- مبلغ: ۸۶,۰۰۰ تومان
- مبلغ: ۹۱,۰۰۰ تومان
Abnormal gene expression, including mRNAs, and microRNAs (miRNA), have been identified in the development of Alzheimer's disease (AD). Although mitofusin2 (mfn2) has been found to be down-regulated in the neurons from hippocampus and cortex in AD patients, little is known about its roles and the regulatory mechanisms in the pathogenesis of AD. This study was performed to investigate the roles of mfn2 protein and its upstream regulatory mechanism in the progression of AD using a senescence accelerated mouse prone-8 (SAMP8) model. The results of quantitative real-time PCR and western blot revealed that mfn2 expression displayed a consistent decrease with aging in the hippocampus of SAMP8 than did age-matched SAMR1 mice. The luciferase activity assay combined with mutational analysis confirmed the binding site of miR-195 to the 3′ -untranslated region (3′-UTR) of mfn2 mRNA. Furthermore, miR-195 inhibitor or antigomir induced the higher level expression of mfn2 protein in vitro and in vivo. In addition, exogenous expression of miR-195 decreased the mitochondrial membrane potential (MMP) of the HT-22 cells by targeting mfn2. In conclusion, these results indicated that deregulation of mfn2 might be involved in mitochondrial dysfunction during the progression of AD, and its decreased expression was regulated at least in part by miR-195 in AD mice. The abnormal expression of miR-195 played a potential role in mitochondrial disorder by targeting mfn2 in hippocampus of SAMP8 mice. Therefore, upregulation of mfn2 protein by inhibiting miR-195 might be a potential new therapeutic strategy for treatment of AD.
4.4. Protein extraction and western blot analysis
Total protein from tissue samples or HT-22 cells transfected with miR-195 mimics/inhibitor were extracted with protein lysis buffer (150 mM NaCl, 1% NP-40 and 50 mM Tris-HCl, pH 8.0) supplemented with a protease inhibitor cocktail (2 μg/mL phenylmethanesulfonyl fluoride, 2 μg/mL pepstain, 2 μg/mL aprotinin, and 2 μg/mL leupeptin). After being lysed on ice for 30 min, lysates were centrifuged at 12,000 rpm for 20 min, and the supernatant was collected. The protein concentration was measured with a BCA kit. Lysates (50 μg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the gel was transferred onto a nitrocellulose membrane. The protein on the membranes was detected with rabbit anti-mfn2 (Cat.ab56889, abcam; diluted 1:1000 in PBS) or a mouse monoclonal β-actin antibody (Cat. A5441, Sigma; diluted 1:1000 in PBS) as a loading control. The membranes were then incubated for 1 h at room temperature with a 1: 5000 dilution of anti-mouse/horseradish peroxidase (Santa Cruz Biotechnology, USA) and developed with the Chemiluminescence Plus Western Blot Analysis kit (Santa Cruz Biotechnology, USA).