Mechanisms Involved In IS Neuroprotection Through Regulation Of GNAL Expression By Long Non-codingRNA AP000654.1-201

Background and ObjectivesStroke,is a neurological impairment caused by interruption of blood supply or rupture of blood vessels in the brain region.Stroke is the second leading cause of mortality and disability in the world,with high incidence,disability and death rates.Globally,stroke affects approximately 13.7 million people each year and causes approximately 6 million deaths worldwide,accounting for approximately 10%of global deaths.The World Health Organization Global Burden of Disease Study System analysis shows that China has the highest incidence of stroke in the world.As the total number of people in China continues to increase,the incidence of stroke continues to rise and China has become the country with the largest stroke burden in the world.Long non-coding RNA(lncRNA)is a non-coding RNA greater than 200 nucleotides in length.lncRNA has a longer nucleotide chain than other non-coding RNAs and has a complex secondary spatial structure inside the molecule,which provides multiple sites for protein binding and can bind to DNA and RNA to form a complex gene expression regulatory network.In this study,we collected peripheral blood from three IS patients and three matched healthy controls,extracted mononuclear cell from peripheral blood,and identified lncRNA and mRNA expression profiles by RNA-Seq technology,we found that lncRNA AP000654.1-201 and its potential target gene GNAL were expressed at different levels between the IS case group and the control group,with statistical significance(P<0.05).By reviewing the literature and databases,we found that lncRNA AP000654.1-201 has not been reported yet,which aroused our attention and interest,we found that lncRNA AP000654.1-201 has trans-regulatory effect on its potential target gene GNAL by raw letter prediction.GNAL gene encodes Gs protein alpha subunit,which couples adenosine A2A receptor and is highly expressed in the CNS,which is highly expressed in cardiovascular tissues,leukocytes,and endothelial cells;involved in the pathological process of various diseases(e.g.ischemia-reperfusion injury,CNS disorders,etc.).A2AR can participate in the transduction of cAMP/PKAc/CREB signaling pathway,thus regulating the expression of anti-apoptotic protein Bcl2 and exerting cytoprotective effects.In this study,we will investigate lncRNA AP000654.1-201 through in vitro and in vivo experiments,respectively.Estimated the level of apoptosis,inflammatory factor secretion and cAMP/PKAc/CREB pathway activation of cell OGD/R model.Utilize mouse MCAO model evaluate the neurological function,brain infarct size,investigate functions and mechanisms of lncRNA AP000654.1-201 action in IS.Methods(1)Screening and identification of lncRNA AP000654.1-201 and potential target gene GNALWe used transcriptome sequencing(RNA-seq)technology to identify lncRNA and mRNA expression profiles in peripheral blood samples from IS patients and normal controls,and statistically analyzed the lncRNA and mRNA genes with significant differences between the two groups.The significantly different lncRNAs and mRNAs were enriched,analyzed and visualized;the target genes of the differential lncRNAs were predicted by bioinformatics methods,and then expanding the sample size to test the accuracy of RNA-Seq results;the localization of lncRNA AP000654.1-201 was explored,so that the possible role of lncRNAs and their mechanisms of action during the process of IS were initially predicted.(2)LncRNA AP000654.1-201 protects neurological function in mice after MCAO modelingIn vivo experiments were conducted to investigate the effect of lncRNA AP000654.1201 on neurological function after MCAO molding in mice.Doppler laser scatter imaging was performed on the brains of mice after MCAO modeling to verify the effect of MCAO modeling and cerebral blood perfusion.The neurological functional performance was assessed using Zea Longa score at 12 and 24 hours after modeling.The motor function of the mice after MCAO modeling was evaluated using the suspension test,and the sensory function of the mice was evaluated using the adhesion test.And the size of infarct area in the brain of mice was evaluated by TTC staining.(4)Identification of target genes for lncRNA AP000654.1-201 and prediction of possible binding transcription factors.ChIRP assay was performed to identify the target genes of lncRNA AP000654.1-201 and the binding proteins,and further RT-qPCR was performed on the DNA fragments that pulled down by ChIRP to verify the targeting relationship between lncRNA AP000654.1-201 and GNAL genes.The possible proteins that may bind to lncRNA AP000654.1-201 were explored by Protein Mass Spectrometry.(3)LncRNA AP000654.1-201 regulates GNAL expression involved in protecting neuronal cell viability and regulating inflammatory responseTo investigate the effects of lncRNA AP000654.1-201 on cell viability,proliferation,apoptosis,and inflammatory factor expression related to cell viability and inflammation levels in vitro.Since GNAL is involved in the transduction process of cAMP signaling pathway,in order to further determine the mechanism of lncRNA AP000654.1-201,we examined the activation level of cAMP pathway to verify lncRNA AP000654.1-201 is involved in regulating cell viability and inflammatory response after OGD/R by regulating the expression of GNAL.And A2AR was selectively inhibited to investigate the role of lncRNA AP000654.1-201 in this regard.Result(1)LncRNA AP000654.1-201 and the potential target gene GNAL are down-regulated in IS patients and have a good specificity for the diagnosis of IS.(2)LncRNA AP000654.1-201 does not encode protein and the vast majority of lncRNA AP000654.1-201 is located in the nucleus.(3)The expression of lncRNA AP000654.1-201 and GNAL genes in the OGD/R cell model show a same trend with RNA-Seq results and also demonstrated similar trends with the result validated in the population samples.(4)LncRNA AP000654.1-201 can rescue the cell viability impairment and reduce the percentage of apoptotic cells in the OGD/R cell model;lncRNA AP000654.1-201 can play a role in maintaining the homeostasis of mitochondrial membrane potential after cells undergo OGD/R injury.(5)LncRNA AP000654.1-201 can down-regulate the expression levels of proinflammatory factors(IL-6,TNF-α,IL-1β),while it can promote the express of proinflammatory factors(IL-10,IFN-γ).(6)LncRNA AP000654.1-201 can promote the signal transduction of cAMP/PKAc/CREB/Bcl2 pathway to play an inhibitory role in apoptosis.(7)We successfully constructed a mouse MCAO model and overexpressed lncRNA AP000654.1-201 in the whole brain of mice injected of AAV-PHP.eB serotype by tail vein to overexpressing adeno-associated virus;GNAL gene expression was downregulated in the brain of MCAO model mice.(8)Overexpress LncRNA AP000654.1-201 improved Zea longa neurological function scores at 12h and 24h after MCAO modeling,and improved forelimb dexterity,sensory sensitivity,and motor function of model mice.(9)Overexpress LncRNA AP000654.1-201 can reduce the area of cerebral infarction in the brain tissue of mice after MCAO modeling and has neuroprotective function.(10)LncRNA AP000654.1-201 can act on GNAL gene in trans and regulate its gene expression,and then exert its biological function.ConclusionsLncRNA AP000654.1-201 is down-regulated in IS patients and its target genes are also down-regulated in the IS population.lncRNA AP000654.1-201 can participate in the transduction of A2AR signaling pathway by regulating GNAL expression,regulate cAMP/PKAc/CREB/Bc12 pathway to perform an anti-apoptotic function.Therefore,lncRNA AP000654.1-201 might be used as a potential biomarker for the treatment of IS.