| Objective: Alzheimer’s disease(AD)is the most common type of dementia in old age,which can be divided into early-onset Alzheimer’s disease(EOAD)and late-onset Alzheimer’s disease(LOAD).Early-onset Alzheimer’s disease(EOAD)is autosomal dominant.The most common causes are amyloid precursor protein(APP)gene,presenilin-1(PSEN-1)gene and presenilin-2(PSEN-2)gene mutation,among which the PSEN-1 gene mutation is the most common cause of early-onset Alzheimer’s disease(EOAD).The main pathological feature of Alzheimer’s disease is the senile plaques(SPs)formed by abnormal aggregation of β-amyloid(Aβ42 and Aβ40).MicroRNAs(miRNAs)are a class of endogenous non-coding small RNAs,which are mainly responsible for regulating gene expression at post-transcription level,and are necessary for the function of neural network and the survival of neurons.Recent studies suggest that multiple microRNAs are involved in the pathological process of Alzheimer’s disease,among which microRNA-34a(miRNA-34 a,miR-34a)is highly expressed in Alzheimer’s disease.Notch signaling pathway is one of the major regulatory factors involved in the behavior of neural stem cells in specific brain regions during development and adulthood,which is highly conserved in evolution and involved in the pathogenesis of AD.Apoptosis is also thought to be closely related to the underlying mechanism of Alzheimer’s disease.We aimed to investigate the effects of microRNA-34 a on apoptosis and Notch signaling pathways in the PSEN-1 mutant EOAD cell model.Materials and methods: We transfected the lentivirus carrying negative no-load control(NC),the lentivirus of wild-type PSEN-1(WT)and the lentivirus of mutant-type PSEN-1 p.Met139Ile(c.417G> C)(MT)to human neuroblastoma cells(SH-SY5 Y cells),the experiments were grouped into normal(N)group,negative no-load control(NC)group,wild-type PSEN-1(WT)group,mutant-type PSEN-1(MT)group.Among them,the SH-SY5 Y cell group infected by lentivirus with pathogenic mutant-type PSEN-1 gene(MT)is the EOAD cell model we want to build.At the same time,gene sequencing confirmed that G→C mutation occurred at the 417 site of PSEN-1 gene in MT group.The mRNA and protein expression levels of PSEN-1,APP,and Jagged1/NOTCH-1/Hes1 in the SH-SY5 Y cells were measured by RT-qPCR and Western blot.The relative expression of Aβ42/Aβ40 was detected by ELISA assay.The expression of microRNA-34 a was detected by RT-qPCR.The target gene of miR-34 a was predicted by bioinformatics analysis and confirmed by double luciferase reporter gene detection.MicroRNA-34 a mimics,microRNA-34 a inhibitors,and their negative controls were transfected into EOAD cell model(MT group)to regulate the expression levels of miR-34 a,and the transfection effect of miR-34 a was verified by qRT-PCR.ELISA method was used to detect the expression of Aβ42/Aβ40 in the EOAD cell model after transfection with miR-34 a.The expressions of Jagged1/NOTCH-1/Hes1 in the EOAD cell model after transfection was measured by RT-qPCR and Western blot.Cell activity was detected by MTT assay.The expression of apoptotic protein Cleaved Caspase-3 was measured by Western blot.Results: Compared to normal(N)group,negative no-load control(NC)group,wild-type PSEN-1(WT)group,the expressions of PSEN-1,APP,Aβ42/Aβ40 and miRNA-34 a in the mutant-type PSEN-1(MT)group were upregulated,the expression of NOTCH-1 was downregulated,the expressions of Jagged1 and Hes1 were upregulated.The dual luciferase reporter gene assay system confirmed that a target gene of microRNA-34 a was NOTCH-1 gene.Upregulation microRNA-34 a expression in MT cells showed increased expression of Aβ42/Aβ40,decreased NOTCH-1 protein expression,and increased Jagged1 and Hes1 expression,decreased cell proliferation,increased Cleaved Caspase-3 expression,increased apoptosis.The reverse trend was observed in the down regulation of microRNA-34 a.Conclusions: Our results suggested that miRNA-34 a increases the amyloid deposition and the apoptosis in the EOAD cell model by targeting the NOTCH signaling pathway. |
PDF Full Text Request