reporter assays verified potential binding sites between miR-200 a and FOXO3 between KCNQ1OT1 and miR-200 a.Chromatin immunoprecipitation experiments confirmed the targeted binding of FOXO3 to the ATG7 promoter region.Results: 1.KCNQ1OT1 was highly expressed in peripheral blood plasma and brain tissue.They were positively correlated with disease severity.miR-200 a was downregulated in brain tissue of tMCAO mice,and negatively correlated with KCNQ1OT1 expression level.Inhibition of KCNQ1OT1 expression in mouse brain can reduce autophagy and brain tissue damage after I/R.2.Autophagy of neurons after OGD/R injury can aggravate apoptosis and reduce cell viability.3.Silencing KCNQ1OT1 can inhibit autophagy induced by OGD/R,increase cell viability and promote neuronal survival.miR-200 a is directly regulated by KCNQ1OT1,and can specifically bind with each other.4.Overexpression of miR-200 a can inhibit OGD/R-induced neuronal autophagy,increase cell viability,and promote cell survival.Silencing miR-200 a can promote OGD/R-induced neuronal autophagy and reduce cell viability.5.FOXO3 was highly expressed in the brain tissue of tMCAO mice and negatively correlated with the expression level of miR-200 a.FOXO3 plays a role in promoting autophagy in OGD/R-damaged neurons.FOXO3 overexpression can reduce cell viability and lead to cell death.6.The specific binding site was confirmed between miR-200 a and the 3’UTR region of FOXO3.KCNQ1OT1 regulates the expression of FOXO3 by regulating the expression level of miR-200 a.7.ATG7 is highly expressed in brain tissue of tMCAO mice.The ATG7 promoter region has a targeted binding site of FOXO3.ATG7 is involved in the regulation of OGD/Rinduced autophagy by the KCNQ1OT1/miR-200a/FOXO3 axis.Conclusion: 1.KCNQ1OT1 is highly expressed in I/R injured brain tissue and neurons.Silencing KCNQ1OT1 can inhibit autophagy induced by I/R injury and promote neuron survival.2.miR-200 a is downrugulated in I/R injured neurons.Overexpression of miR-200 a can inhibit I/R injury-induced autophagy and promote neuronal survival.3.FOXO3 is highly expressed in I/R injured brain tissue and neurons.FOXO3 is a target gene of miR-200 a.ATG7 is a target gene for FOXO3.4.Silencing KCNQ1OT1 enhances the negative regulation of miR-200 a on FOXO3 by promoting the expression of miR-200 a.Reduction of FOXO3 expressing inhibits ATG7 transcription,inhibits I/R injury-induced autophagy,and promotes neuronal survival.