LncRNA LOC103692885/miR-187-3p/Seipin Axis Regulating Endoplasmic Reticulum Stress And Autophagy In Cerebral Ischemia-reperfusion Injury

Background:Ischemia-reperfusion injury induced excessive endoplasmic reticulum stress(ERS),autophagy flow disorder and apoptosis,which played different roles in the development of ischemic stroke.Excessive ERS will eventually initiate apoptotic pathway to terminate cells;ERS could also induce the activation of autophagy flow;the inhibition of autophagy flow increased the risk of apoptosis to a certain extent.There are many kinds of lncRNA expression in nerve cells,which may be associated with the regulation of various phenotypes of nerve cells,but the specific mechanism remains to be studied.Lnc RNA functioned through signal,guide,decoy and scaffold.The subcellular localization of lncRNA determined its functional model.lncRNA acted as an endogenous competitive RNA(ce RNA)to regulates the expression of miRNA,forming lncRNA-miRNA-m RNA regulatory network,which were involved in the pathological changes of ischemic stroke.However,which lncRNAs are significantly changed in ischemic stroke,it is a lack of systematic research.Focusing on the function of differentially expressed lncRNAs in ischemic stroke is of great significance for finding biomarkers and therapeutic targets of cerebral ischemia-reperfusion injury.Objective:(1)To screen the key genes in ischemia-reperfusion injury by RNA-seq,and to analyze the function and pathway are involved in key genes.(2)To investigate the expression of lncRNA LOC103692885 in in vivo and in vitro models of ischemiareperfusion injury,as well as the specific regulatory targets and functions.(3)To explore the relationship of lncRNA LOC103692885/miR-187-3p/Seipin axis in the model of ischemia-reperfusion injury in vivo and in vitro,and to clarify its role in the ischemia-reperfusion injury.Methods:(1)PC12 cells were induced by oxygen glucose deprivation reperfusion(OGD/R),and the ischemia-reperfusion injury was simulated in vitro.The difference expression genes were revealed by RNA-seq between normal control group and OGD/R group.(2)The differentially expressed genes were analyze by GO and KEGG enrichment using Bioinformatics,and the RNA-seq results were verified by q RT-PCR.(3)miR-187-3p was selected as the key miRNA,and its downstream target gene Seipin,the binding relationship was verified between miR-187-3p and Seipin using the dual luciferase reporter gene experiment.(4)miR-187-3p was overexpressed and inhibited in vivo and in vitro models of ischemia-reperfusion injury.The changes of endoplasmic reticulum stress,autophagy,apoptosis and other related molecules were detected by immunohistochemistry,immunofluorescence,q RT-PCR,Western blot and flow cytometry.(5)After infected with LC3 autophagy double labeled adenovirus in OGD/R induced PC12 cells,then miR-187-3p was overexpressed or inhibited.The changes of autophagy flow were observed by laser confocal microscopy.(6)After overexpression and knockdown of Seipin by lentiviral vector,the changes of endoplasmic reticulum stress,autophagy,apoptosis and other related molecules were detected.(7)Cotransfection miR-187-3p inhibitor and si-Seipin lentiviral vector,and the changes of endoplasmic reticulum stress,autophagy,apoptosis and other related molecules were detected.(8)The upstream of miR-187-3p was lncRNA LOC103692885.After PC12 cells and primary neurons were induced by OGD/R,and SD rat models induced by MCAO/R,the change of lncRNA LOC103692885 was detected firstly.To explore the effect of lncRNA LOC103692885 in vitro and in vivo,PC12 cells and primary neurons were infected with overexpression negative control adenovirus or overexpression lncRNA LOC103692885 adenovirus,or microinjected into the hippocampus of SD rats induced by MCAO/R.(9)Cotransfection miR-187-3p mimics and lncRNA LOC103692885 was performed to detect the changes of endoplasmic reticulum stress,autophagy,apoptosis and other related molecules.Results:(1)RNA-seq revealed that 547 lncRNAs were up-regulated and 156 down regulated in OGD / R group;89 miRNAs were up-regulated and 66 miRNAs were down regulated;1858 m RNA were up-regulated and 1095 m RNA were down regulated;Functional enrichment analysis of differential genes showed that they were related to cell cycle,cell death and stress regulation;The results of q RT-PCR and RNA-seq were consistent.