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. 2021 Jun 30;19(1):194.
doi: 10.1186/s12951-021-00940-2.

Mesenchymal stem cell-derived extracellular vesicles prevent the development of osteoarthritis via the circHIPK3/miR-124-3p/MYH9 axis

Shenglong Li #  1   2 , Jie Liu #  3 , Siyu Liu  1 , Weijie Jiao  1 , Xiaohong Wang  4   5
Affiliations

Mesenchymal stem cell-derived extracellular vesicles prevent the development of osteoarthritis via the circHIPK3/miR-124-3p/MYH9 axis

Shenglong Li et al. J Nanobiotechnology. .

Abstract

Background: Extracellular vesicles (EVs) secreted by mesenchymal stem cells (MSCs) may play a vital role in a variety of biological processes, including cartilage regeneration. However, few studies reported their potential in the development of osteoarthritis (OA) previously. In this study, we explored the biological roles and underlying mechanism of MSCs-EVs in OA.

Results: Co-culture experiments revealed that MSCs-EVs could promote the expression of collagen type II alpha 1 chain (COL2A1), SRY-box transcription factor 9 (SOX9) and Aggrecan while negatively regulate the expression of chondrocyte hypertrophy markers matrix metallopeptidase 13 (MMP-13) and RUNX family transcription factor 2 (Runx2) in mouse chondrocytes in the OA model. Besides, the results of cell experiments indicated that MSCs-EVs could notably weaken the suppression of chondrocyte proliferation, migration and the promotion of chondrocyte apoptosis via interleukin1β (IL-1β) induction. In addition, MSCs-circHIPK3-EVs (EVs derived from MSCs overexpressing circHIPK3) considerably improved IL-1β-induced chondrocyte injury. Mechanistically, we elucidated that circHIPK3 could directly bind to miR-124-3p and subsequently elevate the expression of the target gene MYH9.

Conclusion: The findings in our study demonstrated that EVs-circHIPK3 participated in MSCs-EVs-mediated chondrocyte proliferation and migration induction and in chondrocyte apoptosis inhibition via the miR-124-3p/MYH9 axis. This offers a promising novel cell-free therapy for treating OA.

