| Part Ⅰ The function and molecular mechanism of Micro RNA-4443 in ischemic stroke-induced peripheral immunosuppressionBackground and purpose: Stroke-associated infection(SAI)is one of the most common complications of acute stroke,resulting in poor prognosis.Currently,there is no effective prevention and treatment strategy for SAI.Stroke-induced immunodeficiency syndrome(SIDS)is an independent risk factor for SAI,but the molecular mechanisms remain unclear.SIDS is an independent risk factor for SAI.Micro RNAs(miRNAs),a class of non-coding RNAs,have been demonstrated to play crucial regulatory roles in the immune-inflammatory pathogenesis of acute ischemic stroke(AIS).The purpose of this study was to investigate the expression patterns of miRNAs in peripheral blood mononuclear cells(PBMCs)from AIS patients and further explore related molecular mechanisms in stroke-induced immunodeficiency syndrome(SIDS).Methods: The miRNA expression patterns of PBMCs were detected by miRNAs microarray and validated by quantitative real-time polymerase chain reaction(qRT-PCR)in AIS patients and healthy controls.Bioinformatics methods and luciferase reporter assays were used to detect the downstream target genes.Following stimulation with lipopolysaccharide(LPS)and interleukin(IL)-4(IL-4),the expression of miR-4443,tumor necrosis factor receptor-associated factor 4(TRAF4)and the nuclear factor kappa B(NF-κB)pathway were evaluated by qRT-PCR and western blot.Furthermore,transfection with miR-4443 mimic or inhibitor in the monocytes was carried out to observe the expression of TRAF4/NF-κB pathway and gain insight into the mechanisms in SIDS.Results: IL-10 in plasma and PBMCs of AIS patients was significantly higher than that of healthy controls.The miRNAs microarray analysis and qRT-PCR validation showed that only miR-4443 was upregulated expressed in PBMCs from AIS patients,especially in monocytes.Mi R-4443 was shown to directly interact with the 3’ untranslated regions of TRAF4,resulting in suppression of TRAF4 protein expression.Furthermore,the expression of miR-4443 and TRAF4 was regulated by stimulation with LPS or IL-4.Additionally,overexpression of miR-4443 suppressed the TRAF4/IκB/NF-κB signaling pathway to activate the expression of anti-inflammatory cytokines in monocytes.Conclusions: There is a certain degree of immunosuppression in the periphery of AIS patients,and the increased expression of miR-4443 after AIS induced monocyte dysfunction by targeting TRAF4,which may function as a crucial mediator in SIDS.Part Ⅱ The function and molecular mechanism of circular RNA TMCC1 in peripheral immunity after ischemia-reperfusion of strokeBackground and purpose: Endovascular therapy(EVT)is an important reperfusion therapy for the acute phase of large vessel occlusive stroke.SAI is a common clinical complication of EVT patients and affects the prognosis.As an independent risk factor for SAI,SIDS mediated peripheral immune status is closely related to the prognosis of patients.Circular RNAs(circRNAs)are a class of non-coding RNAs that play an important regulatory role in the pathological process of AIS.Therefore,the hypothesis of this study was that ischemia-reperfusion may affect the aberrant expression of circRNAs in peripheral immune cells to regulate the peripheral immune status.The purpose of this study was to analyze the changes of peripheral immune status in EVT patients before and after reperfusion,and to observe the expression characteristics of circRNAs in PBMCs of patients,and then to further explore their related molecular mechanisms in SIDS.Methods: Flow cytometry and qRT-PCR were used to detect the expression of IL-2,IL-4,IL-6,IL-10 and tumor necrosis factor-α(TNF-α)in serum and PBMCs of patients with large vessel occlusive stroke before and after reperfusion.The expression changes of circRNAs in PBMCs before and after reperfusion were assessed by high-throughput sequencing and qRT-PCR,and mouse transient middle cerebral artery occlusion(MCAO)model was constructed for verification.The monocytes in PBMCs were sorted by magnetic beads and their purity was verified by flow cytometry.The expressions of circTMCC1 and IL-4 in lymphocytes and monocytes were compared by qRT-PCR respectively.THP-1 cell stable strains with overexpression or knockdown of circTMCC1 were constructed to analyze the effects of circTMCC1 on the expression of inflammatory factors and NF-κB pathway.Results: Compared with before reperfusion,the expression of IL-4 in serum of endovascular therapy(EVT)patients was decreased,while the expression of IL-6 was increased,and the expression of IL-4 and IL-10 in PBMCs were decreased after reperfusion.At 6h after reperfusion of MCAO mice,the protein expression levels of IL-4 and IL-10 in peripheral blood plasma were decreased,while the protein expression level of IL-6 was increased,and the transcription level of IL-4 in PBMCs was decreased.Circ RNAs high-throughput sequencing and qRT-PCR validation showed that the expression of circTMCC1 was up-regulated in PBMCs of EVT patients before reperfusion,and was significantly down-regulated after reperfusion.Moreover,the expression of circTMCC1 was positively correlated with the expression of IL-4 in serum and PBMCs,and the same results were obtained in MCAO mice.In addition,circTMCC1 was most significantly down-regulated in monocytes of PBMCs.Furthermore,the circTMCC1 in monocytes can suppress the activity of IκB-α/NF-κB signaling pathway,thereby reducing the expression of pro-inflammatory factors and increasing the expression of anti-inflammatory factors.However,knockdown of circTMCC1 expression can activate IκB-α/NF-κB signaling pathway to promote inflammatory response.Conclusions: The expression of circTMCC1 is increased in peripheral monocytes after ischemic in patients with large vessel occlusive stroke,and highly expressed circTMCC1 can inhibit the activation of IκB/NF-κB pathway to promote peripheral immunosuppression,while reperfusion can alleviate peripheral immunosuppression through circTMCC1/IκB /NF-κB pathway. |
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