| BackgroundAbout 10%of all people over the age of 70 years have serious memory loss,and more than half of them are caused by Alzheimer’s disease(AD).It is estimated that caring for a single patient with advanced AD will cost more than $50,000,while family members and caregivers’ emotional toll is immeasurable.AD can be diagnosed as young as 30 years old,commonly,it is prevalent in the elderly.Patients most often lose their episodic memory slowly,and then suffer an increasing dementia over years.In advanced Alzheimer disease,brain imaging reveals a disproportionate atrophy of the hippocampi and a corresponding enlargement of the temporal horns of the lateral ventricles.Microscopically,there are thick,fiber-like strands of silver-staining material(neurofibrillary tangles)and spherical deposits of amorphous material scattered throμghout the cerebral cortex and easily seen with periodic acid-Schiff(neuritic plaques).Neuritic plaques contain amyloid β(Aβ)and neurofibrillary tangles(NFTs)are composed of hyperphosphorylated tau filaments.Aβ accumulation of in blood vessel walls in cortex and leptomeninges may also be observed.The singling out of causative mutations and vulnerability genes for AD has supplied a premise for rapid progress in understanding the biological basis of the disorder.One of the most vulnerable genes for AD is apolipoprotein ε4(Apo ε4).Carrying one E4 allele increases the risk for AD by 2-to 3-fold,whereas two alleles increase the risk 16-fold.The "amyloid hypothesis" for AD mechanisms is based on studies of genetic forms of Alzheimer’s disease,like Down’s syndrome,and research revealing that Aβ42 is toxic to cells.Amyloid hypothesis is the most popular and well-known thesis for the pathogenesis of AD.Aβ peptides are natural products of metabolism constructed by 36 to 43 amino acids.Monomers of AP40 are most prevalent,but the aggregation-prone Aβ42 species are most damaging.β-amyloid peptides generate by proteolysis of the amyloid precursor protein by the β-site amyloid precursor protein-cleaving enzyme 1(BACE-1),a β-secretase,and γ-secretase,a protein complex with presenilin at its catalytic core.A dysregulation between production and clearance,and aggregation of monomers,causes Aβ to accumulate,and this imbalance may be the initiating factor in Alzheimer’s disease.B-site APP cleaving enzyme 2(BACE-2)is the homologue of BACE-1.BACE2 is a kind of APP cleaving enzyme,neither a β-secretase nor a-secretase.Instead,BACE2 functions as a new secretase to cleave APP within the AP domain,thus precluding Aβproduction.Several studies have revealed that BACE-2 is not involved in the AD pathogenesis of DS patients;instead,therapeutic interventions that promote BACE-2 expression may prevent AD pathogenesis.MicroRNAs(miRNAs)are a kind of small non-coding RNAs that have guide function in RNA silencing.In animals,miRNAs are-22 nucleotides in length,and they are produced by Drosha and Dicer—two RNase Ⅲ proteins.MicroRNAs put down gene expression by binding to complementary sequences in the 3’untranslated region(3’UTR)of mRNAs to degrade them thereby prevent their translation.So far more than 1,000 individual miRNA genes have been found,that a single miRNA can target hundreds or thousands of different mRNAs,and that an individual mRNA can be suppressed by multiple different miRNAs,the miRNAs therefore has built up a gene regulatory networks.The biogenesis of miRNAs is under tight temporal and spatial control,and their dysregulation is associated with many human diseases,like neurological degenerative diseases,cancer etc.Early studies have demonstrated that specific miRNAs are expressed in the central nervous system(CNS).These miRNAs regulate neuronal differentiation,synaptic plasticity and neurite outgrowth.Several studies using profiling techniques have revealed that miRNAs dysregulation occurred in AD human brain.Patterns of miRNA expression in cortical GM may contribute to AD pathogenesis.To sum up,microRNAs may have great potential for the treatment of Alzheimer’s disease.ObjectiveIn this study we would like to talk about the role of microRNAs in Alzheimer’s