Exosomal MiR-320e As A Potential Target Of CVSD That Regulates The Wnt2-mediated Wnt/β-Catenin Signaling Pathway

Research objectives: To study the difference in the expression profile of micro RNA(mi RNA)in plasma exosomes between cerebral small vessels disease(CVSD)patients and physical examination control group,It was also verified that mi R-320 e regulates the Wnt/β-Catenin signaling pathway by targeting Wnt2 under oxidative stress.Methods: The plasma of 132 patients admitted to the Affiliated Hospital of Qingdao University was collected and isolated.The plasma exosomes of 16 CVSD patients and 16 healthy controls were sequenced to construct differentially expressed mi RNA libraries,and bioinformatics GO analysis and KEGG analysis were performed.A total of 132 samples were tested by PCR.Bioinformatics and dual luciferase analysis confirmed the relationship between Wnt2 and mi R-320 e.The m RNA and protein levels of Wnt/ β-Catenin pathway components were detected by real-time quantitative PCR and Western blotting.Membrane fluorescence staining was used to detect exosome metastasis.Results: A total of 38 mirnas with significant differentially expressed expression were detected by gene microarray,among which 18 were significantly up-regulated and 20 were down-regulated.Compared with the control group,six mirnas,including mi R-498 and mi R-320 e,were significantly down-regulated,and the difference was statistically significant(the expression difference was more than 1.5 times,P < 0.01).Real-time fluorescence quantitative PCR was performed,and the verification results were consistent with the results of gene microarray.The expression level of plasma exosome mi R-320 e in CVSD patients(0.39286,95%CI=0.2144-0.5713)was significantly lower than that in healthy control group(16.8478,95%CI=7.4456-26.2501),and the difference was statistically significant(P < 0.001).Bioinformatics analysis showed that differentially expressed mi RNA target genes were involved in apoptosis,Alzheimer’s disease,Fox O signaling pathway,Wnt signaling pathway and other processes.Bioinformatics analysis and dual luciferase reporter assay showed that exosome mi R-320 e regulates the expression of Wnt2 by targeting the 3’ noncoding region of Wnt2.The exosome mi R-320 e mediates the Wnt/ β-Catenin signaling pathway in response to oxidative stress through loss-of-function experiments using mimics,inhibitors,and knockdown/overexpressed lentiviruses.Exosome mi R-320 e can target and inhibit the Wnt2/β-Catenin signaling pathway.Conclusion: The expression profile of plasma exosome mi RNA in CVSD patients is significantly different from that in healthy controls.Exosome mi R-320 e is an inhibitor of Wnt/β-Catenin signaling pathway and may play a protective role in the progression of CVSD.mi RNA-320 e may be involved in the occurrence and development of CVSD by regulating downstream proteins and biological signaling pathways.