| Objective:Exosomes are membranous vesicles.Studies have shown that Micro RNAs(mi RNAs)in exosome may play a role in Parkinson’s disease(Parkinson’sdisease,PD).mi RNAs are small coding RNAs.The differential expression of mi RNAs can help diagnose Parkinson’s disease.In this study,gene chips were used to screen out the differentially expressed mi RNAs in the plasma exosomes of patients with Parkinson’s disease.Perform functional enrichment analysis and target gene prediction on mi RNAs with significant differences in expression,and perform q RT-PCR verification on2 mi RNAs with obvious differences in expression to evaluate their value in the diagnosis of PD.Finally,the association of Parkinson’s disease with motor symptoms,depression or rapid eye movement sleep behavior disorder(RBD)was analyzed.Methods:(1)the plasma of 124 patients with PD and 94 healthy subjects in the physical examination center were collected from September 2018 to June 2019;(2)Invitrogen kit was used to isolate exosomes from plasma,and the morphology of exosomes was observed by transmission electron microscopy.The expressions of exosome marker proteins CD63 and CD9 were detected by Westernblot.The size distribution of plasma exosomes was analyzed by QNano to identify them;(3)Then a random sample of 16 patients with PD group outside the plasma exosomes,and 16 cases of control group patients outside the plasma exosomes(to reduce the individual difference,4 cases were mixed into a pool).PD group and control group were divided into 4 express pool,extract its RNA,to carry on the determination of gene chip,using gene chip to detect plasma mi RNAs expression outside secrete body similar between the two groups,the expression of selected difference obvious enrichment of mi RNAs in functional analysis,forecast target genes;(4)After RNA isolation from other patients,q RT-PCR was used to verify whether there was statistical difference in the significantly up-regulated and down-regulated mi RNAs obtained in the preliminary screening;(5)Finally grouping PD patients according to the movement symptoms can be divided into three groups,the non-motor symptoms are divided into: PD-depression group whih PD-non-depressed group,PD-RBD group with PD-non-RBD group,To analyze whether the levels of mi R-6089 and mi R-3613-3p in plasma exosomes were associated with Parkinson’s motor symptoms,Parkinson’s disease combined with depression or RBD.Results:(1)Particles isolated from plasma showed like round vesicles under electron microscopy,and exosome specific marker proteins CD63 and CD9 were expressed in Westernblot.The size of plasma exosomes was concentrated at 20-80 nm by QNano analysis and identified as exosomes.(2)With P<0.05 and FC>2 as the boundary,24 mi RNAs were differentially expressed between PD patients and normal controls,18 of which were up-regulated,6 of which were down-regulated.Heatmaps of these mi RNAs were made.(3)After functional enrichment analysis and target gene prediction of differentially expressed mi RNAs,pathways(Axon guidance)and target genes(GBA、MAPT、GLUD)associated with Parkinson’s disease were found.(4)The most significant differences of mi RNA-3613-3p and mi RNA-6089 were verified by RT-PCR,the differences were statistically significant(P<0.05).(5)There was no significant difference in the relative expression of plasma exocrine mi RNA-3613-3P and mi RNA-6089 in exercise group,but the relative expression of plasma exocrine mi RNA-6089 in PD-depression group was significantly higher than that in exercise group.(6)Finally,the target genes of mi R-6089 were matched with depression-related genes,and the following overlapping genes(GRID2,MAOA,PRKCG,KRAS,and TPH1)were found.We suspected the mi R-6089 may regulate these genes to affect the occurrence and development of depression in PD patients.Conclusion:1.The expression levels of mi R-3613-3P and mi R-6089 in plasma exosomes are associated with Parkinson’s disease and may be potential biomarkers for the diagnosis of Parkinson’s disease.2.The relative expression of mi R-6089 in plasma exocrine is increased in patients with PD complicated with depression,which has the potential to become a biomarker of PD complicated with depression,and more samples are needed to verify it. |
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