【中国声音】#脊髓损伤 MiR-181a-5p通过抑制高迁移率组box-1蛋白的表达，减轻PC12细胞的炎症反应

第一作者：吴至武

通信作者：李美华

作者单位：南昌大学第一附属医院神经外科

Wu Z, Zhang Z, Wang Z, Zhu H, Li M. MiR-181a-5p alleviates the inflammatory response of PC12 cells by inhibiting high-mobility group box-1 protein expression [published online ahead of print, 2022 Mar 10]. World Neurosurg. 2022;S1878-8750(22)00305-9. doi:10.1016/j.wneu.2022.03.025

创伤性脊髓损伤（Traumatic spinal cord injury, TSCI）是由外力引起的脊髓损伤，常伴有脊髓损伤节段以下的运动、感觉和排便障碍。SCI涉及复杂多样的病理生理过程，包括原发性损伤和继发性损伤。继发性损伤包括神经性炎症、氧化应激、神经元凋亡和铁死亡等。SCI后的炎症反应具有重要的病理意义。神经性炎症是SCI后神经性疼痛的主要原因；炎症损伤可引起广泛的神经元死亡，这可能是SCI预后不良的原因。近年来，抑制SCI后的神经炎性损伤已被证明能显著改善神经功能恢复。MicroRNAs（miRNAs）是一种小的非编RNA，可以抑制mRNA的翻译。大量研究发现，SCI后许多miRNAs差异表达，参与调控SCI后的神经性炎症、神经元凋亡、氧化应激等病理过程。我们发现miR-181a-5p在SCI后表达降低，但其具体的作用机制仍不清楚。

高迁移率组box-1蛋白（high-mobility group box-1 protein, HMGB1）是一种核DNA结合蛋白，是损伤相关分子模式的重要成员。HMGB1是炎症反应、肿瘤细胞迁移、铁死亡和自噬的调节因子。本课题组前期研究发现HMGB1在SCI后表达升高，参与调控小胶质细胞的炎症反应。我们通过生物信息学分析发现，HMGB1可能是miR-181a-5p的直接下游靶标。因此，本研究旨在通过采用脂多糖（LPS）诱导PC12细胞构建炎症反应体外神经元细胞模型，探讨miR-181a-5p/HMGB1轴调控LPS诱导的PC12细胞炎症反应的分子机制。结果表明，miR-181a-5p直接靶向HMGB1 3ʹUTR保护PC12细胞免受LPS诱导的炎症，减轻神经元炎症反应，具有神经保护潜能。

结果

损伤脊髓组织的形态学改变和miR-181a-5p、HMGB1的表达水平

H&E染色观察损伤脊髓组织形态，明确脊髓组织损伤情况，可见大量巨噬细胞和炎性细胞聚集，细胞排列紊乱；RT-qPCR实验证实：相比较于假手术组（sham组），SCI组miR-181a-5p表达显著降低，而HMGB1表达显著升高（p< 0.05）。（Fig.1）

Fig.1. Differential expression of miR-181a-5p and HMGB1 in SCI tissues. (A) SCI animal models were made successfully (red arrow). (B) The morphology of the injured spinal cord (red arrow). (C) Hematoxylin-eosin staining confirmed that SCI tissues damaged seriously and showed greater inflammatory cell infiltration after SCI. Scale bar=200μm (magnification:5×) (D) RT-qPCR showed that miR-181a-5p expression was decreased in SCI tissues (compared to the sham group). (E) HMGB1 expression increased in SCI tissues. Error bars are means ± SDs, n=7 biological replicates, 3 independent experiments). *P<0.05 vs. the sham group.

通过LPS诱导PC12细胞，RT-qPCR实验结果发现，LPS诱导PC12细胞24小时后，炎症因子（TNF-α，IL-1β和IL-6）表达升高（Fig.2B-2D），提示成功构建PC12细胞炎症反应模型；同时miR-181a-5p表达显著降低，而HMGB1表达显著升高（Fig.2E-2F）。

Fig. 2. LPS treatment elicited an inflammatory response in PC12 cells. (A) CCK-8 assay of the viability of cells exposed to different concentrations of LPS for 24h. (B–D) RT-qPCR showed that the TNF-α, IL-1β, and IL-6 expression levels increased after LPS treatment (5μg/mL) for 24h. (E) miR-181a-5p expression decreased in PC12 cells after LPS treatment (5μg/mL) for 24h. (F) HMGB1 expression increased in PC12 cells after LPS treatment. Error bars are means ± SDs, n=3 biological replicates, 3 independent experiments). ***P<0.001, **P<0.01, or *P<0.05 vs. control.

