CircERCC2 ameliorated intervertebral disc degeneration by regulating mitophagy and apoptosis through miR-182-5p/SIRT1 axis

Abstract

The molecular mechanism of intervertebral disc degeneration (IVDD) remains unclear. This study aimed to investigate the role of circular RNAs (circRNAs) in the pathogenesis of IVDD. We sued nucleus pulposus (NP) tissues of patients, tert-butyl hydroperoxide (TBHP) stimulated NP cells (NPCs), and IVDD rat model to explore the interaction between circERCC2 and miR-182-5p/SIRT1 axis. The results showed that downregulation of circERCC2 increased the level of miR-182-5p and decreased the level of SIRT1 in degenerative NP tissues in vivo as well as in TBHP-stimulated NPCs in vitro. Treatment of SIRT1-si activated apoptosis and inhibited mitophagy. Moreover, miR-182-5p-si could regulate the mitophagy and the apoptosis of NPCs by targeting SIRT1. The effects of circERCC2 on NPCs and IVDD rat model were mediated by miR-182-5p/SIRT1 axis. In conclusion, this study provides the first evidence that circERCC2 could ameliorate IVDD through miR-182-5p/SIRT1 axis by activating mitophagy and inhibiting apoptosis, and suggests that circERCC2 is a potentially effective therapeutic target for IVDD.

Fig. 1. CircERCC2 was downregulated in IVDD and regulated mitophagy and apoptosis.

a Volcano plots showed differential expression of circRNAs detected by circRNA microarray in IVDD compared with the control. b Volcano plots showed differential expression of circRNAs in GSE67566. c The 9 downregulated circRNAs in IVDD were identified based on the overlap of circRNA microarray and GSE67566. d Heatmap of 9 circRNAs in circRNA microarray and heatmap of 9 circRNAs in GSE67566. e qRT-PCR analysis confirmed the downregulation of circRNAs in IVDD compared with control. *p < 0.05. f circERCC2 is transcribed from 13, 14, and 15 exons of the ERCC2 gene. The expression of circERCC2 was lower in NP tissues from IVDD compared with the control detected by FISH. g FISH detection of circERCC2 in the cytoplasm of NPCs. In (f) and (g), blue fluorescence indicated the nucleus and green fluorescence indicated circERCC2. Scale bar: 20 μm. h Representative plots of apoptosis detected by flow cytometry. circERCC2 inhibited the rate of apoptosis of NPCs. *p < 0.05, **p < 0.01. i NPCs were treated by TBHP or/and circERCC2, and mitophagy and apoptosis related proteins were detected by Western blot analysis

Fig. 2. miR-182-5p was upregulated in IVDD and regulated mitophagy and apoptosis.

a Volcano plot showed differential expression of miRNAs in GSE116726. b The predicted 8 miRNAs of circERCC2 (has_circ_0051470) and heatmap of the 8 miRNAs in GSE116726. (c) NPCs were transfected with miR-182-5p and luciferase constructs of circERCC2 containing wild-type putative miR-182-5p binding sites or mutated sites. *p < 0.05, **p < 0.01. d qRT-PCR analysis confirmed the upregulation of miR-182-5p in the degenerative NP samples from patients with IVDD compared with the control. *p < 0.05, **p < 0.01. e FISH showed that both circERCC2 and miR-182-5p were located in the cytoplasm. Blue fluorescence indicated the nucleus, green fluorescence indicated circERCC2, and red fluorescence indicated miR-182-5p. Scale bar: 20 μm. f FISH analysis of miR-182-5p in NP samples from patients with or without IVDD. Blue fluorescence indicated the nucleus, and red fluorescence indicated miR-182-5p. Scale bar: 20 μm. g Representative plots of apoptosis detected by flow cytometry. miR-182-5p-si inhibited apoptosis induced by TBHP in NPCs. *p < 0.05, **p < 0.01. h NPCs were treated by TBHP or/and miR-182-5p-si, and mitophagy and apoptosis related proteins were detected by Western blot analysis

Fig. 3. Identification of SIRT1 as a target of miR-182-5p.

a Weighted correlation network analysis (WGCNA) of the GEO database (GSE27494, GSE34095, GSE41883, and GSE15227). The topological overlaps of mRNA and their relations to modules were shown in dendrogram. b The turquoise module. c A graphic depiction of the turquoise module using String (https://string-db.org/). d Venn diagram showing targets by different algorithms. e Cystoscope was employed to confirm the targets of miR-182-5p. f NPCs were transfected with miR-182-5p and luciferase constructs of SIRT1 containing wild-type putative miR-182-5p binding sites or mutated sites. *p < 0.05, **p < 0.01. Immunofluorescence double staining for co-localization of SIRT1 (g) and LC3B (h) with TOMM20 in NPCs. i Representative plots of apoptosis detected by flow cytometry. SIRT1-si decreased apoptosis inhibition of circERCC2 in NPCs. *p < 0.05, **p < 0.01. j SIRT1-si decreased apoptosis inhibition of miR-182-5p-si in NPCs. *p < 0.05, **p < 0.01

Fig. 4. miR-182-5p/SIRT1 axial is the target of circERCC2.

a The cell senescence was determined using SA-β-gal staining. SIRT1-si decreased senescence inhibition of circERCC2 in NPCs. *p < 0.05, **p < 0.01. b SIRT1-si decreased senescence inhibition of miR-182-5p-si in NPCs. *p < 0.05, **p < 0.01. c Cell apoptosis was determined by TUNNEL staining. SIRT1-si abolished the inhibitory effect of circERCC2 on the apoptosis of NPCs. *p < 0.05, **p < 0.01. d SIRT1-si abolished the inhibitory effect of miR-182-5p-si on the apoptosis of NPCs. *p < 0.05, **p < 0.01. e SIRT1-si antagonized protective effects of circERCC2 on NPCs. f SIRT1-si antagonized protective effect of miR-182-5p-si on NPCs

Fig. 5. CircERCC2 ameliorated IVDD in vivo.

a T2-weighted MRI of rat tail with punctured disc. MRI grade was significantly lower in circERCC2 group. *p < 0.05, **p < 0.01. b FISH showed that circERCC2 expression was located in the NP region. Blue fluorescence indicated the nucleus and green fluorescence indicated circERCC2. Scale bar: 20 μm. c circERCC2 in IVDD was upregulated in the circERCC2 group. *p < 0.05, **p < 0.01. d miR-182-5p level in IVDD decreased following the injection of circERCC2. *p < 0.05, **p < 0.01. e circERCC2 inhibited ECM degradation, induced mitophagy and inhibited apoptosis in vivo. f–h Immunofluorescence staining showed upregulated collagen II and downregulated MMP13 in circERCC2 group. Scale bar: 25 μm. i H&E staining and Safranin-O/fast green staining showed that IVDD was ameliorated in circERCC2 group. Scale bar: 1000 μm. j The histological grades were significant decreased at week 8 in circERCC2 group. *p < 0.05, **p < 0.01, n = 6

Fig. 6. Schematic representation of the mode of action of circERCC2.

CircERCC2 ameliorates IVDD through targeting miR-182-5p/SIRT1 axis to regulate mitophagy and apoptosis