The Role Of MiR-183 In Amyotrophic Lateral Sclerosis

Introduction: Amyotrophic Lateral Sclerosis（ALS）is a fatal progressive neurodegenerative disease characterized by motor neurons including brain stem motor nuclei and spinal anterior horn neurons and corticospinal tract degeneration,which results in paralysis and death typically within 3 to 5 years from the onset of symptoms.ALS is the most common type of motor neuron disease.Most cases of ALS（about 90% to 95%）do not have known cause,and can be classified as sporadic ALS（s ALS）.However,both genetic and environmental factors are believed to be involved in the pathogenesis of SALS.The remaining 5% to 10% of cases have a definite genetic cause linked to a family history in the pedigree,and these cases are known as familial ALS.Several mechanisms have been implicated in the pathgenesis of ALS,including glutamate-mediated excite-toxicity effects,abnormal astrocyte and microglial activation,deficiency of neurotrophic factor secretion,protein misfolding and aggregations,mitochondrial dysfunction,rupture in the axonal passage,destruction in calcium metabolism,changes in skeletal proteins account for the selective vulnerability of motor neurons,however,the exact mechanism underlying motor neurons death in ALS remians unclear.Accumulating evidences indicate that dysregulation of RNA processing pathway plays an important role in ALS.In our previous study,miRNA Microarray was used to screen miRNA expression profiles of Peripheral Blood Mononuclear cells（PBMCs）in Chinese patients with SALS,and further real-time quantitative reverse transcription polymerase chain reaction（qRTPCR）was used to validate the 4 differentially expressed miRNAs,including miR-183b-3p,which showed extremely high diagnostic accuracy.Therefore,the current study aimed to detect the expression of miR-183 in peripheral blood of ALS patients in different stages of the disease,the changes of miRNA expression in peripheral leukocytes,skeletal muscle and spinal cord of SOD1G93 A mouse model,so as to further explore the role of miR-183 in the development and progression of ALS.Moreover,the possible biological functions of miR-183-3p were further studied to explore the relevant regulatory signaling pathways of miR-183-3p,so as to further clarify the specific molecular mechanism of miR-183b-3p in ALS.The present study consists of three parts:Part Ⅰ: The expression patterns of miR-183 in the development and progression of ALSObjective:By detecting the expression patterns of miR-183 in ALS patients and mouse models with different disease stages and disease phenotypes,to explore the role of miR-183 in the development and progression of ALS.Methods: 1)Peripheral venous blood was collected from 30 s ALS patients（at initial diagnosis and at the 6-month follow up）and 20 healthy controls（HCs）for total RNA extraction.And the expression of miR-183 was detected with qRT-PCR method.Then,the expression of miR-183 between patients and the HCs,between patients at initial diagnosis and at 6-month follow up,and between patients with different subtypes were compared.2)The expression of miR-183 in peripheral blood monocytes,gastrocnemius muscles and lumbar spinal cord of SOD1G93 A transgenic mice with different disease stages was also detected by qRT-PCR.Results: 1)The expression of miR-183 in ALS patients was significantly lower than that in the HCs.Meanwhile,miR-183 was also found to be decreased at the time of 6-month follow up when compared to the baseline in ALS patients.2)In peripheral monocytes,when compared to the wild-type mice at the same age,SOD1G93 A transgenic mice had an increased expression of miR-183 in the presymptomatic（60 days）and early stages（90 days）,while it decreased in the middle（120 days）and late stages（140 days）;meanwhile,the expression of miR-183 in the late stage was lower than that in the middle stage.In the gastrocnemius muscle of mice,when compared to the wild-type mice at the same age,the expression of miR-183 was increased in the presymptomatic and early stage,decreased in the middle stages,but showed no difference with that of wild-type mice in the end stage.In the lumbar spinal cord,when compared to the wild-type mice at the same age,the expression of miR-183 was increased in the presymptomatic and early stages,decreased in the middle and end stages;meanwhile,the expression of miR-183 in the late stage was lower than that in the middle stage.Conclusion: By detecting the expression patterns of miR-183 in ALS patients and mouse models with different disease stages and disease phenotypes,we found that miR-183 was increased in the early stage while decreased in the middle and late stages,which indicated that miR-183 might be involved in the developmennt and progression of ALS.Moreover,we found that the expression of miR-183 in the peripheral monocytes,muscles and spinal cord exhibited a consistent pattern,which indicated that detection of the miR-183 in the peripheral blood could be a noninvasive surrogate for detecting it in the central nervous system.Part Ⅱ: Biological functions of miR-183 Objective:In Part I,we found that miR-183 was differentially expressed in ALS patients and mice,namely increased in the early stage while decreased in the middle and late stages which suggested that miR-183 