Role of the miR-133a-5p/FBXO6 axis in the regulation of intervertebral disc degeneration

Abstract

Objective: Low back pain is a leading cause of disabilities worldwide, and intervertebral disc degeneration (IVDD)-related disorders have been recognised as one of the main contributors. Nevertheless, the underlying mechanism has not yet been fully understood. The aim of this study was to investigate the role of the miR-133a-5p/FBXO6 axis in the regulation of IVDD.

Methods: RT-qPCR, WB and IHC were performed to assess the expression of FBXO6 in human IVD tissues. Nucleus pulposus (NP) cells were treated with IL-1β to induce IVDD cellular model. Silence of FBXO6 was achieved using specific siRNAs. CCK-8 assay, flow cytometry, TUNEL assay, RT-qPCR and WB were used to evaluate the role and mechanism of FBXO6 in the process of IVDD. Online tools, GSE datasets and RT-qPCR were used to search the candidate miRNAs targeting FBXO6. The direct binding sites between FBXO6 and miR-133a-5p were further verified by a dual luciferase assay. RT-qPCR, WB and rescue experiments were conducted to identify the regulatory function of miR-133a-5p on the expression of aggrecan, collagen Ⅱ, MMP3, ADAMTS5, IL-6 and COX2. In addition, the role of the NF-κB pathway in regulating miR-133a-5p was studied using lentiviral shRNA, WB and RT-qPCR.

Results: Results showed that FBXO6 mainly expressed in the NP tissue of IVD and the expression of FBXO6 decreased with the process of IVDD as well as under IL-1β stimulation. The silence of FBXO6 led to the decreased expression of aggrecan and collagen Ⅱ and the increased expression of MMP3, ADAMTS5, IL-6 and COX2, which further induced the degeneration of NP cells. The bioinformatic analysis showed that miR-133a-5p was the candidate miRNA targeting FBXO6. miR-133a-5p was upregulated in IVDD tissues and significantly inhibited the expression of FBXO6. The inhibition of miR-133a-5p ameliorated the acceleration of IVDD induced by the silence of FBXO6 in vitro. Moreover, it was demonstrated that IL-1β regulated the expression of the miR-133a-5p/FBXO6 axis via the NF-κB pathway in NP cells.

Conclusion: miR-133a-5p was upregulated by IL-1β to aggravate intervertebral disc degeneration via sponging FBXO6. Inhibiting miR-133a-5p expression or rescuing FBXO6 expression may be promising strategies for the treatment of IVDD.

The translational potential of this article: This study suggests that the miR-133a-5p/FBXO6 axis could regulate NP cells proliferation, apoptosis, synthesis and degradation of extracellular matrix, which provides a promising therapeutic target and strategy for the treatment of IVDD.

Keywords: FBXO6; Interleukin (IL)-1β; Intervertebral disc; Nucleus pulposus cells; miR-133a-5p.

Figure 1

The expression pattern of FBXO6 in normal and degenerated IVD tissues (A) RT-qPCR analysis showed that FBXO6 mRNA expressed higher in NP than AF tissues (B/C) WB and densitometric analyses showed that the protein level of FBXO6 was higher in NP tissues (D) Specific MRI image of IVDD by Pfirrmann grades (E) The RT-qPCR analysis indicated that FBXO6 mRNA decreased with the severity of IVDD (F/G) The WB and densitometric analyses determined that the protein level of FBXO6 decreased with the progression of IVDD (H/I) IHC staining and quantitative analyses showed that the expression of FBXO6 in NP tissues decreased with the development of IVDD. ∗∗∗P ​< ​0.001.

Figure 2

The expression of FBXO6 in NP cells was reduced by IL-1β (A) The expression level of IL-1β increased with the severity of IVDD (B) A negative correlation was found between the expression of FBXO6 and IL-1β (C–E) The RT-qPCR and WB showed that IL-1β reduced the FBXO6 expression in a dose-dependent manner (F–H) The RT-qPCR and WB analyses showed that IL-1β suppressed the expression of FBXO6 in a time-dependent manner. ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Figure 3

