MiR-495-3p Regulates Apoptosis And Autophagy In Parkinson's Disease Cell Model By Interacting With ?–synuclein

Objective:Parkinson's disease(PD)is the most common neurodegenerative disease after Alzheimer disease.PD's characteristic phathological changes are as follows:First,The progressive death and loss of dopaminergic neurons in the substantia nigra striatum system results in the imbalance between inhibitory and excitatory neurotransmitters.Second,the aggregation of abnormal protein in neurons.The misfolding and aggregation of alpha-synuclein(?-syn)leads to the formation of Lewy body.The pathological changes of dopaminergic neurons can result in the substantia nigra striatum pathway's damage and the reduce of dopareine in putamen and caudate nucleus.Usually,the loss of 70%to 80%of dopaminergic neurons in striatum and loss of 50%dopaminergic neurons in substantia nigra can lead to clinical symptoms,such as static tremor,muscle rigidity,bradykinesia,abnormal gait and some non-motor symptoms,such as autonomic nerve system disorder,mental symptoms and sensory disorder,etc.As the clinical symptoms of PD appear much later than its pathological process,early diagnosis is particularly difficult,and the current treatment can only alleviate the clinical symptoms,but cannot reverse neuronal death.Therefore,it is of great significance to explore the pathogenesis of PD and find out its potential intervention nodes for the control of the process of PD.microRNAs(miRNA)are a class of single-stranded non-codingRNAs,with a length of about 21-23 nucleotides,which are highly conserved.miRNA can inhibit the translation of their target mRNA or promote the degradation of mRNA and regulate gene expression.They are involved in almost all life activities in vivo.The expression levels of miRNA vary under pathological and physiological conditions,and often change in the early stage of disease.Therefore,the study of the expression changes of miRNA is of great importance for the early diagnosis of disease.miR-495-3p has been widely studied in many cancers,and is believed to affect the occurrence and development of tumors by participating in inflammation,autophagy and other pathways.miR-495-3p is widely expressed in brain tissue and is believed to play an important role in the development of nervous system.Differences of miR-495-3p levels were found in blood samples from PD patients compared with health controls.Moreover,there are significant differences in miR-495-3p expression in patients receiving deep brain stimulation(DBS)before and after treatment.Also,miR-495-3p hasn‘t been studied in Parkinson's disease model.Therefore,this study intends to investigate the mechanism of how miR-495-3p affects the development of PD,and provide new ideas for the future treatment of PD.Research methods:Two cell models of PD were used:MPP~+induced human neuroblastoma cells(SH-SY5Y)and fibre-induced human embryonic kidney cells(HEK293-?-syn-GFP)with overexpression of?-synuclein labeled green fluorescent label(GFP).First,CCK8 was used to detect cell viability of SH-SY5Y cells treated by MPP~+and aggregation of?-syn in HEK293-?-syn-GFP was detected under fluorescence microscope.Then Westen blotting was used to detect the expression of apoptotic and autophagy-related proteins in the two cell models.The expression of miR-495-3p in the two cell models was detected by real-time fluorescence quantitative polymerase chain reaction(real-time PCR).The ability of miR-495-3p to bind to the 3‘-UTR region of SNCA mRNA was verified by the Dual Luciferase Reporter Assay.The miR-495-3p mimics were transfected into the two cell models,and the expression of?-syn was detected by Western Blotting and immunofluorescence.The expression of apoptotic and autophagy-related proteins was detected by Western Blotting.Exosomes from clinical samples were extracted using the exosome extraction kit,and the exosomes were identified by electron microscopy,Western Blotting and nanoparticle tracking analysis(NTA).The exosomes from clinical samples were stained with PKH 26,and the fusion with the cells was observed after co-culture with SH-SY5Y cells in vitro.Then,Real-time PCR was used to detect the expression of miR-495-3p in the exosome samples.Finally,the expression of miR-495-3p and the expression of apoptotic and autophagy-related proteins in SH-SY5Y cells were detected after co-culture with the exosomes.Results:1.The expression of miR-495-3p was decreased in MPP~+treated SH-SY5Y cell model and fiber treated HEK293-?-syn-GFP cell model(p?0.05).2.Overexpression of miR-495-3p can decrease the level of?-syn in MPP~+treated SH-SY5Y cells(p?0.05).3.Overexpression of miR-495-3p inhibited?-syn aggregation in fiber-treated HEK293-?-syn-GFP cell model(p?0.05).4.Overexpression of miR-495-3p can reduce apoptosis of MPP~+treated SH-SY5Y cells(p?0.05).5.The overexpression of miR-495-3p can alleviate the inhibition of autophagy in fiber-treated HEK293-?-syn-GFP cells(p?0.05).6.In human plasma derived exosomes,the expression of miR-495-3p in the PD group was lower than that in the health control group(p?0.05).7.After co-culture of exosomes and SH-SY5Y cells,the expression of miR-495-3p in PD group was decreased compared with the health control group,and apoptosis was increased while autophagy was inhibited(p?0.05).Conclusion:1.miR-495-3p can bind to SNCA's mRNA and reduce the expression and aggregation of?-syn.2.miR-495-3p increased the anti-apoptotic ability of SH-SY5Y cells by decreasing the expression of?-syn,and alleviated the inhibition of autophagy in HEK293-?-syn-GFP cells.3.miR-495-3p can be transported to SH-SY5Y cells via exosomes,and may affect their apoptosis and autophagy functions.