MiR-199b-5p inhibits osteogenic differentiation in ligamentum flavum cells by targeting JAG1 and modulating the Notch signalling pathway

Abstract

Ossification of the ligamentum flavum (OLF) is a pathology almost only reported in East Asian countries. The leading cause of OLF is thoracic spinal canal stenosis and myelopathy. In this study, the role of miR-199b-5p and jagged 1 (JAG1) in primary ligamentum flavum cell osteogenesis was examined. MiR-199b-5p was found to be down-regulated during osteogenic differentiation in ligamentum flavum cells, while miR-199b-5p overexpression inhibited osteogenic differentiation. In addition, JAG1 was found to be up-regulated during osteogenic differentiation in ligamentum flavum cells, while JAG1 knockdown via RNA interference caused an inhibition of Notch signalling and osteogenic differentiation. Moreover, target prediction analysis and dual luciferase reporter assays supported the notion that JAG1 was a direct target of miR-199b-5p, with miR-199b-5p found to down-regulate both JAG1 and Notch. Further, JAG1 knockdown was demonstrated to block the effect of miR-199b-5p inhibition. These findings imply that miR-199b-5p performs an inhibitory role in osteogenic differentiation in ligamentum flavum cells by potentially targeting JAG1 and influencing the Notch signalling pathway.

Keywords: JAG1; Notch signalling pathway; miR-199b-5p; ossification of the ligamentum flavum; osteogenic differentiation.

Figure 1

Identification and characterization of OLF patient ligamentum flavum cells. (A) Representative OLF patient ligamentum flavum sample obtained via en bloc resection of the lamina and ligamentum flavum. General sample (a), computed tomography (b) and magnetic resonance imaging (c), including sagittal and cross‐plane scans of the ossification area at the corresponding position. (B) Representative morphology of P0 and P1 OLF patient ligamentum flavum cells. (C) Immunocytochemical detection of vimentin in OLF cells; scale bar represents 200 μm. (D) qRT‐PCR analysis of five osteogenic markers in OLF patient ligamentum flavum cells; *P < 0.05 C compared with day 0. (E) ALP activity and Alizarin red staining of OLF patient ligamentum flavum cells; scale bar represents 200 μm.

Figure 2

MiR‐199b‐5p inhibits osteogenesis in ligamentum flavum cells. (A) Endogenous miR‐199b‐5p expression levels were measured via qRT‐PCR at different time points during osteogenic differentiation in ligamentum flavum cells; *P < 0.05 compared with day 0. (B) MiR‐199b‐5p expression assessed via qRT‐PCR in ligamentum flavum cells transfected with miRNA mimics; *P < 0.05 compared with miR‐NC group. (C) qRT‐PCR analysis of osteogenic marker genes after miR‐199b‐5p overexpression at day 14; *P < 0.05 compared with miR‐NC group. (D) Western blot analysis of osteogenic marker protein expression after miR‐199b‐5p overexpression at day 14. (E) ALP staining and Alizarin Red staining at day 14 showed inhibited ALP activity and calcification after miR‐199b‐5p overexpression when compared with miR‐NC; scale bar represents 200 μm.

Figure 3

JAG1 knockdown down‐regulates Notch signalling and inhibits osteogenic differentiation in ligamentum flavum cells. (A, B) JAG1 mRNA and protein expression levels examined via qRT‐PCR and Western blot at different time points during osteogenic differentiation in ligamentum flavum cells; *P < 0.05 compared with day 0. (C, D) JAG1 mRNA and protein expression level examined via qRT‐PCR and Western blot following siJAG1 transfection in ligamentum flavum cells; *P < 0.05 Compared with si‐NC group. (E) Notch1 and Notch2 protein expression levels examined via Western blot in siJAG1 transfected ligamentum flavum cells. (F, G) Osteogenic marker mRNA and protein expression examined via qRT‐PCR and Western blot at day 14 after JAG1 knockdown; *P < 0.05 Compared with si‐NC group. (H) ALP staining and Alizarin Red staining at day 14 showed inhibited ALP activity and calcification following JAG1 knockdown when compared with miR‐NC group; scale bar represents 200 μm.

