The Role Of MiR-214-3p Affect Dopaminergic Neurons By Regulating Glial Cells CTSB

BackgroundParkinson's disease(PD)is a common neurodegenerative disease,the average age of pathogenesis is 60 years old,the rate of incidence is about 1%above 60 years old,there are more than 5 million patients in the world.The symptoms of this disease include tremors,bradykinesia,muscle rigidity,cognition defects,etc.Its main pathological changes are the death of dopaminergic neurons mainly in the midbrain pedicel.PD is a disease caused by multiple factors associated with genetic factors,environmental factors,age,mitochondria,autophagy,etc.The mechanism is not yet fully understood and needs further study.Now there is still no effective method for clinical early diagnosis and treatment of PD,the study of the pathogenesis is very important to the diagnosis and treatment of PD.Inflammation is involved in the pathogenesis of PD,and the CTSB of glial cell involved in the inflammation of nervous system disease,so the CTSB in development in PD may be important to dopaminergic neuron.This is of great significance for the research on the pathogenesis of PD and the development of treatment methods.MicroRNAs(miRNAs)are single-stranded small non-coding 21–23 nt RNA that when bound to the 3?untranslated region(3?UTR)of mRNA induce mRNAdegradation or inhibition of protein translation.It regulates eukaryotic cell gene expression,cell developmental differentiation and individual development.It has been found that the amount of miR-214-3p in the serum of patients with PD is significantly down-regulated,but the regulatory role of miR-214-3p in the development of PD needs to be further clarified.Bioinformatics analysis revealed that CTSB gene may be the target gene of miR-214-3p,but whether miR-214-3p is associated with PD has not been reported.Our used BV2 cell and U87MG cell as representatives of microglia and astrocyte.SH-SY5Y cell is the dopaminergic neuron cell model of PD.They were used as research objects in vitro to explore the role of miR-214-3p in glial cell acting on dopaminergic neuron.The first is the overexpression of mi R-214-3p Mimics in glial cells,and MPP~+stimulation.Observe the changes of expression of CTSB and miR-214-3p,collect the conditioned medium(CM)of glial cells to culture dopaminergic neurons and detect the activity of dopaminergic neurons.This provides a new theoretical mechanism for the development of PD and a new targets for PD treatment.Materials and MethodPart I miR-214-3p can be combined with the CTSB mRNA 3?UTR1.RecombinantGP-mi RGLO-CTSBwild-typeplasmidand GP-miRGLO-CTSB mutant plasmid.2.The wild-type plasmid of CTSB were co-transfected with Mimics,NC,Inhibitor or Inhibitor NC of miR-214-3p for the luciferase assay;The mutant plasmid of CTSB were co-transfected with Mimics,NC,Inhibitor or Inhibitor NC of miR-214-3p for the luciferase assay.3.Different amount of miR-214-3p Mimics is co-transfected with the same amount of wild-type plasmid into HEK293T cells for luciferase assay.4.Statistics:All statistics tests were performed with SPSS versio 21.0.T test was used to compare the differences between two groups.The P value for significance was set at 0.05.Part II The role of miR-214-3p affects the dopaminergic neuron by regulating glial cells(microglia and astrocyte)CTSB1.Transfected BV2 cell and U87MG cell by mi R-214-3p Mimics,NC,Inhibitor and Inhibitor NC,endogenous CTSB protein is significance Western Blotting.2.Testing the level of CTSB protein and RNA by Western Blotting and qRT-PCR after MPP~+stimulating BV2 cell and U87MG cell.3.After miR-214-3p and MPP~+co-transfecting BV2 cell and U87MG cell,testing the level of CTSB protein and RNA by Western Blotting and qRT-PCR after MPP~+stimulating BV2 cell.4.Culture SH-SY5Y cell and test cell viability by the CM of BV2 cell and U87MG cell.5.Statistics:All statistics tests were performed with SPSS versio 21.0.T test was used to compare the differences between two groups.The P value for significance was set at 0.05.Results1.Test and verify CTSB is the target gene of miR-214-3p.2.mi R-214-3p regulates CTSB by concentration-dependent.3.Compare with miR-214-3p NC,Inhibitor and Inhibitor NC,miR-214-3p Mimics can down-regulate CTSB protein in BV2 cell and U87MG cell(P<0.05)4.MPP~+could significantly increase CTSB protein in BV2 cell and U87MG cell,the mRNA of CTSB did not significant effect,but only MPP~+could significantly down-regulate the expression of miR-214-3p of BV2 cell.5.Both mi R-214-3p and MPP~+cell could down-regulate CTSB protein by co-transfecting BV2 and U87MG cell,but there was no significant change for CTSB mRNA.6.The conditioned media of miR-214-3p and MPP~+co-transfected BV2 cell and U87MG cell can reduce the viability of SH-SY5Y cell.Conclusions1.miR-214-3p can regulate the expression of CTSB gene in glial cell.2.MPP~+can increase the expression of CTSB protein in glial cell.3.The CM of overexpression of mir-214-3p in resting or activated glial cells significantly decreased the activity of dopamine neurons.