miR-21a-5p knockdown promotes the locomotor function recovery of mouse models of spinal cord injury

doi:10.3969/j.issn.2095-4344.0773

Abstract

Abstract:

BACKGROUND: The pathological changes following spinal cord injury (SCI) are complex, and there is a lack of effective treatment method. miRNAs are non-coding RNA molecules that post-transcriptionally regulate gene expression, which provides a novel treatment strategy for SCI.
OBJECTIVE: To investigate the effect of miR-21a-5p on the locomotor function post-SCI in mice and explore the related mechanisms.
METHODS: A total of 48 healthy C57BL/6 adult mice were randomly divided into SCI (SCI model was induced by Allen’s method) and sham 1 groups (n=24 per group). Microarray assay was used to find the differentially expressed miRNAs in the lesion tissues after SCI. An additional 72 mice were randomly divided into sham 2, antagomiR and control groups (n=24 per group). The SCI mice in the later two groups were given the intrathecal injection of antagomir-21a and antagomir, respectively. The locomotor function recovery of hind limbs was evaluated by Basso Mouse Scale score; the expression levels of SCI-related markers in the lesion tissues were detected.
RESULTS AND CONCLUSION: The expression level of miR-21a-5p was significantly up-regulated in the lesion tissues after SCI (P < 0.05). AntagomiR-21a inhibited the expression of miR-21a-5p post-SCI. The Basso Mouse Scale scores showed a significant difference between sham and SCI groups (P < 0.05), the scores in the antagomiR-21a group was higher than those in the control group at 7 days postoperatively, and showed a significant difference on day 4 (P < 0.05). Compared with the control group, antagomiR-21a significantly suppressed the expression levels of fibronectin and CSPGs (P < 0.05), and significantly up-regulated the expression levels of neurotrophic factors (P < 0.05). Our results suggest that there is a up-regulation in the expression level of miR-21a-5p after SCI, and miR-21a-5p knockdown can suppress scar formation and promote secretion of neurotrophic factors, thereby improving motor functional recovery.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: MicroRNAs, Spinal Cord Injuries, Neurturin, Tissue Engineering

CLC Number:

R318

Wang Wen-zhao, Su Yan-lin, Li Hong-fei, Shen Lin, Chen Jia-nan, Pan Xin-da, Jia Tang-hong. miR-21a-5p knockdown promotes the locomotor function recovery of mouse models of spinal cord injury[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(32): 5180-5185.

Figures/Tables 1

2.1 实验动物数量分析第1批实验48只小鼠，分为2组，用于提取脊髓RNA进行miRNAs高通量筛选；第2批72只小鼠分为3组，实验过程无脱失，全部进入结果分析。 2.2 小鼠脊髓损伤后miRNAs表达变化取首批小鼠脊髓损伤3 d后的脊髓组织总RNA与假手术组脊髓组织总RNA进行差异miRNAs高通量筛选，脊髓损伤组与假手术组的miRNAs存在明显的差异表达，其中脊髓损伤组的miR-21a-5p的表达较假手术组显著上调，差异倍率为5.51，见差异表达miRNAs聚类图(图1A)。对总RNA进行qRT-PCR验证，miR-21a-5p在脊髓损伤组的表达显著上调与测序结果一致(图1B)(P < 0.05)。提示在小鼠脊髓损伤后，miR-21a-5p的表达显著增高。 2.3 抑制miR-21a-5p对小鼠脊髓损伤后运动功能的影响取术后14 d观察组与对照组小鼠脊髓组织总RNA行qRT-PCR检测miR-21a-5p表达，观察组小鼠脊髓组织中的miR-21a-5p的表达显著低于对照组(P < 0.05)，见图2A，提示损伤后鞘内注射antagomir?21a可有效抑制小鼠脊髓中miR-21a-5p表达。术后3 h，所有小鼠都完全清醒，假手术组与脊髓损伤组小鼠BMS评分差异明显(P < 0.05)，假手术组小鼠清醒后即可活动，3 d后恢复至BMS主评9分副评11分。观察组与对照组小鼠脊髓损伤后3 d内双后肢完全瘫痪，不能自主排尿，BMS评分0分。3 d后BMS评分逐渐上升，损伤7 d后恢复明显，观察组小鼠术后7 d运动功能逐渐超过对照组，术后14 d观察组小鼠运动功能恢复较对照组显著增强(P < 0.05)，见图2B。提示小鼠脊髓损伤后抑制损伤处脊髓的miR-21a-5p表达可显著提升运动功能的恢复。 2.4 抑制miR-21a-5p对脊髓损伤相关标记物表达的影响取术后14 d观察组与对照组小鼠脊髓组织固定切片，行免疫组织化学DAB染色并于倒置显微镜下观察CSPGs的表达，观察组小鼠CSPGs表达量较对照组显著降低(图3A)，提示抑制miR-21a-5p可以降低脊髓损伤后CSPGs的分泌。取术后14 d观察组与对照组小鼠脊髓组织总RNA行qRT-PCR检测抑制miR-21a-5p后纤维连接蛋白、神经营养因子脑源性神经营养因子以及神经生长因子的表达情况。观察组小鼠总RNA中纤维连接蛋白表达量显著低于对照组(P < 0.05)，提示抑制miR-21a-5p可以下调脊髓损伤后纤维连接蛋白的表达；观察组小鼠总RNA中脑源性神经营养因子、神经生长因子的表达量高于对照组(P < 0.05)，证实脊髓损伤后抑制miR-21a-5p可以提升脑源性神经营养因子、神经生长因子的表达量，见图3B。"

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