(2)Simulating ischemia-reperfusion injury in vivo and in vitro:The expression of miR-187-3p was increased,the expression of Seipin protein was decreased,the area of cerebral infarction was enlarged,and the neurological deficit score was increased,the difference was statistically significant,P < 0.05).Mi R-187-3p is related to cerebral ischemia-reperfusion injury and can regulate the expression of Seipin.(3)In PC12 induced by OGD/R,overexpression of miR-187-3p aggravated endoplasmic reticulum stress(Compared with mimics NC group,GRP78,p-e IF2α and CHOP were upregulated,the difference was statistically significant),hindered autophagy flow(Compared with mimics NC group,autophagy formation and LC3-II expression were decreased,and the difference was statistically significant),promoted apoptosis(Compared with mimics NC group,the expressions of Bax and cleaved-caspase3 were significantly increased);however,inhibition of miR-187-3p could restore the above effects.(4)Compared with the negative control group,the overexpression of Seipin alleviated endoplasmic reticulum stress(the expression of GRP78,p-e IF2α and CHOP were inhibited,the difference is statistically significant)and inhibited apoptosis(the expression of Bax and Cleaved-Caspase3 were decreased,the difference is statistically significant)in PC12 cells induced by OGD / R;however,the knockdown of seipin had the opposite effect,further detection showed that autophagy flow was blocked(LC3-II was decreased,p62 was increased).(5)Compared with miR-187-3p inhibitor group,ER stress further activated(GRP78,p-e IF2α and CHOP were up-regulated),promoted significantly the apoptosis-related indicators of Bax and cleaved-caspase3 and autophagy flow was blocked(the expression of LC3-II decreased,the expression of p62increased)in seipin knockdown PC12 cells.(6)Lnc RNA LOC103692885 and miR-187-3p were co localized in PC12 cells,and mainly located in the cytoplasm.(7)The dual luciferase reporter gene experiment verified that the upstream of miR-187-3p was regulated by lncRNA LOC103692885;lncRNA LOC103692885 was overexpressed in PC12 cells and primary neurons induced by OGD/R,and in SD rat models induced by MCAO/R.It was found that lncRNA LOC103692885 could alleviate excessive endoplasmic reticulum stress(the expression of GRP78,p-e IF2α and CHOP were significantly inhibited,the difference is statistically),recovery autophagy flow(the expression of LC3-II significantly upregulated,the difference is statistically)and inhibit cell apoptosis(the number of TUNEL positive cells decreased significantly)induced by ischemia-reperfusion injury in vitro and in vivo.(8)miR-187-3p mimics could attenuate the neuroprotective function of lncRNA LOC103692885.Conclusions:(1)FOXO,p53,Wnt and NF-κB signaling pathways may be involved in the regulation of ischemia-reperfusion injury in varying degrees;q RT-PCR verified that the differentially expressed genes were basically consistent with the RNA-seq results.Therefore,we will further explore new targets of ischemia-reperfusion injury based on the RNA-seq results.(2)miR-187-3p could play an important role in ischemiareperfusion injury by regulating ERS,autophagy and apoptosis.(3)Seipin is involved in the regulation of ERS,autophagy,apoptosis and other biological processes in ischemia-reperfusion injury.(4)The rescue experiment showed that the regulation of miR-187-3p on ERS,autophagy and apoptosis depended on the existence of Seipin.(5)The function of autophagy activator(rapamycin)is limited in PC12 cells with Seipin knockdown,which indicated that Seipin may play a key role in the activation of autophagy.(6)lncRNA LOC103692885 could competitively bind to miR-187-3p and reduce the expression of miR-187-3p,which caused the up regulation of Seipin expression and promoted Seipin to exert its biological effects: inhibited excessive activated ERS,promoted autophagy and alleviated apoptosis.