disease and Down Syndrome.Furthermore,we aim to identify the mechanisms of microRNA activation of let-7c to the BACE-2 gene leading to reduction ofβ-Amyloid in neurons.Additionally,we intended to provide a foundation for development of microRNA therapy of Alzheimer’s disease.Materials and methods1.MicroRNAs profiling of AD and DS model Extract RNA from DS cell line model UMB1478,then perform reverse transcription to get cDNA.Prepare and run the Taqman Low density Assay.Next we measured the expression level of microRNA let-7c in AD and DS model mice.2.ELISA Assay of β-amyloid in conditioned medium2.1.We measured the concentration of β-amyloid in conditioned medium of 20E2 cell,pswAPP plasmid transfected HEK293,pC99 plasmid transfected HEK293;2.2.Primary cultured neurons from APP/PS1 transgenic mouse brain were infected by AAV9 and conditioned medium were analyzed by β-amyloid 42 ELISA kit;3.Study the effect of let-7c upon BACE-2 activity3.1.2EB2 cells were cultured for 48 hours and the expression of C99 and C83/80 were detected using western blotting;3.2.Plasmid pC83 and plasmid pSuper-BACE-2 or plasmid pC83 and plasmid pSuper-NCT were co-transfected into HEK293 cells using Lipofectamine 2000 regent according to the manufacture’s instruction.After cultured 48 hours,the expression of C83/80 were detected by western blotting;4.The functions of let-7c in transcription of BACE-2 gene4.1.20E2 cells were transfected with pSuper-let-7c plasmid and cultured for 48 hour.Then we performed RNA extraction and detected BACE-2 using RT-PCR and qPCR;4.2.The promotor region of BACE-2 gene obtained by PCR was cloned into the pGL3-Basic vector to construct pB2P1 plasmid.HEK293 cells were co-transfected with plasmid pB2Pl and plasmid pSuper-let-7c and cultured for 48 hours before performing Dual Luciferase Assay;5.Determination of the functional let-7c site in BACE-2 promotor region5.1.A series of deletion plasmids containing various fragments of the 5’ upstream region of BACE2 gene was generated.To investigate the transcriptional regulation of the BACE2 gene by miRNA let-7c,the deletion plasmids were co-transfected into HEK293 cells with pSuper-let-7c plasmid and bioluminescence were measured;5.2.Jaspar software and BLAST were used to determine the putative binding site of miRNA let-7c;6.Detected the effect of Ago2 on regulation of BACE-2 activity by let-7c6.1.Co-transfected pSuper-Ago2 plasmid and pSuper-let-7c plasmid into 2EB2 cells and cultured for 48 hours.RT-PCR was performed to detect the expression of BACE-2,and measured C99 and C83/80 by western blotting;7.2EB2 cells were transfected by pSuper-let-7c plasmid or pSuper empty vector.The proliferation of cell growth was analyzed by MTT assay for five days.Results1.Low density array identified miRNAs differentially expressed in DS1.1.Low density array identified 42 differentially expressed miRNAs in DS cell lines;1.2.Let-7c was increased in DS brain tissues;1.3.Let-7c was increased in AD brain tissues;2.Let-7c decreased A β production2.1.Let-7c decreased Aβ40 in 20E2 cells stably overexpressing APPsw;2.2.Let-7c decreased Aβ40 in HEK293 cells transiently overexpressing APPsw;2.3.Let-7c decreased Aβ40 in HEK293 cells expressing C99;3.Let-7c increased BACE2 activity3.1.Let-7c increased C80 in C99 transfected HEK293 cells;3.2.Knockdown of BACE2 abolished the increasing effect of Let-7c;4.Let-7c increased BACE2 gene transcription4.1.Let-7c increased BACE2 mRNA transcription;4.2.Real time PCR confirmed the increase of BACE2 transcription;4.3.Let-7c markedly increased BACE2 promoter activity;5.A Let-7c responsive element was identified in BACE2 promoter5.1.Let-7c markedly increased BACE2 promoter activity;5.2.Blast identified the putative binding site for Let-7c in BACE2 promoter;6.Knockout of Ago2 decreased BACE2 expression and activity6.1.Knockdown of Ago2 decreased BACE2 gene transcription;6.2.Knockdown of Ago2 decreased BACE2 activity in C99 cleavage;Conclusions1.Let-7c was increased in DS and AD2.Let-7c activated BACE2 gene3.Let-7c reducedβ-amyloid production... |
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