长链非编码RNA NEAT1调控miR-486/FOXO1轴参与椎间盘髓核细胞退变的机制研究

Study on the mechanism of long non-coding RNA NEAT1 regulating miR-486/FOXO1 axis and affects nucleus pulposus cell degeneration of intervertebral disc

中文关键词:  椎间盘退变  长链非编码RNA核富集转录本1  miR-486  FOXO1

英文关键词:Intervertebral disc degeneration  lncRNA NEAT1  miR-486  FOXO1

中文摘要:

  【摘要】 目的：明确长链非编码RNA核富集转录本1（lncRNA NEAT1）、miR-486、FOXO1之间的相互关系，探讨三者在椎间盘退变（intervertebral disc degeneration，IDD）组织中的表达及相关性，阐明NEAT1通过调控miR-486/FOXO1轴参与IDD进展的具体机制。方法：利用双荧光素酶报告基因、RNA结合蛋白免疫沉淀（RNA binding protein immunoprecipitation，RIP）试验等明确NEAT1、miR-486与叉头框蛋白O1（forkhead box protein O1，FOXO1）的生物学关系；采用实时荧光定量聚合酶链式反应（real-time quantitative polymerase chain reaction，RT-qPCR）、免疫印迹（Western blot）检测15例对照和30例IDD髓核组织中NEAT1、miR-486与FOXO1的表达，统计其与Pfirrmann分级的关系；采用白细胞介素1β（interleukin-1β，IL-1β）诱导法构建髓核细胞退变模型，NEAT1小干扰RNA（NEAT1 small interfering RNA，si-NEAT1）和过表达质粒转染髓核细胞构建细胞沉默或过表达模型；细胞计数试剂盒-8（cell counting kit-8，CCK-8）和流式细胞术检测髓核细胞增殖和凋亡；RT-qPCR检测细胞NEAT1与miR-486表达；Western blot检测FOXO1及细胞外基质（extracellular matrix，ECM）中聚集蛋白聚糖（aggrecan）、二型胶原（collagen Ⅱ）、基质金属蛋白酶-13（matrix metalloproteinase, MMP-13）和含Ⅰ型血小板结合蛋白基序的解聚蛋白样基质金属蛋白酶4（a disintegrin-like and metalloproteinase with thrombospondin type l motifs 4，ADAMTS4）的表达。结果：NEAT1可通过碱基互补配对方式海绵吸附miR-486；miR-486靶向FOXO1的3′-非编码区（3′-UTR）抑制FOXO1蛋白表达。在IDD髓核组织中，NEAT1与FOXO1表达上调，与Pfirrmann分级呈显著正相关（P<0.001）；而miR-486表达下调，与Pfirrmann分级呈显著负相关（P<0.001）。分子细胞学实验证实，IL-1β诱导髓核细胞退变后，NEAT1的过表达进一步促进了髓核细胞凋亡（P<0.01），加重了Aggrecan和Collagen Ⅱ的表达抑制（P<0.01），增强了MMP-13和ADAMTS4的表达（P<0.01）；相比之下，NEAT1的沉默则逆转了髓核细胞凋亡（P<0.01），有效恢复了Aggrecan和Collagen Ⅱ表达（P<0.01），同时显著降低了MMP-13和ADAMTS4的表达（P<0.01）。结论：NEAT1调控miR-486/FOXO1分子轴并促进髓核细胞凋亡和ECM降解进而参与IDD病理机制过程。

英文摘要:

  【Abstract】 Objectives: To investigate the relationships among long non-coding RNA nuclear enriched transcript 1(lncRNA NEAT1), miR-486, and FOXO1, and explore their expressions and correlations in intervertebral disc degeneration(IDD) tissues, then clarify the underlying mechanism of NEAT1 in IDD progression by regulating miR-486/FOXO1. Methods: Double luciferase reporter gene and RNA binding protein immunoprecipitation(RIP) assays were used to confirm the relationship among NEAT1, miR-486 and forkhead box protein O1(FOXO1); Real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot were used to detect the expressions of NEAT1, miR-486 and FOXO1 in 15 controls and 30 IDD nucleus pulposus tissues, and their relationships with Pfirrmann grade were statistically analyzed; The degeneration model of nucleus pulposus cells was constructed by interleukin-1β(IL-1β) induction and the cell silencing or overexpression model was constructed by transfection of NEAT1 small interfering RNA(si-NEAT1) or overexpression plasmids into nucleus pulposus cells; Cell counting kit-8(CCK-8) and flow cytometry were used to detect the proliferation and apoptosis of nucleus pulposus cells; RT-qPCR was used to detect the expression of NEAT1 and miR-486; Western blot was used to detect the expressions of FOXO1 and factors in extracellular matrix (ECM), including Aggrecan, Collagen Ⅱ, matrix metalloproteinase(MMP-13) and a disintegrin-like and metalloproteinase with thrombospondin type l motifs 4(ADAMTS4). Results: NEAT1 sponged with miR-486 through complementary base pairing, and miR-486 targeted the 3′-non coding region(3′-UTR) of FOXO1 to inhibit FOXO1 protein expression. In IDD nucleus pulposus tissues, the expressions of NEAT1 and FOXO1 were up-regulated, which were both significantly positively correlated with Pfirrmann grade(P<0.001); the expression of miR-486 was down-regulated, which was significantly negatively correlated with Pfirrmann grade(P<0.001). Molecular and cellular experiments revealed that after IL-1β induced the degeneration of nucleus pulposus cells, the overexpression of NEAT1 further promoted the apoptosis of nucleus pulposus cells(P<0.01), aggravated the expression inhibition of Aggrecan and Collagen Ⅱ(P<0.01), and enhanced the expression of MMP-13 and ADAMTS4(P<0.01); In contrast, the silencing of NEAT1 reversed the apoptosis of nucleus pulposus cells(P<0.01), effectively restored the expression of aggrecan and Collagen Ⅱ(P<0.01), and significantly reduced the expression of MMP-13 and ADAMTS4(P<0.01). Conclusions: NEAT1 can target miR-486/FOXO1 axis and promote nucleus pulposus cell apoptosis and ECM degradation, so as to induce pathological processes of IDD.