MiR-423-5p Regulates Cells Apoptosis and Extracellular Matrix Degradation via Nucleotide-Binding, Leucine-Rich Repeat Containing X1 (NLRX1) in Interleukin 1 beta (IL-1β)-Induced Human Nucleus Pulposus Cells

Abstract

BACKGROUND Disc degeneration is characterized partly by the degradation in the extracellular matrix (ECM) and excess apoptosis of nucleus pulposus (NP) cells. NLRX1 (nucleotide-binding, leucine-rich repeat containing X1) is different from the other nucleotide-binding-domain and leucine-rich-repeat proteins and mainly located to the mitochondrial. It negatively regulates NF-κB (nuclear factor kappa B) and apoptosis inhibition. However, how NLRX1 is regulated and exerts effects in disc degeneration is unclear. Thus, the study aimed to analyze the effects of NLRX1 on NP cells. MATERIAL AND METHODS NLRX1 expression was detected in interleukin (IL)-1β-induced NP cells by western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Then, NLRX1 was overexpressed in IL-1β-induced NP cells to detect apoptosis-related proteins and the extracellular matrix (ECM) by western blot, along with the detection of apoptosis levels using flow cytometry. StarBase predicted miR-423-5p target 3'UTR of NLRX1. Dual luciferase reporter assay showed that miR-423-5p could bind to the 3'UTR of NLRX1. Besides, miR-423-5p significantly affected NLRX1 levels detected by qRT-qPCR. RESULTS The miR-423-5p overexpression markedly, and negatively regulated the protective effects of NLRX1 on IL-1β induced NP cells. Thus, our results suggested that miR-423-5p mediated the regulation of NLRX1 to affect apoptosis and ECM levels in IL-1β induced NP cells. CONCLUSIONS miR-423-5p and NLRX1 could be potential therapeutic targets for patients with disc degeneration.

Figure 1

(A) NLRX1 expression was detected in IL-β induced NP cells by qRT-PCR and WB. *** P<0.001 versus NP cells. (B) NLRX1 expression was significantly increased by transfecting plasmids overexpressing NLRX1. ** P<0.01; versus Ov-NC. (C) Flow cytometry was used to detect the apoptosis levels of NP cells. (D) Apoptosis-related proteins were measured by WB. (E, F) Related type II EMC (collagen and aggrecan) and matrix decomposing enzymes (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5) were detected by WB. IL-1β+Ov-NLRX1: the NP cells were treated with IL-1β and plasmids overexpressing NLRX1. IL-1β+Ov-NC: the NP cells were treated with IL-1β and empty plasmids. ** P<0.01 or *** P<0.001; versus NP cells. ^### P<0.001, ^## P<0.01 or ^# P<0.05; versus IL-1β+Ov-NC. Data shown as mean±SD. NLRX1 – nucleotide-binding, leucine-rich repeat containing X1; NP – nucleus pulposus; qRT-PCR – quantitative real-time polymerase chain reaction; WB – western blot; ECM – extracellular matrix; MMP – matrix metalloproteinase; ADAMTS – a disintegrin and metalloproteinase with thrombospondin motifs; IL – interleukin; SD – standard deviation.

Figure 2

(A) miR-423-5p levels were detected by qRT-PCR in IL-β induced NP cells. *** P<0.001; versus NP cells. (B) miR-423-5p mimic significantly affected NLRX1 expression. (C) The binding sites of miR-423-5p and NLRX1 were predicted by StarBase. (D) luciferase reporter assay analyzed whether miR-423-5p bound to the 3′UTR of NLRX1. Data shown as mean±SD. qRT-PCR – quantitative real-time polymerase chain reaction; IL – interleukin; NP – nucleus pulposus; NLRX1 – nucleotide-binding, leucine-rich repeat containing X1; SD – standard deviation.

Figure 3

(A, B) miR-423-5p regulated the influence NLRX1 on cell apoptosis in IL-1β induced NP cells. * P<0.05, ** P<0.01 or *** P<0.001; versus IL-1β+Ov-NLRX1+miR-NC. Data shown as mean±SD. NLRX1 – nucleotide-binding, leucine-rich repeat containing X1; IL – interleukin; NP – nucleus pulposus; SD – standard deviation.

Figure 4

(A, B) NLRX1 significantly increased ECM and reduced the matrix degradation enzyme levels. * P<0.05, ** P<0.01 or *** P<0.001; versus IL-1β+Ov-NLRX1+miR-NC. Data shown as mean±SD. NLRX1 – nucleotide-binding, leucine-rich repeat containing X1; ECM – extracellular matrix; IL – interleukin; NP – nucleus pulposus; SD – standard deviation.