Effects Of MiR-124 On Dopaminergic Neurons In Parkinson’s Disease And Its Mechanism

Background and Objective:Parkinson’s syndrome(Parkinson s disease,PD)is a common neurodegenerative disease in the clinic,which is accompanied by mitochondrial dysfunction of nerve cells.The reason and mechanisms of PD occurence are not fully understood.Efficient therapy to delay or entirely stop PD progression have not yet found.Since population aging is closely related to the pathogenesis of PD,to explore the causes and prevention strategies of dopaminergic neuron apoptosis becomes a important and urgent target by more and more countries in the world.In neurodegenerative diseases,the dysfunction of autophagy related pathways and mitochondria play a key role in cellular energy metabolism,apoptosis and other life activities.Therefore the regulation of mitochondrial autophagy is the main direction of current researches on the control of Parkinson’s disease.Our study has confirmed that the expression levels of miR-124 decreased in the MPP+ induced nerve cell model due to the increase of MPP+ concentration in a dose-dependent manner.But the regulatory role of miR-124 on autophagy related genes and proteins in Parkinson cell model remains to be studied.The aim of this study was to investigate the potential role of miR-124 in the apoptosis and autophagy of dopaminergic neurons in Parkinson’s disease.Methods:(1)Establishing Parkinson’s disease-like injured cell model of SH-SY5Y cells by MPP+.MTT,flow cytometry cell toxicity detection and Q-PCR were used to assess the effects of MPP+ on SH-SY5Y proliferation and apoptosis and the effect of MPP+ on the expression of miR-124 in SH-SY5Y cells.(2)Construction of Parkinson’s disease-like injured cell model of SH-SY5Y cells was induced by MPP+,miR-124 downregulation cell model by using miR-124 inhibitor vector,miR-124 upregulation cell model by using miR-124 inhibitor vector.The effects of miR-124 downregulation or overexpression on cell apoptosis and autophagy processes were assessed by flow cytometry,Q-PCR,and WB assays.(3)Construction of Parkinson’s disease-like injured cell model of SK-N-SH cells was induced by MPP+,miR-124 up-/down-regulation cell model by using miR-124 mimics and inhibitor vector,respectively.The alteration of cell apoptosis and autophagy in SK-N-SH model were detected.The function of MPP+ and/or miR-124 on AMPK/mTOR signaling pathways were assessed by Q-PCR and WB assays.Results:(1)The establishing of Parkinson like-cell model by MPP+ and the alteration of miR-124 expression.The damage cell model of Parkinson’s disease was established by MPP+.The higher the concentration of MPP+,the greater cytotoxic effects it showed on SH-5YSY cells.When the MPP+ concentration reached 200 uM,the expression of miR-124 is reduced significantly(p<0.05).(2)miR-124 inhibition and MPP+ both induced the autophagy and apoptosis of SH-SY5Y cells,vice versa.miR-124 inhibition and MPP+ function similarly,raising the ration of apoptotic cells,and the rate of LC3 II/LC3 I protein and the expression of Beclin(p<0.05).Up-regulation of miR-124 expression in Parkinson’s disease damaged cells could reduce the increased cellular apoptosis levels triggered by MPP+(p<0.01),stablize the ration of LC3 II/LC3 I expression.(3)miR-124 mediates DA cell autophagy and apoptosis by altering the phosphorylation level of AMPK/mTOR signaling pathway.Compared with the control,the proportion of apoptotic cells increased by 2.5 folds(p<0.05).When miR-124 expression in SH-SY5Y cells was inhibited alone,apoptotic cells increased by 2.44 folds compared with the control group(p<0.05).Once miR-124 was restored by miR-124 mimics in MPP+-induced the Parkinson’s disease cell model,the proportion of apoptotic cells was significantly decreased by 65%(P<0.01).MPP+ and miR-124 inhibitor could affect SH-SY5Y cells similarly,by increasing the expression of p-AMPK and decreasing the expression of p-mTOR.Upregulation of miR-124 expression after MPP+ could restore the expression of p-AMPK and p-mTOR,while the levels of total AMPK and mTOR did not change.Similar results can also be obtained in the SK-N-SH cell model.Conclusions:MPP+-induced PD-like neural cell model were confirmed with high levels of cellular apoptosis as well as autophagy.miR-124,as an important negative regulatory element in Parkinson’s disease was also found down-regulated in MPP+ cell model,which can inhibit the process of autophagy and apoptosis of dopaminergic nerve cells by regulating the phosphorylation levels of AMPK and mTOR.