Figure 4

MiR‐199b‐5p directly targets JAG1 and affects Notch signalling. (A) The miR‐199b‐5p binding site in the JAG1 3′‐UTR is highly conserved among vertebrates. (B) A schematic diagram indicating the wild‐type and mutated‐type miR‐199b‐5p binding sites in the JAG1 3′‐UTR. (C) A wild‐type (WT) JAG1 3′‐UTR or a mutant (MUT) JAG1 3′‐UTR reporter plasmid was co‐transfected into HEK293T cells with either miR‐199b‐5p or mi‐NC and fluorescence was quantified; *P < 0.05 compared with miR‐NC group. (D, E) JAG1 mRNA and protein expression level were examined via qRT‐PCR or Western blot following miR‐199b‐5p transfection in ligamentum flavum cells. (F) Notch1 and Notch2 protein expression levels were examined via Western blot in miR‐199b‐5p transfected ligamentum flavum cells. (G) MiR‐199b‐5p binding site in the 3′‐UTRs of TGFB2 and SOX6 are highly conserved among vertebrates. (H) A TGFB2 3′‐UTR or a SOX6 3′‐UTR reporter plasmid was co‐transfected in HEK293T cells with either miR‐199b‐5p or mi‐NC and fluorescence was quantified; *P < 0.05 compared with miR‐NC group.

Figure 5

JAG1 knockdown could block the effect of miR‐199b‐5p inhibition. (A) MiR‐199b‐5p expression assessed via qRT‐PCR in ligamentum flavum cells transfected with miRNA inhibitor; *P < 0.05 compared with miR‐NC‐I group. (B) JAG1 mRNA expression levels examined via qRT‐PCR following miR‐199b‐5p inhibitor and siJAG1 transfection in ligamentum flavum cells; #P < 0.05 compared with ‘miR‐199b‐5p‐I + si‐NC’ group. (C) JAG1, Notch1 and Notch2 protein expression levels examined via Western blot following miR‐199b‐5p inhibitor and siJAG1 transfection in ligamentum flavum cells. (D, E) Osteogenic marker mRNA and protein expression examined via qRT‐PCR and Western blot at day 14 after miR‐199b‐5p inhibitor and siJAG1 transfection; *P < 0.05 Compared with ‘miR‐NC‐I’ group. #P < 0.05 compared with ‘miR‐199b‐5p‐I + siNC’ group. (F) ALP staining and Alizarin Red staining at day 14 following miR‐199b‐5p inhibitor and siJAG1 transfection; scale bar represents 200 μm.

Figure 6

GSK‐3β/β‐catenin regulates JAG1 and was slightly affected by miR‐199b‐5p in ligamentum flavum cells. (A, B) GSK‐3β and β‐catenin mRNA and protein expression levels examined via qRT‐PCR and Western blot at different time points during osteogenic differentiation in ligamentum flavum cells; *P < 0.05 compared with day 0. (C, D) GSK‐3β and β‐catenin protein expression level examined via Western blot following miR‐199b‐5p mimic and inhibitor transfection in ligamentum flavum cells. (E, F) GSK‐3β, β‐catenin and JAG1 mRNA expression levels examined via qRT‐PCR following GSK‐3β and β‐catenin knockdown in ligamentum flavum cells. *P < 0.05 Compared with si‐NC group. (G, H) GSK‐3β, β‐catenin, JAG1 Notch1 and Notch2 protein expression levels examined via Western blot following GSK‐3β and β‐catenin knockdown in ligamentum flavum cells. (I) The regulatory mechanism of osteogenic differentiation in ligamentum flavum cells in this study. ‘⊥’: inhibit; ‘↓’: promote; ‘×’: weak effect.