Background

Multiple cellular and molecular changes are involved in the etiology of spinal cord injury (SCI) and the recovery from SCI. Accumulating studies showed aberrant expression of microRNAs (miRNAs) after SCI. Here, we established in vivo and in vitro models to analyze the role of miR-212-3p in SCI.

Methods

An in vivo model of SCI was established in Sprague-Dawley rats. SCI-induced histopathological changes of the spinal cord were observed by hematoxylin-eosin staining. Functional recovery of rats with SCI was evaluated using the Basso-Beattie-and-Bresnahan scale. PC12 cells were stimulated by lipopolysaccharide (LPS) to establish SCI model of neuronal apoptosis in vitro. Dual-luciferase reporter assay was performed to validate the potential target of miR-212-3p predicted by TargetScan 7.2. MTT assay and flow cytometry were carried out to measure the viability and apoptosis of PC12 cell, respectively. The expressions of miR-212-3p, PTEN, phosphorylated (p)-AKT, AKT, p-mTOR, mTOR, Cleaved caspase-3 and BCl-2 in spinal cord tissues and PC12 cells were analyzed by qRT-PCR or Western blot.

Results

In the spinal cord of rats with SCI, the expressions of miR-212-3p, p-AKT, p-mTOR and BCl-2 were downregulated, whereas those of PTEN and Cleaved caspase-3 were upregulated. BBB scores were low, and there were histopathological changes, which were all reversed after the injection of agomiR-212-3p. MiR-212-3p directly targeted PTEN. Upregulated miR-212-3p in LPS-injured PC12 cells suppressed apoptosis, downregulated the expressions of PTEN and Cleaved caspase-3, promoted viability and upregulated the expressions of p-AKT, p-mTOR and BCl-2, which were all reversed by overexpressed PTEN.

Conclusion

MiR-212-3p improved functional recovery of SCI rats and inhibited LPS-induced neurocyte apoptosis by targeting PTEN to activate AKT/mTOR pathway.

中文翻译：

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MiR-212-3p 通过 PTEN 下调介导的 AKT/mTOR 通路激活改善大鼠功能恢复并抑制脊髓损伤模型中的神经细胞凋亡

背景

脊髓损伤 (SCI) 的病因和 SCI 的恢复涉及多种细胞和分子变化。越来越多的研究表明 SCI 后 microRNA (miRNA) 的异常表达。在这里，我们建立了体内和体外模型来分析 miR-212-3p 在 SCI 中的作用。

方法

在 Sprague-Dawley 大鼠中建立了 SCI的体内模型。通过苏木精-伊红染色观察脊髓损伤引起的脊髓组织病理学变化。使用 Basso-Beattie-and-Bresnahan 量表评估 SCI 大鼠的功能恢复。脂多糖(LPS)刺激PC12细胞体外建立神经元凋亡SCI模型。进行双荧光素酶报告基因测定以验证由 TargetScan 7.2 预测的 miR-212-3p 的潜在靶标。MTT法和流式细胞仪分别检测PC12细胞的活力和凋亡。通过qRT-PCR或Western印迹分析miR-212-3p、PTEN、磷酸化(p)-AKT、AKT、p-mTOR、mTOR、Cleaved caspase-3和BCl-2在脊髓组织和PC12细胞中的表达.

结果

SCI大鼠脊髓中miR-212-3p、p-AKT、p-mTOR和BCl-2的表达下调，而PTEN和Cleaved caspase-3的表达上调。BBB评分低，有组织病理学改变，注射agomiR-212-3p后全部逆转。MiR-212-3p 直接针对 PTEN。在 LPS 损伤的 PC12 细胞中上调 miR-212-3p 抑制细胞凋亡，下调 PTEN 和 Cleaved caspase-3 的表达，促进活力并上调 p-AKT、p-mTOR 和 BCl-2 的表达，这些都被逆转过表达的 PTEN。

结论

MiR-212-3p 通过靶向 PTEN 激活 AKT/mTOR 通路改善 SCI 大鼠的功能恢复并抑制 LPS 诱导的神经细胞凋亡。