Regulatory effect of FBXO6 in NP Cells (A) High transfection efficiency was found in NP cells after transfection with siRNAs (B) RT-qPCR indicated that the expression of FBXO6 was significantly suppressed by the respective siRNA (C/D) WB and and densitometric analyses results showed that all the three siRNAs have good silence effect on the expression of FBXO6 and siRNA-3 has the highest silence efficiency (E) CCK8 test data determined that IL-1β treatment resulted in low proliferation of NP cells and si-FBXO6 was able to significantly aggravate this effect (F/G) Flow cytometry analysis showed that si-FBXO6 induced the apoptosis of NP cells, PI: propidium iodide, FITC: fluorescein isothiocyanate (H/I) TUNEL assay showed that si-FBXO6 induced the apoptosis of NP cells (J–O) RT-qPCR showed that aggrecan and collagen Ⅱ were decreased by IL-1β treatment and that si-FBXO6 aggravated the suppression of aggrecan and collagen Ⅱ by IL-1β; the expression of MMP3, ADAMTS5, IL-6 and COX2 were increased after IL-1β stimulation, and si-FBXO6 enhanced their downregulation induced by IL-1β (P–V) WB and densitometric analyses showed the regulatory effect of aggrecan, collagen II, MMP3, ADAMTS5, IL-6 and COX2 expressions by si-FBXO6 and IL-1β. The treatment condition of IL-1β was 10 ​ng/ml for 24 ​h; ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Figure 4

miR-133a-5p was the inhibitory regulator of FBXO6 (A) TargetScan, miRDB and miRWalk were used to detect the related microRNAs targeting FBXO6 (B) GSE63492 and GSE116726 datasets were used to evaluate the related microRNAs targeting FBXO6 (C) Compared with normal IVD, miR-133a-5p expression level was upregulated in degenerated IVD specimens (D) RT-qPCR result showed that no significant difference of miR-4635–3p expression was found between normal and degenerated IVD (E) IL-1β induced the expression of miR-133a-5p (F) Negative correlation was found between the expression of FBXO6 and miR-133a-5p in IVD tissues (G/H) miR-133a-5p levels in NP cells after being transfected with the miR-133a-5p mimic or inhibitor were determined by the RT-qPCR (I) Schematic representation of miR-133a-5p's predicted binding sites in the 3′UTR of FBXO6 mRNA (J) The wild- or mutant-type FBXO6 3′UTR reporter plasmid was co-transfected with miR-133a-5p mimics or inhibitors into NP cells (K/L) FBXO6 expressions in NP cells after transfection with miR-133a-5p mimic or inhibitor. ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Figure 5

miR-133a-5p exerted its function in NP cells via sponging FBXO6 (A–F) RT-qPCR showed that the expressions of aggrecan and collagen Ⅱ were increased by the miR-133a-5p inhibitor, and si-FBXO6 alleviated the upregulation of aggrecan and collagen Ⅱ. The expressions of MMP3, ADAMTS5, IL-6 and COX2 were downregulated by the miR-133a-5p inhibitor and si-FBXO6 partly reversed their downregulations (G–M) WB and densitometric analyses showed the expressions of aggrecan, collagen Ⅱ, MMP3, ADAMTS5, IL-6 and COX2 by the miR-133a-5p inhibitor and si-FBXO6. The treatment condition of IL-1β was 10 ​ng/ml for 24 ​h; ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Figure 6

The inflammatory environment regulated miR-133a-5p and FBXO6 via the NF-κB signaling pathway (A) The expression of miR-133a-5p following IL-1β treatment with or without ERK inhibitor (PD98059 (PD)) or JNK inhibitor (SP60025 (SP)) or p38 inhibitor (SB203580 (SB)) or NF-κB inhibitor (SM7368 (SM)) (B) Immunofluorescence detection of GFP in human NP cells transduced with either shp65 or shIkkβ showed a high transfection efficiency (C–D) RT-qPCR showed LV-shp65 and LV-shIkkβ significantly suppressed the expression of p65 and Ikkβ (E–H) A similar result was found by the WB and densitometric analyses (I) IL-1β-dependent induction in miR-133a-5p was significantly blocked by the suppression of the components of the NF-κB pathway (J–L) RT-qPCR and WB showed that the IL-1β-dependent decrease in FBXO6 was significantly relieved by the suppression of the components of the NF-κB pathway (M) Proposed illustration to point out the mechanism by which IL-1β upregulates miR-133a-5p expression to aggravate IVD degeneration via inhibiting FBXO6. The treatment condition of IL-1β was 10 ​ng/ml for 24 ​h; ‘###’ means P ​< ​0.001 when compared with DMSO group; ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.