Molecular mechanism of circ-Tns3/miR-671-5p/sirt1 axis mediating neuronal damage in Alzheimer’s disease

LU Jing; LIU Yating; FENG Xiangqian; WANG Donglin; LIAO Qirong; YANG Hongyan; ZHOU Huadong

doi:10.16016/j.2097-0927.202503077

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Molecular mechanism of circ-Tns3/miR-671-5p/sirt1 axis mediating neuronal damage in Alzheimer’s disease

Monographic Reports | 更新时间：2025-08-25

- Molecular mechanism of circ-Tns3/miR-671-5p/sirt1 axis mediating neuronal damage in Alzheimer’s disease
- Journal of Army Medical University Vol. 47, Issue 16, Pages: 1872-1882(2025)
- 作者机构：
  
  1.蚌埠医科大学第一附属医院神经内科，安徽蚌埠
  2.陆军特色医学中心（第三军医大学大坪医院）神经内科，重庆
- 作者简介：
  
  ZHOU Huadong， E-mail： zhouhuad@tmmu.edu.cn
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81771177)
- DOI：10.16016/j.2097-0927.202503077
  CLC： R341;R394.3;R745.7
- Received：27 March 2025，
  
  Revised：03 April 2025，
  
  Published：30 August 2025
- 稿件说明：
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卢静,刘雅婷,冯祥谦,等.circ-Tns3/miR-671-5p/sirt1轴介导阿尔茨海默病神经元损伤的分子机制[J].陆军军医大学学报,2025,47(16):1872-1882.

LU Jing,LIU Yating,FENG Xiangqian,et al.Molecular mechanism of circ-Tns3/miR-671-5p/sirt1 axis mediating neuronal damage in Alzheimer’s disease[J].J Army Med Univ,2025,47(16):1872-1882.
卢静,刘雅婷,冯祥谦,等.circ-Tns3/miR-671-5p/sirt1轴介导阿尔茨海默病神经元损伤的分子机制[J].陆军军医大学学报,2025,47(16):1872-1882. DOI：10.16016/j.2097-0927.202503077.

LU Jing,LIU Yating,FENG Xiangqian,et al.Molecular mechanism of circ-Tns3/miR-671-5p/sirt1 axis mediating neuronal damage in Alzheimer’s disease[J].J Army Med Univ,2025,47(16):1872-1882. DOI：10.16016/j.2097-0927.202503077.

摘要

目的

探讨环状RNA circ-Tns3在阿尔茨海默病（Alzheimer’s disease，AD）小鼠中的表达水平和在Aβ诱导的神经元损伤中的作用。

方法

选取6月龄雄性体质量23~26 g的APP/PS1转基因AD小鼠和野生型（WT）小鼠各5只。采用Morris水迷宫检测学习记忆能力，免疫组化染色观察脑海马组织Aβ斑块数量。提取小鼠脑总RNA检测circRNA的差异表达，并进行基因本体论（gene ontology， GO）和京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路分析。通过RT-qPCR验证前6位差异显著的circRNA。用Aβ1-42处理神经元细胞建立AD体外细胞模型，转染circ-Tns3小干扰RNA（si-circ-Tns3）进入Aβ1-42处理神经元细胞中达到敲低circ-Tns3的目的。RT-qPCR测定circ-Tns3和miR-671-5p表达水平。CCK-8和TUNEL染色检测细胞活力和凋亡细胞数量，ELISA检测炎性因子IL-6、IL-1β和TNF-α的水平。双荧光素酶报告实验和RNA结合蛋白免疫沉淀法（RIP测定）验证circ-Tns3和miR-671-5p之间的相互作用。Western Blot检测sirt1蛋白表达水平。

结果

6月龄AD小鼠出现明显认知障碍和脑Aβ过度沉积（

0.01）。AD小鼠和WT小鼠鉴定出269个差异表达的circRNA，其中159个上调，110个下调。GO和KEGG的富集分析显示，差异表达的circRNA主要参与突触传递、记忆功能和胆碱能突触等信号通路。circ-Tns3表达不仅在AD小鼠脑组织中显著增加，也在Aβ1-42处理的神经元中表达增加。在细胞实验中，敲低circ-Tns3显著增加了Aβ1-42处理的神经元的细胞活力和减少了凋亡细胞，降低MDA含量，增加SOD活性，降低了IL-6、IL-1β和TNF-α的含量（

