中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (9): 1603-1609.doi: 10.4103/1673-5374.255979

• 原著:退行性病与再生 • 上一篇    下一篇

miR-15b-5p参与姜黄素对阿尔茨海默病的神经保护

  

  • 出版日期:2019-09-15 发布日期:2019-09-15
  • 基金资助:

    广东省检计划项目（2016A020226022），广州市医药卫生技术项目（20161A011068），广州市科技创新委员会项目（201704020043）

miR-15b-5p targeting amyloid precursor protein is involved in the anti-amyloid eflect of curcumin in swAPP695-HEK293 cells

Hong-Ying Liu, Xian Fu, You-Fu Li, Xian-Liang Li, Zhen-Yu Ma, Ying Zhang, Qing-Chun Gao   

  1. Department of Neurology, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong Province, China
  • Online:2019-09-15 Published:2019-09-15
  • Contact: Qing-Chun Gao, MD, qcgao@263.net.
  • Supported by:

    his study was supported by the Science and Technology Planning Project of Guangdong Province of China, No. 2016A020226022 (to HYL); the Medical and Health Technology Project of Guangzhou of China, No. 20161A011068 (to HYL); the Guangzhou Science Technology and Innovation Commission of China, No.201704020043 (to QCG).

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摘要：

姜黄素对阿尔茨海默病存在神经保护作用，但是miRNA是否参与姜黄素的保护作用仍不确定。为此，实验设计为：(1)实验首先以swAPP695-HEK-293细胞作为阿尔茨海默病的细胞模型，以定量实时PCR、Western blot以及酶联免疫吸附反应检测淀粉样前体蛋白，Aβ40和Aβ42mRNA和蛋白的表达；（2）分别以0，0.5，1，2，5，10 μM姜黄素干预swAPP695-HEK-293细胞24h，以定量实时PCR评估miR-15b-5p，miR-19a-3p，miR-195-5p，miR-101-3p，miR-216b-5p，miR-16-5p以及miR-185-5p表达的变化，以定量实时PCR、Western blot以及酶联免疫吸附反应检测淀粉样前体蛋白，Aβ40和Aβ42mRNA和蛋白的表达；(3)以miR-15b-5p模拟物转染swAPP695-HEK-293细胞，或在miR-15b-5p抑制物转染前24h以1 μM姜黄素干预swAPP695-HEK-293细胞。以Western blot和免疫荧光评估miR-15b-5p表达对淀粉样前体蛋白，Aβ40和Aβ42水平的影响，以荧光素酶测定法用于分析miR-15b-5p与淀粉样前体蛋白3'非翻译区之间的相互作用。结果显示，swAPP695-HEK-293细胞中淀粉样前体蛋白和Aβ表达水平较高，姜黄素能够抑制APP和Aβ的表达，并上调miR-15b-5p的表达。此外，姜黄素干预的swAPP695-HEK-293细胞中miR-15b-5p表达与淀粉样前体蛋白和Aβ表达水平呈负相关。miR-15b-5p减少了淀粉样前体蛋白3'非翻译区的荧光素酶质粒的荧光强度，而miR-15b-5p模拟物则能恢复荧光反应。说明姜黄素下调swAPP695-HEK-293细胞中淀粉样前体蛋白和Aβ表达，其作用可能部分由姜黄素诱导miR-15b-5p靶向淀粉样前体蛋白3'非翻译区介导的效果。

orcid: 0000-0003-0643-1808 (Qing-Chun Gao)

关键词: 阿尔茨海默病, 姜黄素, 天然植物药物, miRNA, miR-15b-5p, 淀粉样前体蛋白, A&beta, 3'非翻译区, 神经元, 神经再生

Abstract:

Curcumin exerts a neuroprotective effect on Alzheimer’s disease; however, it is not known whether microRNAs are involved in this pro¬tective effect. This study was conducted using swAPP695-HEK293 cells as an Alzheimer’s disease cell model. swAPP695-HEK293 cells were treated with 0, 0.5, 1, 2, 5, and 10 μM curcumin for 24 hours. The changes in miR-15b-5p, miR-19a-3p, miR-195-5p, miR-101-3p, miR-216b-5p, miR-16-5p and miR-185-5p expression were assessed by real-time quantitative polymerase chain reaction. The mRNA and protein levels of amyloid precursor protein, amyloid-β40 and amyloid-β42 were evaluated by quantitative real-time polymerase chain re¬action, western blot assays and enzyme-linked immunosorbent assays. swAPP695-HEK293 cells were transfected with miR-15b-5p mimic, or treated with 1 μM curcumin 24 hours before miR-15b-5p inhibitor transfection. The effects of curcumin on amyloid precursor protein, amyloid-β40 and amyloid-β42 levels were evaluated by western blot assays and enzyme-linked immunosorbent assay. Luciferase assays were used to analyze the interaction between miR-15b-5p and the 3′-untranslated region of amyloid precursor protein. The results show that amyloid precursor protein and amyloid-β expression were enhanced in swAPP695-HEK293 cells compared with HEK293 parental cells. Curcumin suppressed the expression of amyloid precursor protein and amyloid-β and up-regulated the expression of miR-15b-5p in swAPP695-HEK293 cells. In addition, we found a negative association of miR-15b-5p expression with amyloid precursor protein and amyloid-β levels in the curcumin-treated cells. Luciferase assays revealed that miR-15b-5p impaired the luciferase activity of the plasmid harboring the 3′-untranslated region of amyloid precursor protein. These findings indicate that curcumin down-regulates the expression of amyloid precursor protein and amyloid-β in swAPP695-HEK293 cells, which was partially mediated by miR-15b-5p via targeting of the 3′-untranslated region of amyloid precursor protein.

Key words: nerve regeneration, Alzheimer’s disease, natural plant drug, curcuminoids, miRNAs, amyloid precursor protein, amyloid-β, 3′-untranslated region, Luciferase assays, neurons, neural regeneration