The Effect And Mechanism Of MicroRNA-708 In Cerebral Ischemia Reperfusion Injury

Cerebral ischemia-reperfusion injury(ischemia reperfusion injury,IRI)refers to blood flow recanalization after cerebral ischemia in some cases,which can lead to further tissue damage and dysfunction,it is a complex pathophysiological process of multiple factors involved in.Following the ischemic insult,the reperfusion of damaged tissues induces both local and systemic inflammation.Tissular and cellular damage after reperfusion of previously viable ischemic tissues is defined as ischemia/reperfusion injury(IRI).IRI causes widespread microvascular dysfunction and altered tissue barrier function.If severe enough,the inflammatory response after IRI may even induce a systemic inflammatory response or multiple organ dysfunction syndromes,which account for up to 30-40%of intensive care unit mortality?But the existing treatment of cerebral IRI is unsatisfactory,so we need to study on the mechanism of IRI for searching new way to prevent and treat cerebral IRI.MicroRNAs(miRNAs)is an important post transcriptional regulator,which bind with multiple target messenger RNA(mRNA)to regulate the expression of target genes.MicroRNA-708(miR-708)is the recently discovered miRNA,whose function is not yet clear.Currently known functions are associated with tumor progression.The target genes of miR-708 include cyclin2B,survivin,Rap lb,neuronatin and AKT2.Additionally,miR-708 was found to decrease the expression of cell surface protein CD38 in the trachea smooth muscle cells by down regulation of JNK/MAP and PTEN/AKT signaling pathways.Recently,in the rat model of middle cerebral artery ischemia(MCAO),the expression of miR-708 was decreased 50%by 24 and 72 hours after IRI,compared with control group.Jaunus kinases(JAK)is a cytoplasmic tyrosine kinase,JAK-cytokine receptor complex with receptor tyrosine kinase transfer the extracellular cytokine signaling into cell.JAK kinase mediates multiple cytokine signaling pathways,which primarily affect cell growth,differentiation and survival in hematopoiesis and immune responses.Recent studies found that JAK/STAT signaling pathway was involved in IRI.Cerebral ischemia activates JAK/STAT signal pathway including JAK1 and STAT3 activation.JAK1 mediates the translocation of STAT3 and participates in focal cerebral ischemia of astrocytes.In addition,in mice IRI model,the expression of phosphorylated JAK1 and phosphorylated STAT1 was elevated significantly at 24 h after IRI.By using astragaloside,ginsenoside Rgl,ginsenoside Rbl or ginsenoside R1 alone or in combination to treat IRI mice,the expression of phosphorylated JAK1 and STAT1 were significantly decreased,indicating that JAK1 and STAT1 participate in the process of IRI mice.In this article,we will investigate the role of miR-708 in cerebral IRI,identify the target of miR-708,and explore the mechanism of miR-708 in cerebral IRI.Part 1 The expression of miR-708 in cerebral ischemia reperfusion in ratsObjective:To observe the expression of miR-708 in the MCAO model of rats,and to investigate the relationship between the expression of miR-708 and ischemia-reperfusion injury.Methods:We used 10 of SD rats to establish right middle cerebral artery ischemia reperfusion model(MCAO/R),and 10 of SD rats as sham operation control.Brain tissues were taken after perfusion 24h.A portion of brain tissue was used to perform TTC staning and the other part of brain tissue was used to extract RNA.The expression of miR-708 was detected by real-time PCR.The whole blood of rats were collected and the total mRNA was extracted by a kit.The expression of miR-708 was determined by real-time PCR.Results:1.TTC staining showed that the staining of brain tissue(subcortical white matter)in rats in the control group was red,while the TTC staining in the brain tissue(subcortical white matter)of the IRI model group was grey,indicating that the model was successful.2.Comparing with the control group,the expression of miR-708 decreased significantly in MCAO rats,indicating that the expression of miR-708 was decreased in IRI.Conclusion:The expression of miR-708 was decreased in cerebral IRI.Part2 The role of miR-708 in the cell model of ischemia-reperfusion in vitroObjective:To elucidate the role of miR-708 in in vitro oxygen and glucose deprivation and reoxygenation(OGD/R)model,including cell proliferation,apoptosis,cell cycle distribution,migration,invasion and expression of