The Mechanism Study Of Circ?0025984-miR-143-3p-TET1 Signal Shaft Protects Neurons And Improves Endoplasmic Reticulum Stress In Ischemic Stroke

Background: Cerebrovascular disease is one of the major diseases that can endanger human health and life.There are about 2.5 million new patients with cerebrovascular diseases in China every year.Ischemic stroke(IS)refers to ischemic necrosis of brain tissue caused by hypoxia and ischemia due to the blockage of blood circulation in the brain.IS accounts for about 80% of stroke,which can cause high death rate,consume a lot of social and medical resources,and bring great physical and mental pain to the patients.At present,the core treatment for IS is vascular recanalization,that is,intravenous thrombolytic therapy was conducted in an effective time,which can reduce the death of cells in the ischemic penumbra at the critical state of perfusion,thereby reducing the neurological dysfunction and mortality of patients.However,the early diagnosis and treatment time of stroke patients are limited,and only a few patients can receive vascular recanalization surgery.Therefore,it is particularly important to explore the early diagnosis and treatment targets of IS on the molecular level.Based on the development of high-throughput sequencing technology,most of the studies are based on small RNAs,such as miRNA,lnc RNA and circ RNA.Mi R-143-3p is a miRNA associated with a variety of diseases.Studies have proved that the expression level of miR-143-3p in extracellular vesicles of patients with IS was significantly higher than that of healthy controls,suggesting that miR-143-3p may have some relationship with stroke.While it is too one-sided to consider the relationship between miRNA and stroke only in the study.Moreover,clinical trials have shown that the efficacy of miRNA alone is not ideal in the treatment of stroke.In recent years,researchers have discovered thatthere are binding sites between circ RNA and miRNA that can competitively bind miRNA to mRNA,thereby inhibiting the regulation of miRNA on mRNA.A large number of data showed that the imbalance of circ RNA-miRNA-mRNA regulatory network can affect the occurrence and development of neurological diseases.At the same time,stroke will cause endoplasmic reticulum stress(ERS),and lead to the apoptosis and necrosis of nerve cells,which can directly aggravate brain damage.Aims: In this study,bioinformatics technology was applied to screen the circ RNA and mRNA bound with miR-143-3p in nerve cells to establish the circ RNA-miRNA-mRNA regulatory network in nerve cells.meanwhile,we also explored the functions of this pathway in stroke cells and animal models.In addition,ERS and autophagy were combined to investigate the mechanism of circ RNA-miRNA-mRNA regulation network in stroke injury,which might provide a theoretical basis for the early diagnosis and clinical treatment of stroke.Methods:1.The circ RNAs and mRNAs combined with miR-143-3p were screened through bioinformatics.SK-N-SH cells were applied as the research object,and the control group and the miR-143-3p group were set up;q RT-PCR assay was adopted to verify the candidate circ RNAs and mRNAs in SK-N-SH cells,and further confirm the circ RNA and mRNA combined with miR-143-3p.2.A172 and SK-N-SH cells were adopted to construct Oxygen and glucose deprivation(OGD)model,and Middle cerebral artery occlusion(MCAO)mice model was also established in SD rat.Circ?0025984 and miR-143-3p expressions were regulated through the transfections of miR-143-3p inhibitor lentivirus,circ?0025984lentivirus,miR-143-3p mimics lentivirus+circ?0025984 lentivirus.The expression and role of circ?0025984/miR-143-3p/TET1 regulatory network were verified through q RT-PCR,western blot,immunofluorescence and Annexin V-FITC/PI double staining assays in stroke cell model.3.A172 was taken as the research object,FISH,AGO2 pull down and dual-luciferase reporter gene assays were applied to verify the targeted bindings between circ?0025984 and miR-143-3p,and between miR-143-3p and TET1.4.A172 cells were applied as the research object,and the OGD model was constructed.The expressions of circ?0025984 and miR-143-3p were regulated through the transfections of miR-143-3p inhibitor lentivirus,circ?0025984 lentivirus or miR-143-3p mimics lentivirus+circ?0025984 lentivirus in A172 cells.The expressions of autophagy and autophagy-related