Keywords: Circular RNA HIPK3 (circHIPK3); Extracellular vesicles; MYH9; Mesenchymal stem cells (MSCs); MiR-124-3p; Osteoarthritis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Isolation and characterisation of MSC-derived EVs. A After the culture of mesenchymal stem cells (MSCs), microscopic observation showed a typical spindle shape of cells. B Extracted MSCs had osteogenic, adipogenic and chondrogenic potentials by Alizarin Red, Oil Red O and Alcian Blue staining. C MSC positive markers (CD73, CD90 and CD105) and negative markers (HLA-DR, CD11b, CD19, CD34 and CD45) were analyzed by flow cytometry. D The morphology of EVs was observed under a transmission electron microscope. E The particle size distribution of MSC derived EVs was measured by nanometer size analyzer. F Zeta potential measurements of the surface charge of MSCs-EVs (mV). G EVs markers (CD9, CD63, CD81 and GM130) were analyzed by Western blot
Fig. 2
Fig. 2
Influence of EVs-circHIPK3 on the proliferation, migration and apoptosis of chondrocytes. A With different doses of MSCs-EVs (50 μg, 100 μg, 200 μg, 300 μg and 400 μg), qRT-PCR experiment was performed to detect the expression of circHIPK3 post co-culture with chondrocytes. With MSCs-circHIPK3-EVs or a vector (PBS) as the control, B EdU experiment was conducted to detect the proliferation after co-culture with chondrocytes. With a vector (PBS) as the control, C–D Transwell experiment and cell wound scratch assay were performed for the detection of cell migration. With a vector (PBS) as the control, E Flow cytometry was utilized for the detection of cell apoptosis post co-culture with chondrocytes. *P < 0.05, **P < 0.01
Fig. 3
Fig. 3
MSCs-circHIPK3-EVs up-regulated COL2A1, Aggrecan, and SOX9 and decreased MMP-13 and Runx2 expression. A After the treatment of OA mouse derived chondrocytes with MSCs-circHIPK3-EVs, the expression changes in these key cartilage genes (COL2A1, Sox9, Runx2, Aggrecan and MMP-13) by qRT-PCR. B After OA mouse derived chondrocytes were treated with MSCs-circHIPK3-EVs, Western blot was performed to detect the expression change in these key cartilage genes (Sox9, COL2A1, Aggrecan, Runx2 and MMP-13). *P < 0.05
Fig. 4
Fig. 4
CircHIPK3 promoted MYH9 expression by binding to miR-124-3p. A Subcellular localization of circHIPK3 in chondrocytes was detected by nucleoplasmic separation experiment. B Binding sites between circHIPK3 and miR-124-3p were predicted by Bioinformatics website, and circHIPK3 wild plasmids (circHIPK3 wt) and circHIPK3 mutant plasmids (circHIPK3 mut) were constructed. C The results of dual luciferase reporter gene revealed that miR-124-3p bound to circHIPK3. D Bioinformatics website was searched to predict target genes that might bind to miR-124-3p (microT, PITA, PicTar, miRanda, Targetscan and miRmap). E Bioinformatics website was searched to predict binding sites of MYH9 to miR-124-3p, and construct MYH9 wild plasmids (MYH9 wt) and circHIPK3 mutant plasmids (MYH9 mut). F MiR-124-3p bound to MYH9 based on the dual luciferase reporter gene. G MRNA expression of MYH9 was detected by qRT-PCR after upregulation or suppression of miR-124-3p in chondrocytes. H Protein expression of MYH9 was detected by Western blot post upregulation or suppress of miR-124-3p in chondrocytes. *P < 0.05, **P < 0.01
Fig. 5
Fig. 5
EVs-circHIPK3 derived from MSCs attenuated IL-1β induced chondrocyte injury. A Overexpressed circHIPK3 was observed in MSC EVs based on qRT-PCR. B With chondrocytes treated with IL-1β and MSCs-circHIPK3-EVs, the expression of miR-124-3p and MYH9 was detected by qRT-PCR. C MYH9 expression was detected through Western blot post treating chondrocytes with IL-1β and MSCs-circHIPK3-EVs. D With chondrocytes treated with IL-1β and MSCs-circHIPK3-EVs, the expression change in cartilage genes (Aggrecan, COL2A1, Runx2, Sox9 and MMP-13) was detected by qRT-PCR. E The expression change in key cartilage genes (Runx2, MMP-13, COL2A1, Aggrecan, and Sox9) was detected through Western blot after chondrocytes were treated with IL-1β and MSCs-circHIPK3-EVs. F Cell proliferation was detected through EdU experiment with chondrocytes treated with IL-1β and MSCs-circHIPK3-EVs. G Transwell experiment was conducted for the detection of cell proliferation after treating chondrocytes with IL-1β and MSCs-circHIPK3-EVs. H A flow cytometer was employed for the detection of cell apoptosis after chondrocytes were treated with IL-1β and MSCs-circHIPK3-EVs. *P < 0.05, **P < 0.01
Fig. 6
Fig. 6
MiR-124-3p overexpression reversed MSCs-circHIPK3-EVs mediated attenuation of chondrocyte injury. A The results of qRT-PCR demonstrated that miR-124-3p expression was considerately upregulated in chondrocytes transfected with miR-124-3p mimic. B–F MSCs-circHIPK3-EVs was used to co-culture chondrocytes transfected with miR-124-3p mimic, and the chondrocytes were pre-stimulated with IL-1β (10 ng/ml, 24 h). The expression of miR-124-3p and MYH9 was then detected through qRT-PCR (B); MYH9 expression was detected via Western blot (C); EdU experiment (D), Transwell experiment (E) and flow cytometry (F) were performed for the detection of cell proliferation, migration and apoptosis. *P < 0.05, **P < 0.01
Fig. 7
Fig. 7
MYH9 knockdown reversed circHIPK3-EVs mediated attenuation of chondrocyte injury. A The results of qRT-PCR proved that MYH9 expression was repressed in sh-MYH9 transfected chondrocytes. B–F The co-culture was performed by using MSCs-circHIPK3-EVs and sh-MYH9 transfected chondrocytes and the prestimulation was conducted with IL-1β (10 ng/ml, 24 h). MYH9 mRNA expression was then detected by qRT-PCR (B); MYH9 protein expression was detected by Western blot (C); EdU experiment (D), Transwell experiment (E) and flow cytometry (F) were performed for the detection of cell proliferation, migration and apoptosis. *P < 0.05, **P < 0.01
Fig. 8
Fig. 8
sh-MYH9 decreased COL2A1, Aggrecan and Sox9,  increased MMP-13 and Runx2. A After the treatment of OA mouse derived chondrocytes with sh-MYH9, MSCs-EVs and MSCs-circHIPK3-EVs, the expression changes in these key cartilage genes (COL2A1, Sox9, Runx2, Aggrecan and MMP-13) by qRT-PCR. B After OA mouse derived chondrocytes were treated with sh-MYH9, MSCs-EVs and MSCs-circHIPK3-EVs, Western blot was performed to detect the expression change in these key cartilage genes (Sox9, COL2A1, Aggrecan, Runx2 and MMP-13). *P < 0.05, **P < 0.01
Fig. 9
Fig. 9
MSCs-circHIPK3-EVs inhibited cartilage degradation. We established the OA model, and divided them into five groups: OA model, Normal, OA + MSCs-EVs, OA + MSCs-circHIPK3-EVs and OA + circHIPK3. A HE staining and Mankin score assessment. B the number of PCNA-positive cells using immunohistochemistry. C–D The mRNA and protein levels of Sox9, COL2A1, Aggrecan, Runx2 and MMP-13 were detected by qRT-PCR and western blot
Fig. 10
Fig. 10
A schematic diagram of MSCs-circHIPK3-EVs in OA
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