0.01）。通过starBase数据库预测circ-Tns3和miR-671-5p具有互补序列，双荧光素酶报告实验和RIP测定证实circ-Tns3和miR-671-5p之间存在相互作用。生信数据库预测miR-671-5p和sirt1具有互补序列。Western Blot检测显示，在Aβ1-42处理的神经元中，敲低circ-Tns3后，sirt1蛋白表达增加（

0.01）。此外，在Aβ1-42处理的神经元中，敲低circ-Tns3后再加入miR-671-5p抑制剂，sirt1蛋白表达水平明显下降（

0.01）。

结论

circ-Tns3在AD小鼠和AD体外细胞模型中高表达，敲低circ-Tns3改善了神经元损伤，circ-Tns3可能通过结合miR-671-5p调节sirt1蛋白表达参与神经元的损伤机制。

Abstract

Objective‍　‍To investigate the expression level of circular RNA circ-Tns3 in Alzheimer’s disease (AD) mice and its role in Aβ-induced neuronal damage. Methods‍　‍Five APP/PS1 transgenic AD mice and 5 wild-type (WT) mice

weighting of 23~26 g and aged 6 months were subjected in the study. Morris water maze test was used to assess learning and memory abilities

and immunohistochemical staining was performed to observe the number of Aβ plaques in the hippocampal tissue. Subsequently

total RNA was extracted from the brains to detect the differential expression of circRNAs between AD and WT mice

and the results were further analyzed with Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The top 6 differentially expressed circRNAs were validated by real-time quantitative PCR (RT-qPCR). In

in vitro

experiments

Aβ1-42 was used to treat neuron

al cells to establish AD cell model

and si-circ-Tns3 was transfected into Aβ1-42-treated neuronal cells to knock down circ-Tns3. RT-qPCR was used to determine the expression levels of circ-Tns3 and miR-671-5p. Cell viability and apoptotic rate were detected by CCK-8 assay and TUNEL staining

respectively. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured using corresponding kits

and the levels of inflammatory factors IL-6

IL-1β

and TNF-α were detected with ELISA. The interaction between circ-Tns3 and miR-671-5p was verified by dual-luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay. The expression level of Sirt1 protein was detected by Western blotting. Results‍　‍The 6-month-old AD mice exhibited significant cognitive impairment and Aβ deposition (

0.01). There were 269 differentially expressed circRNAs identified between AD and WT mice

of which 159 were up-regulated and 110 down-regulated. GO and KEGG enrichment analyses showed that these differentially expressed circRNAs were mainly involved in synaptic transmission

memory

and cholinergic synapse signaling pathways. The expression of circ-Tns3 was significantly increased not only in the brain tissue of AD mice but also in neuronal cells after Aβ1-42 treatment. In cellular experiments

knockdown of circ-Tns3 significantly reduced cell viability and number of apoptotic cells in Aβ1-42-treated neuronal cells

decreased MDA content

increased SOD activity

and reduced the levels of IL-6

IL-1β

and TNF-α (

0.01). The starBase database predicted that circ-Tns3 and miR-671-5p have complementary sequences

and dual-luciferase reporter and RIP assays confirmed their interaction. The bioinformatics database predicted that miR-671-5p and sirt1 have complementary sequences. Western blotting indicated that in neuronal cells treated with Aβ1-42

the expression of sirt1 was increased after knockdown of circ-Tns3 (

0.01). In Aβ1-42-treated neuron

al cells

after knockdown of circ-Tns3

addition of miR-671-5p inhibitor significantly decreased the expression level of sirt1 protein (

0.01). Conclusions‍　‍circ-Tns3 is highly expressed in AD mice and cell model of AD. Knocking circ-Tns3 down improves neuronal damage. circ-Tns3 may be involved in the neuronal damage through regulating sirt 1 protein by binding to miR-671-5p.

关键词

Keywords

references

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