通过在LPS组PC12细胞中转染miR-181a-5p mimics、miR-181a-5p inhibitor上调或下调miR-181a-5p的表达，RT-qPCR和Western blot结果显示上调miR-181a-5p后HMGB1表达显著降低，同时炎症因子（TNF-α，IL-1β和IL-6）表达降低，下调miR-181a-5p后得到相反的结果（Fig.3），结果表明miR-181a-5p抑制HMGB1表达，具有抗炎效应。

Fig. 3. Anti-inflammatory effects of miR-181a-5p in LPS-treated PC12 cells. (A) miR-181a-5p expression after transfection (measured via RT-qPCR). (B) HMGB1 expression decreased in the miR-181a-5p-mimics group, but increased in the miR-181a-5p inhibitor group. (C-E) RT-qPCR revealed that the TNF-α, IL-1β, and IL-6 levels were significantly attenuated in the miR-181a-5p mimics group, but increased in the miR-181a-5p inhibitor group. (F) Western blotting revealed parallel changes in the protein expression levels of HMGB1, TNF-α, IL-1β, and IL-6. Error bars are means ± SDs. n=3 biological replicates, 3 independent experiments). ***P<0.001, **P<0.01, *P<0.05 vs. LPS group.

通过在miR-181a-5p mimics+LPS组PC12细胞中转染过表达的HMGB1质粒，RT-qPCR和Western blot结果显示HMGB1过表达后能逆转miR-181a-5p的抗炎效应（Fig.4）。

Fig. 4. HMGB1 overexpression reversed the anti-inflammatory effects of miR-181a-5p. (A) RT-qPCR revealed that HMGB1 expression was significantly increased on transfection of an HMGB1-overexpressing plasmid into LPS-treated PC12 cells. (B-D) RT-qPCR indicated that the expression levels of TNF-α, IL-1β, and IL-6 were increased when HMGB1 was overexpressed. (E) Western blotting confirmed increases in the levels of the HMGB1, TNF-α. IL-1β, and IL-6 proteins. Error bars are means ± SDs. n=3 biological replicates, 3 independent experiments). ***P<0.001, **P<0.01 or P>0.05 vs. the miR-181a-5p mimics+LPS group.

双荧光素酶检测结果证实HMGB1是miR-181a-5p的直接下游靶标（Fig.5A-5B）；核质分离实验结果提示miR-181a-5p和HMGB1在PC12细胞的细胞质中均有分布，为二者的相互作用提供了场所（Fig.5C）。

Fig. 5. miR‐181a-5p targeted HMGB1 to negatively regulate the expression thereof. (A) The predicted sequences of miR‐181a-5p matching the 3ʹ‐UTR of HMGB1 (as revealed by bioinformatics). (B) Dual-luciferase assays of PC12 cells co-transfected with miR‐181a-5p mimics and a luciferase reporter containing the HMGB1 3ʹ‐UTR WT or MUT. (C) MiR-181a-5p and HMGB1 distributions in the nuclear and cytoplasmic fractions of PC12 cells. GAPDH, U6, and Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) served as reference.

结论

我们的实验结果得出：（1）在SCI组织和LPS诱导的PC12细胞中，miR-181a-5p表达降低，HMGB1表达增加；（2）miR-181a-5p的上调可以抑制LPS诱导的PC12细胞的炎症反应和HMGB1的表达；（3）HMGB1过表达逆转了miR-181a-5p的抗炎作用；（4）双荧光素酶检测证实HMGB1是miR-181a-5p的直接靶点。

综上所述，体外实验中发现miR-181a-5p通过直接靶向HMGB1抑制LPS诱导的PC12细胞的炎症反应，为进一步的体内实验研究提供了基础，miR-181a-5p可能作为SCI的治疗靶点。

通讯作者简介

李美华教授

南昌大学第一附属医院

神经外科主任、主任医师、二级教授，博士研究生导师

江西省卫生系统有突出贡献的中青年专家、江西省百千万人才、江西省骨干教师和中青年学科带头人

2016年“侧颅底入路治疗复杂颅底肿瘤的临床应用”获科技进步奖二等奖（第一完成人）和2020年“颅咽管瘤诊疗新体系的构建及推广应用”获江西省科技进步奖一等奖（第二完成人）

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