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(collagenⅡ)、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CollagenⅡ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CollagenⅡ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。
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 upregulated, 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-13and 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.
作者
柴星宇
宋将
朱静华
郝清海
袁崇明
刘涛
CHAI Xingyu;SONG Jiang;ZHU Jinghua(Department of Spine Surgery,the Affiliated Tengzhou Hospital of Xuzhou Medical University,Tengzhou,277500,China)
出处
《中国脊柱脊髓杂志》
CAS
CSCD
北大核心
2022年第7期639-647,共9页
Chinese Journal of Spine and Spinal Cord
基金
徐州医科大学附属医院发展基金(优秀人才基金项目)(项目编号:XYFY2020039)