文章摘要
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)
作者单位E-mail
刘涛 同济大学附属上海市第四人民医院脊柱外科上海 200434  
支中正 同济大学附属上海市第四人民医院脊柱外科上海 200434  
王英杰 同济大学附属上海市第四人民医院脊柱外科上海 200434  
李富平 同济大学附属上海市第四人民医院脊柱外科上海 200434  
周付超 同济大学附属上海市第四人民医院脊柱外科上海 200434  
康健 同济大学附属上海市第四人民医院脊柱外科上海 200434 fmspine@163.com 
何志敏 同济大学附属上海市第四人民医院脊柱外科上海 200434  
摘要点击次数: 287
全文下载次数: 213
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.
查看全文   下载PDF阅读器
关闭