Isolation And Identification Of Plasma Exosomes And The Study Of Microrna Function In Patients With Parkinson's Disease

Background:Parkinson's disease(PD)is the second most common neurodegenerative disorder,and its typical pathological feature is the presence of Lewy bodies in surviving dopaminergic neurons.Lewy bodies are insoluble protein aggregates consisting primarily of?-synuclein(?-syn).At present,the diagnosis of PD is mainly based on clinical symptoms and signs,Parkinson's Disease Rating Scale and drug test response.However,its early symptoms are not typical,and it is difficult to distinguish it from multiple system atrophy and dementia with Lewy bodies.The rate of misdiagnosis of PD is likely as high as 20%.Therefore,it is important to look for biomarkers for early diagnosis of PD.Exosomes have been isolated from various body fluids such as urine,saliva,breast milk,plasma and cerebrospinal fluid.It is a small extracellular vesicle whose size is ranging from 30 to 150 nm,and it is free to pass through the blood-brain barrier.These features ensure that the bioactive substances in the exosomes are relatively stable.The study found that the expression of multiple miRNAs in plasma exosomes of PD patients was significantly different compared with healthy controls.Therefore,it is important to further explore these differentially expressed plasma exosomal miRNAs for finding biomarkers for early diagnosis of PD.Objective:To study the feasibility of plasma exosomal miRNAs as a diagnostic biomarker for PD,and the pathogenesis of PD in which these miRNAs may be involved.Methods:1.Thirty patients with PD who were hospitalized in neurosurgery at Anhui Provincial Hospital were collected,and thirty age-and sex-matched healthy individual from the medical examination center were selected as controls.After centrifugation,the plasma samples were obtained from the blood of the elbow veins in two groups of members.2.In order to complete the identification of exosomes,exosomes in plasma samples were isolated by using the exoEasy Maxi Kit of QIAGEN,and the size and morphology of exosomes were observed under a transmission electron microscopy;the exosomal marker protein CD63 was detected by western blotting;the size of plasma vesicles were measured by dynamic light scattering measurements.3.The plasma exosomal RNA was extracted by exoEasy Maxi Kit and miRNeasy Mini Kit of QIAGEN.The expression levels of 30 miRNAs in exosome of plasma samples were detected by qRT-PCR,and the miRNAs which were distinctive were selected.The ROC curve was used to analyze the specificity and sensitivity of the differentially expressed miRNAs as biomarkers for the diagnosis of PD.4.SH-SY5Y cells were induced by different concentrations of MPP~+to obtain the PD cellular model.The proliferation of PD cellular model in each concentration group was detected by CCK-8;the expression of miRNAs in each PD cellular model group was detected by qRT-PCR;the expression of?-synuclein protein in each PD cellular model group was detected by Western blotting.5.The target genes of differentially expressed miRNAs were predicted by target gene prediction softwares,meanwhile,the Gene Ontology and KEGG pathways in which these target genes involved were analyzed,and the PD-related pathways were selected for further study.Results:1.The expression levels of plasma exosomal hsa-miR-15b-5p,hsa-miR-138-5p,hsa-miR-338-3p,hsa-miR-106b-3p and hsa-miR-431-5p in PD patients were significantly lower than that in the control group,and the expression level of hsa-miR-30c-2-3p was significantly higher than that in the control group,and the difference was statistically significant(P<0.05).2.When SH-SY5Y cells were induced by different concentrations of MPP~+for 24h,with the increase of MPP~+concentration,the levels of hsa-miR-15b-5p,hsa-miR-138-5p,hsa-miR-338-3p,hsa-miR-106b-3p,hsa-miR-146a-5pand hsa-miR-431-5p decreased gradually,and hsa-miR-30c-2-3p and hsa-miR-411-5p increased gradually;meanwhile,the levels of?-synuclein protein was also gradually increasing.Compared with the control group,the difference was statistically significant(P<0.05).3.The ROC curve analysis showed that compared with the control group,the area under the curve(AUC)values of hsa-miR-15b-5p,hsa-miR-30c-2-3p,hsa-miR-138-5p,hsa-miR-431-5p,hsa-miR-338-3p and hsa-miR-106b-3p were all greater than 0.6,and the specificity and sensitivity of these miRNAs were good,meanwhile,the specificity andsensitivityofthecombinationofhsa-miR-15b-5p,hsa-miR-30c-2-3p,hsa-miR-138-5p and hsa-miR-106b-3p was better for the diagnosis of PD.4.The target genes of hsa-miR-15b-5p,hsa-miR-30c-2-3p,hsa-miR-138-5p and hsa-miR-338-3p were enriched in dopaminergic synapse pathway,and the target gene of hsa-miR-15b-5p were enriched in Parkinson's disease pathway.Conclusion:1.The small vesicles isolated from plasma were identified as exosomes.2.The hsa-mi R-15b-5p,hsa-mi R-30c-2-3p,hsa-mi R-138-5p,hsa-mi R-106b-3p,hsa-mi R-338-3p and hsa-mi R-431-5p may be used as potential biomarkers for the diagnosis of PD,and the combined diagnostic accuracy of hsa-mi R-15b-5p,hsa-mi R-30c-2-3p,hsa-mi R-138-5p and hsa-mi R-106b-3p was better.3.The target genes of hsa-mi R-15b-5p,hsa-mi R-30c-2-3p,hsa-mi R-138-5p and hsa-mi R-338-3p may regulate the expression of dopamine by dopaminergic synapse and Parkinson's disease pathway.