The Expression Of MiR-34c In The Hippocampus Of Senescence Accelerated Mouse Prone8and The Validation Of Its Target Gene

Objective: Alzheimerˊs disease (AD), the most prevalent neurodegen-erative disorder, with a decline of memory and cognitive functions in a broadrange, is a major public health issue all over the world. AD is known forplacing a great burden on caregivers. The mechanisms underlying thepathogenesis of memory impairment in AD includ oxidative stress theory,gene mutations theory, β-amyloid cascade theory, Tau protein hypothesis,mitochondrial dysfunction and autophagy. MicroRNAs (miRNAs) aresingle-stranded non-coding small RNAs, approximately19-22nucleotides (nt)in length. It is reported that miRNAs play key roles in regulating proteinexpression by binding completely or partially to target protein-coding mRNAsat the posttranscriptional level for degradation or translational inhibition. Inthe past few years, a number of studies of miRNAs in AD have emerged tosupport that deregulated miRNAs, such as miR-18b, miR-325and miR-615,play important roles in the development and prognosis of AD. In present study,miRNAs array, bioinformatic analysis, Real-time PCR, Western blot, and theimmunostaining were performed to screen miRNAs and its correspondingtargets gene. Luciferase assay was used to verify syt1expression, which isnegative regulated by miR-34c. The present study was performed to provide abasis for clarifying the brain aging and the pathogenesis of Alzheimer’sdisease and the development of miRNA-targeting drugs.Methods:(1) Healthy senescence accelerated mouse-prone8(SAMP8)and senescence accelerated-resistant mouse1(SAMR1) were obtained fromthe Animal Center of Beijing University Medical Department.(2) Thealterations of miRNAs profiles were detected by microarray analysis fromhippocampus of SAMR1and SAMP8.(3) The possible singnal pathway of target gene regulated by deregulated miRNAs was predicted by KyotoEncyclopedia of Genes and Genomes (KEGG).(4) The alterations of miR-34cexpression in the hippocampus of3month old SAMR1and SAMP8werevalidated by real-time RT-PCR. And then we futher identified miR-34c levelsin hippocampus, cortex, heart, liver and kidney tissues of3month old SAMR1.The expression of miR-34c in hippocampus of3,6and9month-old SAMP8and SAMR1was determined by real-time RT-PCR assay.(5) The possiblebiological function of target gene refulated by miR-34c was predicted by GOanalysis.(6) Predicted target gene of miR-34c were to be chosen thoughTargetScan, PicTar, miRanda database and affirmed target gene mRNA levelby Real-time PCR in hippocampus of3,6and9-month-old SAMR1andSAMP8. Potential target protein of selected were analyzed by Western blotand immunohistochemistry.(7) We performed luciferase activity assay tovalidate that the3ˊU TR of syt1contains a conserved miR-34c binding site.The pre-miR-34c or inhibitor of miR-34c was transfected into HEK-293cells.(8) Statistical analysis: All data were presented as mean±SD. Statisticalanalysis was determined by the independent Student t test and one-wayANOVA using SPSS13.0software. P value cut-off0.05was consideredsignificant.Results:(1) The result showed that there were15deregulated miRNAschanged above1.5-fold, in which7miRNAs were up-regulated and8miRNAs down-regulated and miR-34c were mostly differentially upregulation reach2.52-fold.(2) The results of pathway enrichment analysisprovided by KEGG showed that these miRNAs might be involved in theregulation of neurotrophin signaling pathway, renal cell carcinoma,melanogenesis, bacterial invasion of epithelial cells, adherens junction, axonguidance and synaptic vesicle cycle signaling pathway.(3) Real-time PCRresults corresponding miRNArray revealed that compared with SAMR1mice,the miR-34c level increased in3month old SAMP8(P<0.05). The expressionof miR-34c was significantly up-regulated in the hippocampus and cortexcompared with the heart, liver, kidney tissues (P<0.05). Real-time PCR results revealed that the miR-34c level increased in SAMP8mice compared withSAMR1, and the increase in SAMP8was age-related (P<0.05).(4) The resultsof Gene Ontology provided by GO analysis showed that the miR-34c may beinvolved in axon guidance, development of nervous system and the structureof synaptic vesicle.(5) We searched for potential targets of miR-34c usingthree algorithms (TargetScan, Pictar and miRanda) and we focused on syt1and foxg1, and then detected the expression of the potential targets.(6) Wefound the expression of syt1in the hippocampus of3,6and9month-oldSAMP8by western blot and immunohistochemistry staining. Analyses of theexpression of syt1protein used by western blotting showed that syt1proteinwas significantly decrease in the hippocampus of SAMP8compared withSAMR1（P＜0.05); The immunohistochemistry result showed that the SYT1protein were high expressed in CA3of hippocampus and low expressed inCA1and DG.(7) We generated the dual-luciferase reporter vectors of syt1mRNA3ˊUTR to identify the binding of miR-34c by luciferase assay．Therelative luciferase activity was decreased in pre-miR-34c group and increasedin the inhibitor group compared with negative group.Conclusion:(1) There were some deregulated miRNAs in thehippocampus of SAMP8, which might play a potential role in the pathogenesisof neurodegenerative diseases, such as Alzheimer’s disease, throughneurotrophin signaling pathway, axon guidance and synaptic vesicle cyclesignaling pathway.(2) The translation of SYT1protein might be suppressedby miR-34c, which might play an important role in the pathogenesis of AD.