Silencing SUMO2 promotes protection against degradation and apoptosis of nucleus pulposus cells through p53 signaling pathway in intervertebral disc degeneration

Abstract

Objective: Intervertebral disc degeneration (IDD), as a common cause of back pain, is related to the promotion of cellular senescence and reduction in proliferation. Based on recent studies, small ubiquitin-related modifier (SUMO) proteins have been implicated in various biological functions. Therefore, in the present study, we investigated the effects of SUMO2 on proliferation and senescence of nucleus pulposus cells (NPCs) via mediation of p53 signaling pathway in rat models of IDD.

Methods: After the establishment of rat models of IDD for the measurement of positive expression of SUMO2/3 protein, the mRNA and protein levels of SUMO2, molecular phenotype [matrix metalloproteinase-2 (MMP-2) and hypoxia-inducible factor-1α (HIF-1α)] and p53 signaling pathway-related genes [p21, murine double minute-2 (MDM2), growth arrest and DNA-damage-inducible protein 45 α (GADD45α), cyclin-dependent kinase 2/4 (CDK2/4), and CyclinB1] were determined, followed by the detection of cell proliferation, cell cycle, apoptosis, and cell senescence.

Results: The rat models of IDD were successfully constructed. The results obtained showed that there was a higher positive expression of SUMO2/3 protein in IDD rats. Moreover, the silencing of the SUMO2 gene decreases the levels of SUMO2, p53, p21, MDM2, GADD45α, MMP-2, and HIF-1α expressions and p53 phosphorylation level while it increases the levels of CDK2/4 and CyclinB1 expressions. In addition, SUMO2 gene silencing enhances proliferation and suppresses apoptosis and cell senescence of NPCs.

Conclusion: In conclusion, SUMO2 gene silencing promotes proliferation, and inhibits the apoptosis and senescence of NPCs in rats with IDD through the down-regulation of the p53 signaling pathway. Thus, SUMO2 is a potential target in the treatment of IDD.

Keywords: Intervertebral disc degeneration; Nucleus pulposus cells; Proliferation; SUMO2 gene silencing; p53 signaling pathway.

Figure 1. The HE staining reflects pathological changes in NPCs of IDD rats under the optical microscope and electron microscope

Under the optical microscope, the chondrocytes were irregularly arranged, and the NPCs were disrupted in the rats with IDD. In addition, under the electron microscope, degenerated NPCs, some microcalcifications with high electron density, and sheath or wool-like collagen fibrils were observed in the rats with IDD; HE, hematoxylin and eosin; IDD, intervertebral disc degeneration; NPC, nucleus pulposus cell.

Figure 2. SUMO2/3 protein expressed in NPCs between the sham and IDD group, determined by immunohistochemistry

(A) SUMO2/3-positive cells were brown-stained, which were pointed by black arrows (×400). (B) Quantitative analysis of SUMO2/3-positive cells; *P<0.05, compared with the sham group; IDD, intervertebral disc degeneration; NPC, nucleus pulposus cell; SUMO, small ubiquitin-related modifier.

Figure 3. Expressions of type II collagen in NPCs in the sham and IDD groups, determined by optical microscope

Notes: IDD, intervertebral disc degeneration; NPC, nucleus pulposus cell.

Figure 4. Obvious changes of growth morphology were observed in IDD rats under the transmission electron microscope

Notes: Primary sham group (×40). Primary IDD group (×40). Passage sham group (×100). Passage IDD group (×100); NPC, nucleus pulposus cells; IDD, intervertebral disc degeneration.

Figure 5. GFP gene report shows higher expression of green fluorescent protein in the three PLL-SUMO2-shRNA groups by fluorescence microscopy

Notes: GFP, green-fluorescent protein; SUMO, small ubiquitin-related modifier.

Figure 6. The results from RT-qPCR and Western blotting demonstrate that SUMO2 gene silencing decreases the levels of SUMO2, p53, p21, MDM2, GADD45α, MMP-2, and HIF-1α expressions and p53 phosphorylation level but increases the levels of CDK2/4 and CyclinB1 expression

Notes: (A) mRNA level of SUMO2 in the six groups. (B) mRNA levels of SUMO2/3, p53, p21, MDM2, CDK2/4, CyclinB1, MMP-2, and HIF-1α in the sham, model, NC and shRNA group. (C) Gray values of SUMO2/3, p53, p21, GADD45α, MDM2, CDK2/4, CyclinB1, MMP-2, and HIF-1α. (D) Protein levels of SUMO2/3, p53, p21, GADD45α, MDM2, CDK2/4, CyclinB1, MMP-2, and HIF-1α in the sham, model, NC and shRNA groups; *P<0.05, compared with the sham group; ^#P<0.05, compared with the model and NC groups; CDK2/4, cyclin-dependent kinase 2/4; GADD45α, growth arrest and DNA damage inducible 45α; HIF-1α, hypoxia-inducible factor-1α; MDM2, mouse double minute 2; MMP-2, matrix metalloproteinase-2; NC, negative control; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SUMO2/3, small ubiquitin-related modifier 2/3.

Figure 7. The MTT assay shows that SUMO2 gene silencing enhances proliferation of NPCs

Notes: *P<0.05, compared with the sham group; ^#P<0.05, compared with the model and NC groups; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide; NC, negative control; OD, optical density.

Figure 8. Flow cytometry analysis of PI staining shows that SUMO2 gene silencing promotes cell cycle entry of NPCs

Notes: (A) Cell cycle entry of NPCs in the sham, model, NC, and shRNA groups. (B) Percentage chart of cell cycle of NPCs in the sham, model, NC, and shRNA groups; *P<0.05, compared with the sham group; ^#P<0.05, compared with the model and NC groups; NC, negative control; NPC, nucleus pulposus cell; PI, propidium iodide.

Figure 9. Flow cytometry analysis of Annexin V-APC staining shows that SUMO2 gene silencing inhibits apoptosis

Notes: (A) Apoptosis of NPCs in the sham, model, NC, and shRNA groups. (B) Apoptotic rate of NPCs in the sham, model, NC, and shRNA groups; *P<0.05, compared with the sham group; ^#P<0.05, compared with the model and NC groups; Annexin V-APC, Annexin V- adenomatous polyposis coli; NC, negative control; NPC, nucleus pulposus cell.

Figure 10. The SA-β-gal staining revealed that SUMO2 gene silencing suppresses senescence of NPCs

Notes: (A) SA-β-gal staining observation of NPCs in the sham, model, NC, and shRNA groups (×400). (B) SA-β-gal staining positive rate of NPCs in the sham, model, NC, and shRNA groups; *P<0.05, compared with the sham group; #P<0.05, compared with the model and NC groups; NC, negative control; NPC, nucleus pulposus cell; SA-β-gal, senescence-associated β-galactosidase.