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J Zhejiang Univ (Med Sci)  2018, Vol. 47 Issue (5): 473-479    DOI: 10.3785/j.issn.1008-9292.2018.10.05
    
Osthole suppresses amyloid precursor protein expression by up-regulating miRNA-101a-3p in Alzheimer's disease cell model
LIN Ying(),YAO Yingjia,LIANG Xicai,SHI Yue,KONG Liang,XIAO Honghe,WU Yutong,NI Yingnan,YANG Jingxian*()
College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, Liaoning Province, China
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Abstract  

Objective: To investigate the effect of osthole on the expression of amyloid precursor protein (APP) in Alzheimer's disease (AD) cell model and its mechanism. Methods: The SH-SY5Y cell with over expression of APP was established by transfection by liposome 2000. The cells were treated with different concentrations of osthole, and the cell viability was determined by MTT and lactate dehydrogenase (LDH) assay. The differentially expressed miRNAs with and without osthole treatment were detected by miRNA array, and the target genes binding to the differentially expressed miRNAs were identified and verified by databases and Cytoscape. After the inhibitor of the differentially expressed miRNA was transduced into cells, the changes of APP and amyloid β (Aβ) protein were determined by immunofluorescence cytochemistry, and the mRNA expression of APP was determined by RT-PCR. Results: The AD cell model with over expression of APP was established successfully. The results of MTT and LDH assay showed that osthole had a protective effect on cells and alleviated cell damage. miR-101a-3p was identified as the differentially expressed miRNA, which was binding to the 3'-UTR of APP. Compared with APP group, the expression of APP and Aβ protein and APP mRNA increased in the miR-101a-3p inhibitor group (all P < 0.01), while the expression of APP and Aβ protein and APP mRNA decreased in the cells with osthole treatment (all P < 0.01). Conclusion: Osthole inhibits the expression of APP by up-regulating miR-101a-3p in AD cell model.



Key wordsAlzheimer disease      Osthole/pharmacology      MicroRNAs      Amyloid beta-protein precursor      Protein array analysis      Microchip analytical procedures     
Received: 05 July 2018      Published: 23 January 2019
CLC:  R742  
Corresponding Authors: YANG Jingxian     E-mail: lnutcm_ly@163.com;jingxianyang@yahoo.com
Cite this article:

LIN Ying,YAO Yingjia,LIANG Xicai,SHI Yue,KONG Liang,XIAO Honghe,WU Yutong,NI Yingnan,YANG Jingxian. Osthole suppresses amyloid precursor protein expression by up-regulating miRNA-101a-3p in Alzheimer's disease cell model. J Zhejiang Univ (Med Sci), 2018, 47(5): 473-479.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2018.10.05     OR     http://www.zjujournals.com/med/Y2018/V47/I5/473


蛇床子素通过上调微RNA-101a-3p抑制阿尔茨海默病细胞淀粉样前体蛋白表达

目的: 研究阿尔茨海默病细胞模型中蛇床子素抑制淀粉样前体蛋白(APP)的作用及机制。方法: 脂质体2000转染建立APP高表达的SH-SY5Y细胞模型。利用MTT和乳酸脱氢酶(LDH)法检测蛇床子素对APP高表达细胞的存活率和损伤程度的影响。利用基因芯片技术筛选给予蛇床子素治疗后差异表达的微RNA(miRNA),然后通过生物信息学分析预测与差异表达miRNA靶向结合的基因。细胞内转入差异表达miRNA的抑制剂,然后采用免疫荧光细胞化学法观察细胞中APP、β淀粉样蛋白(Aβ)表达情况,RT-PCR法检测细胞中APP mRNA表达。结果: 实验成功构建了APP高表达的阿尔茨海默病细胞模型。MTT和LDH法检测结果显示,蛇床子素对于APP高表达的细胞具有一定的保护作用,可以减轻细胞的损伤。miRNA-101a-3p是蛇床子素调控较明显的miRNA,其与APP的3'非翻译区靶向结合。与模型对照组比较,miR-101a-3p抑制剂组APP和Aβ蛋白荧光强度增加,APP mRNA表达量增加(均P < 0.01);加入蛇床子素后细胞中APP和Aβ蛋白荧光强度降低,APP mRNA表达量减少(均P < 0.01)。结论: 在阿尔茨海默病细胞模型中,蛇床子素通过上调miR-101a-3p抑制APP表达。