neuronal markers after OGD/R.Methods:1.To construct an in vitro cell IRI model by OGD/R;2.The RNA of the control group and the OGD/R group were extracted,and the expression of miR-708 was detected by real-time PCR;3.By using Annexin V and PI double staining,the apoptosis of OGD/R cells was detected by flow cytometry;4.The effects of miR-708 on OGD/R induced apoptosis(Annexin,V)and cell cycle(flow cytometry)were detected by transfection of miR-708 mimics and control;5.To detect the effects of miR-708 mimics on cell motility and cell invasion after OGD/R by transwell assay;6.To detect the expression of neuronal marker MAP2 by immunofluorescence assay.Results:1.We found that the number of early apoptotic cells was increased with the duration of OGD treatment.After 6 hours of OGD treatment,apoptosis increased significantly,reaching nearly 10 times of the control;2.The expression of miR708 was significantly decreased after OGD/R treatment;3.miR-708 mimics significantly increased the expression level of miR-708 in OGD/R and significantly enhanced the cell proliferation.In addition,miR-708 mimics significantly reduced the cell apoptotic level of OGD/R cells(from 29.19%to 16.19%).miR-708 mimics reduced the percentage of G1 phase cells and significantly increased the percentage of S phase cells;4.By using tranwell and wound healing assay,we found that miR-708 mimics significantly enhanced the cell migration and cell invasion;5.miR-708 mimics induced the expression of MAP2 in OGD/R cells.Conclusion:1.The apoptosis of SH-SY5Y cells induced by OGD/R;2.The expression of miR-708 was significantly decreased after OGD/R;3.miR-708 mimics significantly enhanced SH-SY5Y cell proliferation,and decreased cell apoptosis;4.miR-708 minmcs significantly increased cell migration and invasion ability of OGD/R cells;5.miR-708 mimics increased the expression of neuronal marker MAP2 in OGD/R cells.Part3 miR-708 protects OGD/R by targeting JAK1Objective:To clarify the target gene of miR-708 and to elucidate the mechanism of miR-708 in protecting cerebral IRI.Methods:1.The target gene of miR-708 was identified by bioinformatic analysis;2.Luciferase reporter gene system was used to detect the role of miR-708 in luciferase activity;3.Real-time PCR,immunofluorescence and Western blot were used to detect the expression of target gene;4.To detect the apoptosis of OGD/R cells by transfection of target gene siRNA,and to detect the expression changes of apoptosis related signal pathway proteins by Western blot;5.By using immunofluorescence and real-time PCR to detect the changes of neuronal markers after interfering with the expression of target genes after OGD/R;6.Real-time PCR was used to detect the expression of JAK1 in cerebral IRI model and the control;The expression of JAK1 and cleaved Caspase3 were detected by Western blot.and to investigate the correlation between the expression of JAK1 and miR-708.Results:1.JAK1 was the target gene of miR-708 and we found that miR-708 significantly reduced the luciferase activity of wild-type JAK1;2.miR-708 mimics significantly decreased the expression of JAK1;3.In OGD/R SH-SY5Y cells,the expression of JAK1 was increased significantly;4.JAK1 siRNA significantly decreased the apoptosis level of OGD/R cells;inhibition of JAK1 expression reduced the expression of STAT3 and Mcl-1 and induced the expression of cleaved caspase3;miR-708 mimics showed the similar results;5.miR-708 mimics and suppression of JAK1 significantly increased NeuN and MAP2 expression in OGD/R cells;6.Comparing with control,the mRNA and protein levels of JAK1 and cleaved caspase3 in the rat IRI model increased significantly.Correlation analysis showed that the expression of miR-708 was negatively correlated with JAK1.Conclusion:1.JAK1 was the target gene of miR-708,and miR-708 significantly reduced the luciferase activity of wild-type JAK1;2.In OGD/R cells,the transcription(mRNA)and the translation(protein)level of JAK1 were significantly increased;3.The mRNA and protein expression of JAK1 were significantly decreased by transfection with miR-708 mimics in OGD/R treated SH-SY5Y cells;4.Inhibition of JAK1 expression decreased.the cell apoptosis of OGD/R cells and the expression of apoptosis related proteins cleaved caspase3 was also significantly decreased;5.Suppression of JAK1 increased the expression of neuronal markers MAP2 and NeuN in OGD/R cells;6.miR-708 was negatively correlated with JAKI expression in cerebral IRI.