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

English Keywords:Intervertebral disc degeneration  lncRNA NEAT1  miR-486  FOXO1

English Abstract:

  【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.