was involved in the development and progression of ALS.Therefore,in the current Part of our study,we aimed to detect whether miR-183 affects certain biological function of motor neurons,to explore how it participates in the development and progression of ALS.Methods:The mimics and inhibitors of miR-183 were constructed,and were transfected into the motor neuron like cells（NSC-34）with the Hiper FEC transfection reagent.Then,1).apoptosis was detected by flow cytometry（FITC/PI staining）,cell viability was detected by CCK-8 method,cell cycle was also detected by flow cytometry,with aim to explore the influence of miR-183 on cell apoptosis,cell viability and cell cycle.2).the plasmid TARDBP^G294V was constructed and was cotransfected into NSC-34 cells with the mimics or inhibitors of miR-183,with aim to observe whether miR-183 could strengthen or reverse the cytotoxicity caused by TARDBP^G294V.Results:With transfection of mimics and inhibitors of miR-183 into NSC-34 cells,we found that:1).miR-183 mimics could significantly reduce the apoptosis of NSC-34 cells and maintain the survival of cells,while miR-183 inhibitors increased cell apoptosis.Moreover,miR-183 mimics led to an increase of cell viability while miR-183 inhibitors showed an opposite effect.In the cell cycle analysis,miR-183 mimics reduced the percentage of cells in the G0-G1 phases while increased the percentage of cells in the S phase,while miR-183 inhibitors showed an opposite effect.2).In the cells co-transfected with TARDBP^G294V,miR-183 mimics could partially reverse the cytotoxicity caused by TARDBP^G294V,while cell death increased when cotransfected with miR-183 inhibitors.Conclusion: miR-183 plays a protective role by inhibiting apoptosis,enhancing cell viability and shorten the interphase of cell devison.In the early stage of ALS,miR-183 was upregulated and exhibited a neuroprotective role;while in the middle and late stages of ALS,miR-183 was downregulated,which increased the selective death of motor neurons and acclerated disease progression.Part Ⅲ: The molecular mechanisms of miR-183 inducing the selective death of motor neuronsObjective: In the second part,we found that downregulation of miR-183 could lead to the accelerated death of motor neurons,but the specific mechanisms was unclear.In this part,we used RNA expression profile combined with bioinformatics and experiments findings to identify the most possible cadidate target genes for miR-183.Furthermore,we explored the molecular mechanism of miR-183 inducing the selective death of motor neurons.Methods:1).NSC34 cells were transfected with mimics and inhibitors of miR-183,and the changes in gene expression level caused by miR-183 mimics and inhibitors were detected by Affymetrix HTA 2.0 RNA microarray.Then we idetified the most likely target genes and pathways of miR-183 by combining bioinformatics analysis and phenotypic results of part II.2).We verified the corresponding candidate target genes by combining previous studies and corresponding cell experiments;3).The pathway of miR-183 b was studied with qRT-PCR,Western blot and cell biological experiments.Results: 1).With Affymetrix HTA 2.0 RNA microarray and previous studies,we found that Early Growth Response 1 gene（EGR1）was an important target gene of miR-183,which was verified by previous experiments and subsequent qRT-PCR and Western blot.Moreover,we also found that miR-183 was involved in altering the expression of EGR1 downstream genes including Phosphatase and tensin（PTEN）,Phosphatidylinositol 3-kinase（PI3K-Akt）,p53,cleaved Casp-3、cleaved Casp-9.2).Cells overexpressing EGR1 induced the same phenotype of cells transfected with miR-183 inhibitor,namely increasing cell apoptosis and decreaing cell viability;while cells having a downexpression of EGR1 induced the same phenotype of cells transfected with miR-183 mimics,namely reducing cell apoptosis and increasing cell viability;3).In cells treated with miR-183 inhibitors,inhibiting the expression of EGR1 could reverse the cell apoptosis induced by miR-183 inhibitors.Moreover,cell cytoxity induced by overexpression of EGR1 could partially be rescued with the supplement of miR-183;4).With western blot,we found that upregulation of EGR1 could induce the same effect as the miR-183 inhibitor,namely increasing the expression of PTEN,inhibiting the PI3K/AK pathway and increasing the expression of the apoptosis related proteins;while downregulation of EGR1 could induce the same effect as the miR-183 mimic,namely decreasing the expression of PTEN,facilating the PI3K/AK pathway and decreasing the expression of the apoptosis related proteinsConclusion: In the progression of ALS,miR-183 might affect the survival and apoptosis of motor neurons through the regulation of EGR1/PTEN/PI3K-Akt and EGR1/p53 signaling pathways.The upregulation of miR-183 in the early stage of ALS could be a reactive protective mechiam of the body;while in the middle and late stages,the protective feedback loop was destroyed,and down-regulated of miR-183 can inhibit the PI3K-Akt pathway,enhacing the expression of p53,thus promoting the apoptosis and inducing the damage of motor neurons.Therefore,supplement of miR-183 in the middle and late stages of ALS might provide a new idea for the precision treatment of ALS.