MiR-214-3p Regulates α-synuclein Protein Aggregation And Affects The Development Of Parkinson’s Disease

BackgroundParkinson’s disease(PD)is a chronic neurodegenerative disease that occurs in middle-aged and elderly people.α-synuclein(α-syn)is abundantly expressed in the nervous system.α-syn protein can be misfolded into oligomers or further aggregated to form aggregates.Both oligomers and aggregates are toxic to nerve cells,resulting in neurodegenerative diseases.microRNA(miRNA)is a small molecular RNA with an endogenous length of about 22 nucleotides.Its main function is to reduce the expression of protein by targeting the 3’untranslated region of a specific mRNA sequence to cause mRNA degradation or inhibit the process of translation.In recent years,the role of miRNA in Parkinson’s disease has attracted more and more attention of researchers.Studies found that in the brain cells of Parkinson’s disease cell model and animal model,compared with the normal control group,the expression level of miR-214-3p decreased,and α-syn protein aggregates increased in the cell model.Based on the above results,we suggest whether miR-214-3p can affect the aggregation of α-syn and the occurrence and development of Parkinson’s disease.α-syn aggregates are mainly degraded by Autophagy-Lysosome Pathway(ALP).Imaging studies have found that most of the α-syn aggregates entering the cells are transported to lysosomes for clearance.However,α-syn aggregates can be cut off the C-terminal by cathepsin B(CTSB)in lysosome,and α-syn aggregates with cut C-terminal are more likely to aggregate than full-length α-syn aggregates.It is suggested that CTSB is involved in the process of triggering the formation of aggregates of α-syn.In the early stage of the class,it was verified that miR-214-3p could target the 3’untranslated region of CTSB gene mRNA to down-regulate the protein level of CTSB.So in this study,we explored whether miR-214-3p affects the aggregation of α-syn by regulating the expression of CTSB protein.Studies have shown that miR-214-3p negatively regulates the expression of Atg12 by targeting the 3 ’untranslated region of Atg12 mRNA,which inhibits autophagy and reduces apoptosis of hippocampal neurons.So we suggest whether miR-214-3p can reduce the accumulation of aggregates and alleviate the deterioration of disease by inhibiting the level of autophagy in Parkinson’s cell model.This study suggests what role miR-214-3p plays in the process of Parkinson’s disease.Is it possible to inhibit the level of CTSB protein and affect the aggregation ofα-syn protein?Whether it can inhibit the level of autophagy affects the progression of Parkinson’s disease.For this reason,we overexpressed α-syn protein in cultured cells and inoculated α-syn fiber seeds to form α-syn aggregates in the cells,which were used to construct the cell model of Parkinson’s disease.Then transfect miR-2143p mimics and inhibitor,to elucidate the molecular mechanism of miR-214-3p affecting α-syn protein aggregation.Materials and Method1.miR-214-3p down-regulates the level of CTSB and affects the aggregation of α-syn1.The α-syn overexpression plasmid was transfected into the cells,and the efficiency of plasmid transfection was verified by Western blot.2.The α-syn monomer protein was oscillated at 37℃ for 14 days under aseptic condition,then the supernatant was discarded at 100000g for 30 minutes,and the supernatant was fully mixed by ultrasonic treatment for several seconds after re-suspension.the α-syn fiber seeds were prepared and stored at-80℃.3.Mimics,a mimic of miR-214-3p,and antagomir,an inhibitor,were transfected into the cells,and the transfection efficiency and miR-214-3p expression level were detected by real-time fluorescence quantitative PCR.4.The levels of CTSB and α-syn aggregates were detected by cellular immunofluorescence.5.siRNA knocked down the expression level of CTSB protein in cells,and the knockdown effect of siRNA was verified by cellular immunofluorescence and Western blot.2.miR-214-3p regulates the level of autophagy1.Low glucose stimulation increased the level of autophagy,and the level of LC3B protein was detected by Western blot.2.Mimics,a mimic of miR-214-3p,and antagomir,an inhibitor,were transfected into the cells,and the autophagy level of the cells was detected by Western blot and immunofluorescence.Results1.When the α-syn fiber seeds were inoculated into the cells,the α-syn aggregates in the cells increased with the increase of seed concentration.When α-syn fiber seeds were inoculated into cells,with the increase of inoculation time,the number of α-syn aggregates in cells increased at first and then decreased.2.Overexpression of miR-214-3p can significantly reduce the number of α-syn aggregates and reduce the number of aggregates.Inhibiting the expression of miR-214-3p can increase the number of aggregates and increase the size of aggregates.3 miR-214-3p can down-regulate the expression of CTSB,while inhibition of miR-214-3p can increase the protein level of CTSB.4.The CTSB protein in antagomir group of SH-SY5Y cells was knocked down by siRNA,and the aggregation of α-syn protein was significantly decreased.5.Overexpression of miR-214-3p in SH-SY5 Y cells leads to a decrease in the level of autophagy,and inhibition of the expression of miR-214-3p can increase the level of autophagy.Conclusions1.miR-214-3p can reduce the aggregations of α-syn by down-regulating the expression level of CTSB protein,and inhibiting the expression of miR214-3p can increase the expression level of CTSB protein and promote the aggregation of α-syn.2.miR-214-3p can down-regulate the level of autophagy in SH-SY5Y cells,while inhibition of miR-214-3p can induce the increasing of autophagy level.