关键词: 阿尔茨海默病,  蛇床子素/药理学,  微RNAs,  淀粉样β蛋白前体,  蛋白质阵列分析,  芯片分析技术 
Fig 1 Green fluorescent picture of SH-SY5Y cells transfected with APP and GFP
Fig 2 Survival rate in each group (n=3)
Fig 3 Differential expression of microRNAs demonstrated by clustering heat map
Fig 4 Expression of APP and Aβ determined by immunofluorescence cytochemistry
Fig 5 Protein expression of APP and Aβ in each group
Fig 6 miRNA-101a-3p and APP mRNA expression in each group
[1]   傅秋黎, 戴海斌, 沈耀 et al. 组胺对β淀粉样蛋白诱导PC12细胞阿尔茨海默病体外模型的保护作用[J]. 浙江大学学报(医学版), 2007, 36 (2): 146- 149
FU Qiuli , DAI Haibin , SHEN Yao et al. Reversing effect of histamine on neurotoxicity induced by β-amyloid1-42[J]. Journal of Zhejiang University(Medical Sciences), 2007, 36 (2): 146- 149
doi: 10.3785/j.issn.1008-9292.2007.02.008
[2]   REVETT T J , BAKER G B , JHAMANDAS J et al. Glutamate system, amyloid β peptides and tau protein:functional interrelationships and relevance to Alzheimer disease pathology[J]. J Psychiatry Neurosci, 2013, 38 (1): 6- 23
doi: 10.1503/jpn
[3]   SWOMLEY A M , F?RSTER S , KEENEY J T et al. Abeta, oxidative stress in Alzheimer disease:evidence based on proteomics studies[J]. Biochim Biophys Acta, 2014, 1842 (8): 1248- 1257
doi: 10.1016/j.bbadis.2013.09.015
[4]   查芹芹, 阮燕, 刘中华 et al. β-淀粉样前体蛋白第二、三外显子删除突变抑制β-淀粉样蛋白分泌[J]. 北京大学学报(医学版), 2003, 35 (3): 261- 265
ZHA Qinqin , RUAN Yan , LIU Zhonghua et al. Deletion mutation of β-amyloid precursor protein decreases the secretion of β-amyloid in SH-SY5Y neuroblastoma cells[J]. Journal of Peking University(Medical Sciences), 2003, 35 (3): 261- 265
doi: 10.3321/j.issn:1671-167X.2003.03.009
[5]   胡昱, 赵丹, 孙东, 等.蛇床子素抑制Aβ25-35诱导的神经细胞毒性的研究[C]//第三次中华中医药科技成果论坛论文集.中华中医药学会, 2013: 6.
HU Yu, ZHAO Dan, SUN Dong, et al. Study on osthole against Aβ25-35-induced neural cells cytotoxicity[C]//Proceeding of the Third Chinese Traditional Medicine Science and Technology Achievements Forum. Chinese Medicine Association, 2013: 6. (in Chinese)
[6]   HU Y , WEN Q , LIANG W et al. Osthole reverses beta-amyloid peptide cytotoxicity on neural cells by enhancing cyclic AMP response element-binding protein phosphorylation[J]. Biol Pharm Bull, 2013, 36 (12): 1950- 1958
doi: 10.1248/bpb.b13-00561
[7]   YAO Y , GAO Z , LIANG W et al. Osthole promotes neuronal differentiation and inhibits apoptosis via Wnt/β-catenin signaling in an Alzheimer's disease model[J]. Toxicol Appl Pharmacol, 2015, 289 (3): 474- 481
doi: 10.1016/j.taap.2015.10.013
[8]   KONG L , HU Y , YAO Y et al. The coumarin derivative osthole stimulates adult neural stem cells, promotes neurogenesis in the hippocampus, and ameliorates cognitive impairment in APP/PS1 transgenic mice[J]. Biol Pharm Bull, 2015, 38 (9): 1290- 1301
