Downregulation of microRNA-125a is involved in intervertebral disc degeneration by targeting pro-apoptotic Bcl-2 antagonist killer 1

Abstract

Objectives: To investigate the role of the microRNA-125a (miR-125a) and BAK1 in intervertebral disc degeneration (IDD).

Materials and methods: Degenerative lumbar nucleus pulposus (NP) tissues were obtained from 193 patients who underwent resection, and normal controls consisted of normal NP tissues from 32 patients with traumatic lumbar fracture in our hospital. All patients were graded according to the Pfirrmann criteria. QRT-PCR was used to detect the expression of miR-125a and BAK1, and apoptosis of NP tissues detected by TUNEL staining. After isolation of non- degenerative and degenerative nucleus pulposus cells (NPCs), the targeting relationship between miR-125a and BAK1 was verified by dual luciferase reporter gene assay. Flow cytometry was determined the NPCs apoptosis, and Western blot to measure the expressions of BAK1, Caspase-3, Bax and Bcl-2.

Results: MiR-125a was reduced while BAK1 was elevated in IDD patients with the increase of Pfirrmann grade. Besides, miRNA-125a was negatively correlated to the NPCs apoptosis, while BAK1 mRNA was positively correlated to cell apoptosis. Additionally, BAK1 is the target gene of miRNA-125a. When transfection of miR-125a mimics in vitro, the apoptosis of NPCs were inhibited, with the down-regulation of BAK1, Caspase-3, and Bax, and the upregulation of Bcl-2. In addition, siBAK1 can reverse the pro-apoptosis function of miR-125a inhibitors in NPCs.

Conclusion: miRNA-125a may regulate the apoptosis status of the NPCs by inhibiting the expression of its target gene BAK1, which provided a potential strategy for further development of IDD therapies.

Keywords: Apoptosis; BAK1 protein; Intervertebral disc - degeneration; MicroRNA; Mir-125a; Nucleus pulposus.

Figure 1

Expression levels of microRNA-125a and pro-apoptotic Bcl-2 antagonist killer 1 in patients with intervertebral disc degeneration and normal controls

Figure 2

Cell apoptosis of nucleus pulposus tissues from patients with intervertebral disc degeneration and normal controls detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining A: The cell apoptosis of NP tissues from patients with IDD and normal controls detected by TUNEL staining. Black arrows indicate TUNEL-positive (apoptotic) cells, red arrows indicate healthy cells. B: Comparison of the apoptotic index (AI) in NP tissues from IDD patients with different Pfirrmann grading and normal controls. Different letters indicate statistical significance (P<0.05), whereas the same letter means no significantly different (P>0.05). C: The correlation analysis of miRNA-125a expression and cell apoptosis. D: The correlation analysis of the mRNA level of BAK1 and cell apoptosis

Figure 3

Pro-apoptotic Bcl-2 antagonist killer 1 is the target gene for microRNA-125a A: miR-125a targeted to the 3’-UTR of the BAK1 mRNA predicted by microRNA.org. B: Results of luciferase reporter gene assay; *, P<0.05 compared with NC group

Figure 4

Effect of microRNA-125a on the apoptosis rate of nucleus pulposus cells A: The apoptosis rate of NPCs in each group was detected by flow cytometry. B: The relative protein expressions of BAK1, Caspase-3, Bax and Bcl-2 in each group were detected by Western blot. 1, Normal group, 2, Mock group, 3, NC group, 4, miR-125a mimics group, 5, miR-125a inhibitors group, 6, miR-125a inhibitors + siBAK1 group; Different letters indicate statistical significance (P<0.05), whereas the same letter means no significantly different (P>0.05)