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.
Abstract
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.
基金项目
上海市虹口区卫生健康委项目(虹卫2102-15)
上海市第四人民医院人才助推计划(SY-XKZT-2021-3001)
上海市虹口区卫生健康委重大项目(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)
维普
万方数据