Exosomal MiR-320e As A Novel Potential Biomarker For Cerebral Small Vessel Disease

Background and Objective:Cerebral small vessel disease(CSVD)may have a hidden onset and can lead to extensive neurological dysfunction and dementia,causing a huge burden on individuals and society.However,due to the complex pathogenesis of CSVD,there are currently no reliable non-invasive biomarkers to assist in diagnosis.In recent years,a large body of evidence has shown that exosomal miRNAs are one of the important factors involved in the pathogenesis of stroke.Therefore,our study aims to explore the abnormal expression profile of exosomal miRNAs through microarray analysis and to identify candidate biomarkers for CSVD.Methods:Plasma samples were collected from 150 CSVD patients and 80 non-CSVD controls enrolled in the study between July 2018 and January 2020.Plasma exosomes were extracted using a kit method and identified by transmission electron microscopy(TEM),western blot and nanoparticle tracking analysis(NTA).16 CSVD patients and16 controls were chosen randomly to create 4 CSVD pools and 4 control pools(each pool containing 4 blood samples).Total RNA was extracted from these samples and micro RNA microarray combined with bioinformatics methods were used to analyze the differential expression profile of plasma exosomal miRNAs between the two groups.Target gene prediction and functional enrichment analysis were performed on miRNAs that met the criteria of |fold change| ≥ 1.5 and p<0.05.Fluorescent quantitative PCR(q RT-PCR)was used to verify the expression trend of selected differential miRNAs,and ROC curves were drawn to evaluate the diagnostic value of miRNAs for CSVD.Finally,we analyzed the correlation between exosomal miR-320 e levels and CSVD imaging features.Results:(1)Through transmission electron microscopy observation,we found that the isolated microvesicles were anuclear,oval or cup-shaped.Particle size analysis showed that the size distribution of these microvesicles ranged from 30-150 nm.western blot analysis detected three exosome-specific protein markers,namely CD9,CD63,and TSG101.In summary,the results of TEM,NTA,and western blot were consistent with the characteristics of exosomes.(2)Under the criteria of |fold change| ≥ 1.5 and p<0.05,micro RNA microarray analysis identified a total of 14 significantly differentially expressed miRNAs.Among them,12 miRNAs were downregulated and 2 miRNAs were upregulated.A total of2752 target genes were predicted for these 14 differentially expressed miRNAs.Further enrichment analysis showed that these target genes were related to tumor,inflammation regulation,metabolism,and endocrine function.(3)The results of RT-PCR analysis showed that the expression of miR-320 e in plasma exosomes from CSVD patients was significantly downregulated compared to the control group(p<0.0001).ROC curve analysis showed that the area under the curve for exosomal miR-320 e was 0.752.According to the results of multivariate analysis,exosomal miR-320 e was an independent predictor of moderate to severe white matter hyperintensity([a OR]= 0.452,95% [CI]= 0.258-0.792,p=0.006)and enlarged perivascular spaces([a OR]= 0.493,95% [CI]= 0.302-0.805,p=0.005).Its expression level was correlated with deep white matter hyperintensity(p=0.0018),periventricular white matter hyperintensity(p=0.0021),and severity of enlarged perivascular spaces(p=0.0059).In addition,the level of exosomal miR-320 e was significantly negatively correlated with the total imaging burden of CSVD(r=-0.276,p=0.001).Conclusion:We investigated the expression profile of abnormal miRNAs in plasma exosomes of CSVD patients.The results showed that miR-320 e in plasma exosomes has a certain diagnostic value for CSVD,and its level is significantly correlated with the imaging burden of CSVD patients.Therefore,exosomal miR-320 e may become a new biomarker for CSVD.However,further research is still needed to evaluate its clinical utility.