MSC-derived exosomes promote proliferation and inhibit apoptosis of chondrocytes via lncRNA-KLF3-AS1/miR-206/GIT1 axis in osteoarthritis

Abstract

Background: Exosomes secreted by human mesenchymal stem cells (hMSCs) have been shown to promote cartilage regeneration. This study aimed to explore whether exosomal lncRNA-KLF3-AS1 derived from hMSCs can promote chondrocyte proliferation via miR-206/GIT1 axis in osteoarthritis (OA).

Methods: hMSCs and MSC-derived exosomes (MSC-exo) were prepared for morphological observation and identification by transmission electron microscopy (TEM) and flow cytometry. IL-1β-induced OA chondrocytes and collagenase-induced mouse OA model were established for the further experiments. Luciferase activity assay was performed to test whether miR-206 could bind to KLF3-AS1 or GIT1. Cell proliferation and apoptosis were evaluated by CCK-8 assay and flow cytometry, respectively.

Results: MSC-Exos increased chondrogenic genes Col2a1 (type II collagen alpha 1) and aggrecan, decreased hondrocyte hypertrophy markers MMP-13 (matrix metalloproteinase-13) and Runx2 (runt-related transcription factor 2) in chondrocytes isolated from OA model mice. Furthermore, MSC-Exos attenuated IL-1β-induced chondrocyte proliferation inhibition and apoptosis induction. Moreover, MSC^KLF3-AS1-Exos (exosomes derived from KLF3-AS1-overexpressing-MSCs) ameliorated IL-1β-induced chondrocyte injury. Results also demonstrated that KLF3-AS1 acted as a competitive endogenous RNA (ceRNA) by sponging miR-206 to facilitate GIT1 expression. In addition, miR-206 overexpression and GIT1 knockdown reversed MSC^KLF3-AS1-Exos-mediated attenuation of chondrocyte injury.

Conclusion: Exosomal KLF3-AS1 derived from MSCs involved in MSC-Exos-mediated chondrocyte proliferation induction and chondrocyte apoptosis inhibition via miR-206/GIT1 axis. Abbreviation: G-protein-coupled receptor kinase interacting protein-1 (GIT1).

Keywords: GIT1; Mesenchymal stem cells; exosomes; lncRNA-KLF3-AS1; miR-206; osteoarthritis.

Figure 1.

Morphological observation and identification of MSCs and MSC-Exo. (a) Phase-contrast microscopy of human bone marrow-derived MSCs in the third passage (scale bar: 100 μm). (b) Analysis of the immunophenotype of mouse bone marrow-derived MSCs by flow cytometry. MSCs were positive for CD29, CD106, and CD44, while negative for CD34, CD45. (c) Morphology of MSC-derived exosomes (MSC-Exos) under an Transmission electron microscope. Scale bar, 500 nm. (d) MSC-Exos were positive for TSG101 and CD63 as indicated by flow cytometry analysis.

Figure 2.

MSC-Exos increased Col2a1 and aggrecan, decreased MMP-13 and Runx2. The chondrocytes were divided into four groups: normal group (chondrocytes isolated from normal mice), OA group (chondrocytes isolated from OA model mice), MSC-Exos group (10 μg/ml MSC-Exos were added into OA mice-derived chondrocytes) and PBS group (as control of MSC-Exos). The relative mRNA (a) and protein (b) levels of Col2a1, aggrecan, MMP-13, and Runx2 were examined by qRT-PCR and Western blot, respectively. GAPDH served as the loading control. *p < 0.05 vs. the normal group; ^#p < 0.05 vs. the PBS group.

Figure 3.

