Hsa_circ_0001946 Ameliorates Mechanical Stress-induced Intervertebral Disk Degeneration Via Targeting miR-432-5p and SOX9

Abstract

Study design: Experimental analysis of circular RNA in intervertebral disk degeneration (IDD).

Objective: This study aimed to explore the roles of hsa_circ_0001946 (circ-CDR1as) in mechanical stress-induced nucleus pulposus cell injury in IDD.

Summary of background data: Mechanical stress is an important pathogenic factor for IDD. Excessive compression stress leads to nucleus pulposus (NP) cell apoptosis and extracellular matrix (ECM) degradation and accelerated IDD. Circ-CDR1as is associated with various degenerative conditions, but its role in IDD is not clear. Herein, we explored the roles and mechanisms of circ-CDR1as in IDD in vitro.

Materials and methods: An in vitro model of IDD was constructed by treating NP cells with 1.0 MPa compression stress. Quantitative real-time polymerase chain reaction assay was used for detecting the expression of circ-CDR1as and miR-432-5p. Immunofluorescent analysis was performed for MMP13 detection. Western blot assay was performed for detecting apoptosis and ECM-related protein expression. Flow cytometry analysis was used for cell apoptosis analysis. The dual-luciferase reporter was used to analyze the interaction between miR-432-5p and circ-CDR1as or SOX9. Differences in means between groups were evaluated using the Student t test or one-way analysis of variance.

Results: In compression-treated human NP cells, we found that circ-CDR1as was significantly downregulated. Functional experiments showed that circ-CDR1as overexpression reduced the compression-induced apoptosis and ECM degradation in NP cells. Further research indicated that circ-CDR1as could act as a molecular sponge for miR-432-5p, a miRNA that enhanced compression-induced damage of NP cells by inhibiting the expression of SOX9. The luciferase reporter experiments also showed that the mutual dialogue between circ-CDR1as and miR-432-5p regulated the expression of SOX9.

Conclusions: Circ-CDR1as binds to miR-432-5p and plays a protective role in mitigating compression-induced NP cell apoptosis and ECM degradation by targeting SOX9. Circ-CDR1as may provide a novel therapeutic target for the clinical management of IDD in the future.

Figure 1

Effects of circ-CDR1as on compression-induced NP cell apoptosis and extracellular matrix degradation. A and B, The quantitative real-time polymerase chain reaction analysis was used to determine the expression level of circ-CDR1as in human NP cells treated with circ-CDR1as siRNA (si) or circ-CDR1as OE vector. C, The western blot analysis was applied to detect the protein levels of aggrecan, collagen II, Bax, and Bcl-2 in human NP cells transfected with circ-CDR1as siRNA under compression treatment. GAPDH served as an internal control. D, The protein levels of aggrecan, collagen II, Bax, and Bcl-2 in human NP cells transfected with circ-CDR1as OE vector under compression treatment. GAPDH served as an internal control. Data were shown as means with error bars representing SD. *P<0.05, **P<0.01, ***P<0.001, n=3. circRNA indicates circular RNA, GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NP, nucleus pulposus; OE, overexpression.

Figure 2

Circ-CDR1as functioned in the human nucleus pulposus cells under compression via targeting miR-432-5p. The human nucleus pulposus cells under compression were pretreated with circ-CDR1as overexpression vector, or circ-CDR1as overexpression with NC mimics, or circ-CDR1as overexpression with miR-432-5p mimics. The MMP13 protein level in the nucleus pulposus cells from each group was determined by immunofluorescence staining, with relative intensity of fluorescence quantified. Original magnification: ×200. Data were presented as means with error bars representing SD. **P<0.01, ***P<0.001, n=3.

Figure 3

MiR-432-5p facilitated compression-induced NP cell apoptosis and extracellular matrix degradation. A and B, The human NP cells were treated with miR-432-5p inhibitor or miR-432-5p mimics, and the relative expression levels of miR-432-5p were validated by quantitative real-time polymerase chain reaction analysis. C and D, The protein levels of aggrecan, collagen II, Bax, and Bcl-2 in human NP cells treated with miR-432-5p inhibitor or miR-432-5p mimics under compression. GAPDH served as an internal control. Data were presented as means with error bars representing SD. **P<0.01, ***P<0.001, n=3. GAPDH indicates glyceraldehyde 3-phosphate dehydrogenase; NP, nucleus pulposus.

Figure 4

SOX9 was the direct target gene of miR-432-5p in human nucleus pulposus cells. The human nucleus pulposus cells under compression were pretreated with miR-432-5p inhibitor, or miR-432-5p inhibitor with si-NC, or miR-432-5p inhibitor with si-SOX9. The MMP13 protein level in the nucleus pulposus cells from each group was determined by immunofluorescence staining. Original magnification: ×200. Data were shown as means with error bars representing SD. ***P<0.001, n=3.

Figure 5

Circ-CDR1as mitigated the extracellular matrix degradation induced by compression via targeting miR-432-5p and SOX9. The human nucleus pulposus cells under compression were pretreated with circ-CDR1as overexpression vector, or circ-CDR1as overexpression with si-NC, or circ-CDR1as overexpression with si-SOX9. The protein expression level of MMP13 in the nucleus pulposus cells from each group was determined by immunofluorescence staining, with relative intensity of fluorescence quantified. Original magnification: ×200. Data were shown as means with error bars representing SD. **P<0.01, ***P<0.001, n=3.