miR-31 from Mesenchymal Stem Cell-Derived Extracellular Vesicles Alleviates Intervertebral Disc Degeneration by Inhibiting NFAT5 and Upregulating the Wnt/ β-Catenin Pathway

Abstract

In this study, we explored the regulatory mechanism of intervertebral disc degeneration (IDD) that involves miR-31 shuttled by bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) and its downstream signaling molecules. Nucleus pulposus cells (NPCs) were isolated and treated with TNF-α to simulate IDD in vitro. The TNF-α-exposed NPCs were then cocultured with hBMSCs or hBMSC-EVs in vitro to detect the effects of hBMSC-EVs on NPC viability, apoptosis, and ECM degradation. Binding between miR-31 and NFAT5 was determined. A mouse model of IDD was prepared by vertebral disc puncture and injected with EVs from hBMSCs with miR-31 knockdown to discern the function of miR-31 in vivo. The results demonstrated that hBMSC-EVs delivered miR-31 into NPCs. hBMSC-EVs enhanced NPC proliferation and suppressed cell apoptosis and ECM degradation, which was associated with the transfer of miR-31 into NPCs. In NPCs, miR-31 bound to the 3'UTR of NFAT5 and inhibited NFAT5 expression, leading to activation of the Wnt/β-catenin pathway and thus promoting NPC proliferation and reducing cell apoptosis and ECM degradation. In addition, miR-31 in hBMSC-EVs alleviated the IDD in mouse models. Taken together, miR-31 in hBMSC-EVs can alleviate IDD by targeting NFAT5 and activating the Wnt/β-catenin pathway.

Figure 1

Characterization of hBMSC-EVs: (a) transmission electronic microscopic image of EVs, scale bar = 200 nm; (b) size of EVs determined by NanoSight; (c) protein expression of ALIX, CD63, TSG101, and GRP94 in the isolated hBMSC-EVs determined by Western blot.

Figure 2

miR-31 expression in the isolated hBMSC-EVs: (a) miR-31 expression in the isolated hBMSC-EVs after TNF-α treatment determined by RT-qPCR; (b) miR-31 expression in the isolated hBMSC-EVs after RNAse or proteinase K treatment determined by RT-qPCR. ^∗p < 0.05 vs. hBMSC-EVs or proteinase K+RNAse. Cell experiments were repeated three times.

Figure 3

hBMSC-EVs deliver miR-31 to NPCs: (a) microscopic images of NPCs. Scale bar = 50 μm. (b) Immunofluorescence detection of Col II, KRT-19, HIF-1α, SOX-9, and ACAN proteins in the NPCs. Scale bar = 25 μm. (c) Diagram showing coculture of hBMSCs or hBMSC-EVs with NPCs. (d) Confocal microscopy was used to observe the uptake of PKH-67 labeled EVs by NPCs after coculture for 24 h. Scale bar = 25 μm. (e) Representative fluorescence micrographs in NPCs cocultured with Cy3-miR-31-BMSCs (red). Scale bar = 25 μm. (f) miR-31 expression in NPCs cocultured with BMSC-EVs or TNF-α+BMSC-EVs determined by RT-qPCR. ^∗p < 0.05. Cell experiments were repeated three times.

Figure 4

hBMSC-EVs inhibit NPC apoptosis and ECM degradation and promote cell proliferation in NPCs. (a) miR-31 expression in TNF-α-treated NPCs cocultured with BMSCs or further treated with GW4869 determined by qRT-PCR. (b) Proliferation of TNF-α-treated NPCs cocultured with BMSCs or further treated with GW4869 determined by CCK-8 assay. (c) Apoptosis of TNF-α-treated NPCs cocultured with BMSCs or further treated with GW4869 determined by flow cytometry. (d) Protein expression of apoptosis-related genes cleaved caspase-3, Bax, and Bcl-2 in TNF-α-treated NPCs cocultured with BMSCs or further treated with GW4869 determined by Western blot. (e) Protein expression of ECM-related genes in TNF-α-treated NPCs cocultured with BMSCs or further treated with GW4869 determined by Western blot. ^∗p < 0.05 vs. PBS; ^#p < 0.05 vs. TNF-α; ^&p < 0.05 vs. TNF-α+hBMSCs. Cell experiments were repeated three times.

Figure 5

miR-31 in hBMSC-EVs augments NPC proliferation and inhibits cell apoptosis and ECM degradation in NPCs. (a) miR-31 expression in EVs from hBMSCs transduced with lentivirus carrying anti-miR-31 (left) and in TNF-α-treated NPCs cocultured with BMSCs-EVs+anti-miR-31 (right) determined by RT-qPCR. (b) Proliferation of TNF-α-treated NPCs cocultured with BMSCs-EVs+anti-miR-31 determined by CCK-8 assay. (c) Apoptosis of TNF-α-treated NPCs cocultured with BMSCs-EVs+anti-miR-31 determined by flow cytometry. (d) Protein expression of apoptosis-related genes cleaved caspase-3, Bax, and Bcl-2 in TNF-α-treated NPCs cocultured with BMSCs-EVs+anti-miR-31 determined by Western blot. (e) Protein expression of ECM-related genes in TNF-α-treated NPCs cocultured with BMSCs-EVs+anti-miR-31 determined by Western blot. ^∗p < 0.05. Cell experiments were repeated three times.

