Identification And Functional Validation Of Specific MiRNAs In Peripheral Blood Small Extracellular Vesicle Of Moyamoya Disease

Objective:Currently there is a lack of specific biomarkers for the diagnosis of moyamoya disease(MMD).Studies have shown that micro RNAs(miRNAs)within small extracellular vesicles(s EV)of peripheral blood are valuable for the diagnosis of several diseases.The aim of this study was to sequence and analyze miRNAs within peripheral blood s EVs from MMD patients,from which to identify MMD-specific miRNAs and to perform functional validation of the target miRNAs,in order to explore the potential role of miRNAs in the pathogenesis of MMD and to provide a specific target for the diagnosis of MMD.Methods:Sixty patients with MMD attending the Department of Neurosurgery at the Second Affiliated Hospital of Nanchang University between November 2020 and December2022 and 26 matched healthy volunteers in the same time period were recruited.Clinical data of MMD patients were collected.Peripheral blood was collected from MMD patients and healthy volunteers and isolated s EV was stored at-80°C.Sequence analysis was preformed to identify differentially expressed miRNAs in the peripheral blood s EVs of MMD patients,which were validated by quantitative reverse transcription PCR(q RT-PCR).Efficacy in diagnosing MMD was evaluated by receiver operating characteristic(ROC)curves for the target miRNAs.The relationship between target miRNAs and preoperative stroke in MMD was explored based on q RT-PCR validation results.The effects of target miRNAs on the angiogenesis function of human cerebral microvascular endothelial cell(hCMEC/D3)were investigated by wound healing assay,cell proliferation assay and tube formation assay.Results:Baseline data such as age,c-reactive protein and percentage of neutrophils were not statistically different between the two groups.Sequencing results showed that 78 miRNAs were expressed up-regulated and 41 down-regulated within the peripheral blood s EV in MMD group compared to healthy controls.In the ROC curve analysis,the AUCs of miR-377-5p,miR-433-5p and miR-1264 were 0.85,0.85 and 0.95,respectively.miR-433-5p was significantly highly expressed in the ischemic stroke group with MMD.The migration of hCMEC/D3 was inhibited by miR-377-5p,miR-433-5p and miR-1264.The proliferation of hCMEC/D3 was inhibited by miR-1264,but miR-377-5p and miR-433-5p had no significant effect on its proliferation.miR-377-5p,miR-433-5p and miR-1264 had no significant effect on the tube-forming function of hCMEC/D3.Conclusion:There is significant differential expression of miRNAs in peripheral blood s EV in MMD patients.miR-377-5p,miR-433-5p and miR-1264 may be potential biomarkers for the diagnosis of MMD.miR-1264 may be involved in the development of MMD by inhibiting the migration and proliferation of hCMEC/D3.