doi: 10.1248/bpb.b15-00142
[9]   MIRANDA K C , HUYNH T , TAY Y et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes[J]. Cell, 2006, 126 (6): 1203- 1217
doi: 10.1016/j.cell.2006.07.031
[10]   教亚男, 姚璎珈, 孔亮 et al. 蛇床子素对转染APP595/596基因的SH-SY5Y细胞的保护作用[J]. 中国病理生理杂志, 2015, 31 (11): 2053- 2058
JIAO Yanan , YAO Yingjia , KONG Liang et al. Protective effects of osthole on SH-SY5Y cells transfected with APP595/596 gene[J]. Chinese Journal of Pathophysiology, 2015, 31 (11): 2053- 2058
doi: 10.3969/j.issn.1000-4718.2015.11.021
[11]   YANG J , YAN Y , XIA Y et al. Neurotrophin 3 transduction augments remyelinating and immunomodulatory capacity of neural stem cells[J]. Mol Ther, 2014, 22 (2): 440- 450
doi: 10.1038/mt.2013.241
[12]   LING Y , MORGAN K , KALSHEKER N . Amyloid precursor protein (APP) and the biology of proteolytic processing:relevance to Alzheimer's disease[J]. Int J Biochem Cell Biol, 2003, 35 (11): 1505- 1535
doi: 10.1016/S1357-2725(03)00133-X
[13]   JI H J , HU J F , WANG Y H et al. Osthole improves chronic cerebral hypoperfusion induced cognitive deficits and neuronal damage in hippocampus[J]. Eur J Pharmacol, 2010, 636 (1-3): 96- 101
doi: 10.1016/j.ejphar.2010.03.038
[14]   LI K , DING D , ZHANG M . Neuroprotection of osthole against cerebral ischemia/reperfusion injury through an anti-apoptotic pathway in rats[J]. Biol Pharm Bull, 2016, 39 (3): 336- 342
doi: 10.1248/bpb.b15-00699
[15]   HUA K F, YANG S M, KAO T Y, et al. Osthole mitigates progressive IgA nephropathy by inhibiting reactive oxygen species generation and NF-κB/NLRP3 pathway[J/OL]. PLoS One, 2013, 8(10): e77794.
[16]   JIAO Y , KONG L , YAO Y et al. Osthole decreases beta amyloid levels through up-regulation of miR-107 in Alzheimer's disease[J]. Neuropharmacology, 2016, 108:332- 344
doi: 10.1016/j.neuropharm.2016.04.046
[17]   LI S H , GAO P , WANGL T et al. Osthole stimulated neural stem cells differentiation into neurons in an Alzheimer's disease cell model via upregulation of MicroRNA-9 and rescued the functional impairment of hippocampal neurons in APP/PS1 transgenic mice[J]. Front Neurosci, 2017, 11:340
doi: 10.3389/fnins.2017.00340
[18]   LI S , YAN Y , JIAO Y et al. Neuroprotective effect of osthole on neuron synapses in an Alzheimer's disease cell model via upregulation of microRNA-9[J]. J Mol Neurosci, 2016, 60 (1): 71- 81
doi: 10.1007/s12031-016-0793-9
[19]   LONG J M , LAHIRI D K . MicroRNA-101 downregulates Alzheimer's amyloid-β precursor protein levels in human cell cultures and is differentially expressed[J]. Biochem Biophys Res Commun, 2011, 404 (4): 889- 895
doi: 10.1016/j.bbrc.2010.12.053
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