Figure 6

miR-31 targets NFAT5 and activates the Wnt/β-catenin pathway in NPCs: (a) a PPI network of the genes related to the key downstream genes constructed through GeneMANIA; (b) binding of miR-31 to NFAT5 in 293T cells determined by dual luciferase reporter assay; (c) expression of miR-31 in NPCs after transfection with miR-31 mimic or miR-31 inhibitor determined by RT-qPCR; (d) expression of NFAT5 in NPCs after transfection with miR-31 mimic or miR-31 inhibitor determined by RT-qPCR; (e) expression of NFAT5 and β-catenin in NPCs after transfection with miR-31 mimic or miR-31 inhibitor determined by Western blot; (f) the transcription activity of TCF/LEF in NPCs after transfection with miR-31 mimic or miR-31 inhibitor determined by TOPFlash; (g) protein expression of NFAT5 and β-catenin in TNF-α-treated NPCs determined by Western blot; (h) the transcription activity of TCF/LEF in TNF-α-treated NPCs determined by TOPFlash; (i) expression of NFAT5 and β-catenin in TNF-α-treated NPCs cocultured with BMSCs-EVs or BMSCs-EVs+anti-miR-31 determined by Western blot; (j) the transcription activity of TCF/LEF in TNF-α-treated NPCs cocultured with BMSCs-EVs or BMSCs-EVs+anti-miR-31 determined by TOPFlash. ^∗p < 0.05. Cell experiments were repeated three times.

Figure 7

miR-31 in hBMSC-EVs targets NFAT5 to inhibit NPC apoptosis and ECM degradation in NPCs: (a) protein expression of NFAT5 and β-catenin in TNF-α-exposed NPCs treated with oe-NFAT5 or combined with BMSCs-EVs determined by Western blot; (b) the transcription activity of TCF/LEF in TNF-α-exposed NPCs treated with oe-NFAT5 or combined with BMSCs-EVs determined by TOPFlash; (c) proliferation of TNF-α-exposed NPCs treated with oe-NFAT5 or combined with BMSCs-EVs determined by CCK-8 assay; (d) apoptosis of TNF-α-exposed NPCs treated with oe-NFAT5 or combined with BMSCs-EVs determined by flow cytometry; (e) protein expression of apoptosis-related genes cleaved caspase-3, Bax, and Bcl-2 in TNF-α-exposed NPCs treated with oe-NFAT5 or combined with BMSCs-EVs determined by Western blot; (f) protein expression of ECM-related genes in TNF-α-exposed NPCs treated with oe-NFAT5 or combined with BMSCs-EVs determined by Western blot. ^∗p < 0.05. Cell experiments were repeated three times.

Figure 8

miR-31 in hBMSC-EVs relieves IDD in mice: (a) representative fluorescence micrographs showing PKH26-labeled EVs in IVD tissues of IDD mice treated with BMSCs-EVs or combined with anti-miR-31. Scale bar = 50 μm. (b) miR-31 expression in IVD tissues; (c) serum levels of inflammatory factors in IDD mice treated with BMSCs-EVs or combined with anti-miR-31 determined by ELISA; (d) protein expression of NFAT5 and β-catenin in IVD tissues of IDD mice treated with BMSCs-EVs or combined with anti-miR-31 determined by Western blot; (e) protein expression of cleaved caspase-3, Bax, and Bcl-2 in IVD tissues of IDD mice treated with BMSCs-EVs or combined with anti-miR-31 determined by Western blot; (f) protein expression of ECM-related genes in IVD tissues of IDD mice treated with BMSCs-EVs or combined with anti-miR-31 determined by Western blot; (g) cell apoptosis determined by TUNEL assay in IVD tissues of IDD mice treated with BMSCs-EVs or combined with anti-miR-31 9 weeks after puncture. Blue fluorescence (DAPI) indicates the total number of cells; green fluorescence (FITC) indicates TUNEL-positive cells. Scale bar = 50 μm. (h) HE staining of IVD tissues of IDD mice treated with BMSCs-EVs or combined with anti-miR-31. Scale bar = 500 μm. ^∗p < 0.05.n = 8.

Figure 9

Schematic diagram of the mechanism by which miR-31 in hBMSC-EVs affects IDD. hBMSC-EVs transferred miR-31 to NPCs where miR-31 targets NFAT5 and reduces the expression of NFAT5 and activates the Wnt/β-catenin pathway, thus promoting the proliferation of NPCs, inhibiting their apoptosis, and reducing the release of cellular inflammatory factors, ultimately alleviating IDD in mice.