KLF3-AS1 acts as a ceRNA by sponging miR-206 to facilitate GIT1. (a) Results of luciferase activity assay verified the direct binding between KLF3-AS1 and miR-206. ^$p < 0.05 vs. the mimic NC+ KLF3-AS1 WT group. (b) Results of luciferase activity assay showed that 3ʹUTR of GIT1 is directly targeted by miR-206. ^$p < 0.05 vs. the mimic NC+ GIT1 WT group. (c-d) KLF3-AS1 overexpression significantly inhibited miR-206 expression, whereas induced GIT1 expression at both mRNA (c) and protein (d) levels. In contrast, KLF3-AS1 knockdown exerted the opposite effects. ^@p < 0.05 vs. the vector group; ^%p < 0.05 vs. the scramble group.

Figure 4.

Exosomal KLF3-AS1 derived from MSCs attenuated IL-1β-induced chondrocyte injury. (a) PCR revealed that KLF3-AS1 expression can be detected in both MSCs and MSC-Exos. (b) qRT-PCR results confirmed the overexpression efficiency of KLF3-AS1 expression in MSCs. ^$p < 0.05 vs. the vector group. (c-d) Normal mouse chondrocytes were prestimulated by IL-1β (10 mg/ml, 24 h, normal medium as control) before being co-cultured with MSC^vector-Exos, MSC^KLF3-AS1-Exos, or equal volume of PBS as control for 24 h. Results of qRT-PCR and Western blot revealed that MSC^vector-Exos treatment significantly reversed IL-1β-mediated effects on expression of miR-206, GIT1 (c-d), Col2A1, aggrecan, MMP-13, Runx2 (e-f). (G-H) Results of CCK-8 assay and flow cytometry showed that MSC^vector-Exos treatment significantly reversed the IL-1β-mediated chondrocyte proliferation inhibition and apoptosis induction. MSC^KLF3-AS1-Exos treatment further enhanced the MSC^vector-Exos-mediated effects. *p < 0.05 vs. the control group; ^#p < 0.05 vs. the PBS group; ^$p < 0.05 vs. the MSC^vector-Exos group.

Figure 5.

miR-206 overexpression reversed MSC^KLF3-AS1-Exos-mediated attenuation of chondrocyte injury. (a) qRT-PCR results confirmed that miR-206 expression was significantly upregulated in miR-206 mimic-transfected chondrocytes. *p < 0.05 vs. the mimic NC group. The miR-206 mimic-transfected normal mouse chondrocytes were prestimulated by IL-1β (10 mg/ml, 24 h) before being co-cultured with MSC^vector-Exos or MSC^KLF3-AS1-Exos. (b-c) Results of qRT-PCR and Western blot revealed that miR-206 mimic significantly reversed MSC^KLF3-AS1-Exos-mediated decrease in miR-206 expression and increase in mRNA and protein expression of GIT1. (d-e) Results of CCK-8 assay and flow cytometry showed that miR-206 mimic significantly reversed MSC^KLF3-AS1-Exos-mediated cell proliferation induction and cell apoptosis inhibition. ^#p < 0.05 vs. the mimic NC + MSC^vector-Exos group; ^$p < 0.05 vs. the mimic NC + MSC^KLF3-AS1-Exos group.

Figure 6.

GIT1 knockdown reversed MSC^KLF3-AS1-Exos-mediated attenuation of chondrocyte injury. (a) qRT-PCR results confirmed that GIT1 mRNA expression was significantly downregulated in si-GIT1-transfected chondrocytes. *p < 0.05 vs. the scramble group. The si-GIT1-transfected normal mouse chondrocytes were prestimulated by IL-1β (10 mg/ml, 24 h) before being co-cultured with MSC^vector-Exos or MSC^KLF3-AS1-Exos. (b-c) Results of qRT-PCR and Western blot revealed that GIT1 knockdown significantly reversed MSC^KLF3-AS1-Exos-mediated increase in mRNA and protein expression of GIT1. (d-e) Results of CCK-8 assay and flow cytometry showed that GIT1 knockdown significantly reversed MSC^KLF3-AS1-Exos-mediated cell proliferation induction and cell apoptosis inhibition. ^#p < 0.05 vs. the scramble + MSC^vector-Exos group; ^$p < 0.05 vs. the scramble + MSC^KLF3-AS1-Exos group.