| 刘涛,支中正,王英杰,等.LncRNA GAS5通过miR-21调控脊髓损伤后神经细胞凋亡的机制研究.骨科,2024,15(2): 145-154. |
| LncRNA GAS5通过miR-21调控脊髓损伤后神经细胞凋亡的机制研究 |
| LncRNA GAS5 Regulates Nerve Cell Apoptosis through miR-21 after Spinal Cord Injury |
| 投稿时间：2023-12-21 |
| DOI：10.3969/j.issn.1674-8573.2024.02.009 |
| 中文关键词: 脊髓损伤 长链非编码RNA 生长阻滞特异性转录因子5 微小RNA-21 同源性磷酸酶-张力蛋白 凋亡 |
| 英文关键词: Spinal cord injury LncRNA GAS5 miR-21 PTEN Apoptosis |
| 基金项目:上海市虹口区卫生健康委员会课题资助项目（虹卫2102-15）；上海市第四人民医院人才助推计划项目（SY-XKZT-2021-3001）；上海市虹口区卫生健康委员会重大项目（虹卫2001-03）；上海市第四人民医院人才助推重点项目（SY-XKZT-2020-1003） |
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| 中文摘要: |
| 目的 明确长链非编码RNA生长阻滞特异性转录因子5（LncRNA GAS5）与微小RNA-21（miR-21）的关系，阐明LncRNA GAS5调控miR-21参与脊髓损伤后神经细胞凋亡的机制。方法 建立脊髓损伤大鼠和氧糖剥夺复氧（OGD/R）处理的大鼠肾上腺嗜铬细胞瘤细胞（PC-12）模型；沉默和过表达GAS5，构建下调shGAS5表达的慢病毒，通过生物信息学分析预测LncRNA GAS5与miR-21存在结合位点等。通过双荧光素酶报告基因实验等探究GAS5与miR-21的结合位点；采用实时荧光定量聚合酶链式反应（RT-qPCR）检测miR-21、GAS5、同源性磷酸酶-张力蛋白（phosphatase and tensin homolog，PTEN）基因、半胱天冬氨酸蛋白酶3（Caspase 3）、B淋巴细胞瘤（Bcl）-2相关X基因（Bax）、Bcl-2和蛋白激酶B（AKT）的RNA表达水平；Western blot检测Cleaved caspase 3、Bax、Bcl-2、AKT和磷酸化AKT（p-AKT）的蛋白表达水平；TUNEL技术检测神经细胞凋亡程度。结果 大鼠脊髓损伤后脊髓中miR-21、Bcl-2及p-AKT表达水平降低（P＜0.05），GAS5、PTEN、Bax及Cleaved caspase-3表达均升高（P＜0.05）。PC-12体外培养与OGD/R损伤后出现与大鼠脊髓损伤模型类似结果，且于OGD/R处理后2 h最显著。GAS5可通过碱基互补配对方式结合miR-21，进而调控下游PTEN信号通路。下调GAS5或过表达miR-21可抑制PC-12细胞凋亡（P＜0.05）。结论 GAS5通过结合miR-21，上调PTEN并抑制AKT磷酸化，进而促进脊髓损伤诱导的神经细胞凋亡。 |
| 英文摘要: |
| Objective To clarify the relationship between long non coding RNA growth arrest-special transcript 5 (LncRNA GAS5) and microRNA-21 (miR-21), and explore the mechanism of neuronal apoptosis regulated by LncRNA GAS5 via miR-21 after spinal cord injury. Methods The models of spinal cord injury of rats and adrenal pheochromocytoma cells (PC-12) with oxygen glucose deprivation reoxygenation (OGD/R) were established. After silencing or overexpressing GAS5, a lentivirus with downregulated shGAS5 expression was constructed, and the binding site between LncRNA GAS5 and miR-21 was predicted through bioinformatics analysis. The binding sites of GAS5 and miR-21 were explored by dual luciferase reporter gene. The real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the RNA expression levels of miR-21, GAS5, homologous phosphatase and tensin homolog (PTEN) genes, caspase 3, Bcl-2 related X genes (Bax), Bcl-2, and protein kinase B (AKT). Western blotting was used to detect the protein expression levels of Cleaved caspase 3, Bax, Bcl-2, AKT, and phosphorylated AKT (p-AKT). TUNEL technology was used to detect the degree of neuronal apoptosis. Results The expression levels of miR-21, Bcl-2, and p-AKT decreased (P＜0.05), while the expression of GAS5, PTEN, Bax, and Cleaved caspase-3 increased (P＜0.05) in rats with spinal cord injury. PC-12 cells showed similar results to the rat spinal cord injury model after in vitro culture and OGD/R injury, and the results were most significant 2 h after OGD/R treatment. GAS5 could bind to miR-21 through complementary base pairing, thereby regulating the downstream PTEN signaling pathway. Downregulation of GAS5 or overexpression of miR-21 could inhibit the apoptosis of PC-12 cells (P＜0.05). Conclusion GAS5 upregulates PTEN and inhibits AKT phosphorylation by binding to miR-21, thereby promoting neuronal apoptosis induced by spinal cord injury. |
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