proteins were assessed using RFP-LC3,transmission electron microscope and western blotting analysis.Meanwhile,the circ?0025984-overexpressed A172 cells were transfected with ATG7 plasmid or ATG7 si RNAs,respectively.The expressions of GRP78,LC3-II/LC3-I and ORP78 were determined by western blotting analysis.We further verified whether the circ?0025984/miR-143-3p/TET1 regulatory network could mediate the apoptosis of nerve cells through endoplasmic reticulum stress and autophagy in stroke.5.TET1 expression was regulated by transfections of TET1 plasmid or TET1 si RNAs in A172 cells.The regulatory relationship between TET1 and ORP150 was confirmed by q RT-PCR,western blot and CHIP experiments.Biological information was applied to predict the promoter sequence of ORP150 gene and the Cp G island region,and ORP150 methylation was detected by MSP.After the methylation level was regulated by 5-azac,western blotting analysis was adopted to determine the expressions of ORP150,LC3-II/LC-I,Caspase-3 and Caspase-9.And Annexin V-FITC/PII double staining was applied to examine cell apoptosis.Moreover,we verified the effects of TET1 on the functions and related mechanisms in stroke.Results:1.Top 10 circ RNAs and mRNAs combined with miR-143-3p were screened by bioinformatics analysis.The results of q RT-PCR revealed that the overexpression of miR-143-3p significantly decreased the expressions of circ?0025984 and TET1(P<0.001).2.Compared with the normal control group,the expression of miR-143-3p was significantly increased,and the expression levels of circ?0025984 and TET1 genes were dramatically decreased in the OGD model cells(P<0.001).Mi R-143-3p knockdown or circ?0025984 overexpression significantly increased the expression of TET1 protein(P<0.05)and decreased apoptosis(P<0.001).In addition,overexpression bothmiR-143-3p and circ?0025984 decreased TET expression,and increased cell apoptosis.3.Circ?0025984 and miR-143-3p were mainly distributed in the cytoplasm of A172 cells with high expression abundance.Both circ?0025984 and miR-143-3p were expressed in AGO2 group,and the expressions were significantly higher than that in Ig G group(P<0.001).After overexpression of miR-143-3p,the fluorescence signals of circ?0025984 3 'UTR WT and TET13' UTR WT were decreased,while the fluorescence signals of the MUT group remained unchanged.4.After miR-143-3p knockdown or circ?0025984 overexpression,the levels of autophagy and endoplasmic reticulum stress-related proteins decreased;Both miR-143-3p and circ?0025984 overexpression increased the levels of autophagy and endoplasmic reticulum stress-related genes and proteins.We further discovered that circ?0025984 overexpression and with knockdown,the expression level of intracellular LC3-II remained unchanged,while the overexpression of miR-143-3p decreased the expression of ORP150;overexpression of circ?0025984 upregulated ORP150 expression.5.Overexpression of TET1 could prominently promote ORP150 expression,while the downregulation of TET1 could observably reduce the expression of ORP150.CHIP results exhibit that TET1 protein could bind to the ORP150 promoter region.The promoter sequence of ORP150 gene and Cp G island region were predicted by biological information.MSP results displayed that the methylation level of ORP150 was memorably increased in the OGD group compared with the control group,while the overexpression of TET1 dramatically reduced the methylation level of ORP150.overexpression of TET1 markedly downregulated GRP78,LC3-II,Caspase-3 and Caspase-9,and upregulated ORP150,while 5-cytidine treatment were consistent with the results of overexpression of TET1.Besides,overexpression of TET1 could markedly reduce cell apoptosis,while overexpression of ORP150 can greatly improve cell apoptosis.Conclusion: This study showed that circ?0025984 and TET1 combined with miR-143-3p were selected by bioinformatics analysis.Meanwhile,the OGD A172 and SK-N-AS cell models and the MCAO SD rat models were successfully constructed.Moreover,we confirmed that the expression of miR-143-3p significantly increased,and the expressions of circ?0025984 and TET1 notably decreased in the IS models.TET1 knockdown could block the demethylation process of ERS molecule chaperone ORP150 promoter,induced the autophagy and apoptosis of nerve cells through ATG7,and further promoted the occurrence and development of IS.