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. 2024 Nov 6;19(1):729.
doi: 10.1186/s13018-024-05164-2.

METTL3 accelerates staphylococcal protein A (SpA)-induced osteomyelitis progression by regulating m6A methylation-modified miR-320a

Ding Gao  1 , Jian Shi  2 , Siyu Lu  2 , Junyi Li  2   3 , Kehan Lv  2   3 , Yongqing Xu  4 , Muguo Song  5   6
Affiliations

METTL3 accelerates staphylococcal protein A (SpA)-induced osteomyelitis progression by regulating m6A methylation-modified miR-320a

Ding Gao et al. J Orthop Surg Res. .

Abstract

Osteomyelitis (OM) is an inflammatory disease of bone infection and destruction characterized by dysregulation of bone homeostasis. Staphylococcus aureus (SA) has been reported to be the most common pathogen causing infectious OM. Recent studies have demonstrated that N6-methyladenosine (m6A) regulators are associated with the development of OM. However, the molecular mechanism of m6A modifications in OM remains unclear. Here, we investigated the function of methyltransferase-like 3 (METTL3)-mediated m6A modification in OM development. In this study, human bone mesenchymal stem cells (hBMSCs) were treated with staphylococcal protein A (SpA), a vital virulence factor of SA, to construct cell models of OM. Firstly, we found that METTL3 was upregulated in OM patients and SpA-induced hBMSCs, and SpA treatment suppressed osteogenic differentiation and induced oxidative stress and inflammatory injury in hBMSCs. Functional experiments showed that METTL3 knockdown alleviated the inhibition of osteogenic differentiation and the promotion of oxidative stress and inflammation in SpA-treated hBMSCs. Furthermore, METTL3-mediated m6A modification upregulated miR-320a expression by promoting pri-miR-320a maturation, and the mitigating effects of METTL3 knockdown on SpA-mediated osteogenic differentiation, oxidative stress and inflammatory responses can be reversed by miR-320 mimic. In addition, we demonstrated that phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) was a downstream target of miR-320a, upregulation of PIK3CA alleviated miR-320a-induced inhibition of osteogenic differentiation, and upregulation of oxidative stress and inflammatory responses during SpA infection. Finally, we found that silencing METTL3 alleviated OM development by regulating the miR-320a/PIK3CA axis. Taken together, our data demonstrated that the METTL3/m6A/miR-320a/PIK3CA axis regulated SpA-mediated osteogenic differentiation, oxidative stress, and inflammatory responses in OM, which may provide a new therapeutic strategy for OM patients.

Keywords: staphylococcus aureus; Inflammatory response; Osteogenic differentiation; Osteomyelitis; Oxidative stress.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
METTL3 is aberrantly highly expressed in osteomyelitis and SpA-induced hBMSCs. A: The expression of METTL3 in patients with osteomyelitis (n = 24) and healthy individuals (n = 17) was detected by RT-qPCR. B: The expression of METTL3 in SpA-treated hBMSCs was measured by RT-qPCR. C: The protein expression of osteogenesis markers (Runx2, OCN and ALP) in SpA-treated hBMSCs was assessed by Western blot. D: The mineralization of hBMSCs was measured using ARS staining after treated with or without SpA (scale bars, 50 μm). E-G: The protein levels of SOD, CAT and GSH in SpA-treated hBMSCs were measured by ELISA. H: The inflammatory cytokines (IL-1β, IL-6 and TNF-α) in SpA-treated hBMSCs was detected by ELISA. *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 2
Fig. 2
Silencing of METTL3 reverses the effects of SpA on osteogenic differentiation, oxidative stress and inflammatory responses in hBMSCs. A: The knockdown efficiency of silencing METTL3 in hBMSCs was examined by RT-qPCR. B: Western blot analysis was performed to measure the expression of Runx2, OCN and ALP in hBMSCs of Control, SpA, SpA + NC-kd and SpA + METTL3-kd groups. C-D: The calcium deposition was detected by ARS staining in Control, SpA, SpA + NC-kd and SpA + METTL3-kd groups (scale bars, 50 μm). E: The protein levels of SOD, CAT and GSH in hBMSCs of Control, SpA, SpA + NC-kd and SpA + METTL3-kd groups were measured by ELISA. F: The expression of IL-1β, IL-6 and TNF-α in Control, SpA, SpA + NC-kd and SpA + METTL3-kd groups of hBMSCs was assessed by ELISA. *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 3
Fig. 3
METTL3 regulates miR-320a expression in an m6A-dependent manner. A: The expression of six m6A-modificated miRNAs in SpA-treated hBMSCs were measured by RT-qPCR. B: The expression of miR-320a and pri-miR-320a in hBMSCs after silencing of METTL3 was measured by RT-qPCR. C-D: Me-RIP assay was performed using anti-DGCR8/m6A in METTL3 downregulation hBMSCs. *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 4
Fig. 4
METTL3 regulates SpA-mediated osteogenic differentiation, oxidative stress and inflammatory responses via miR-320a. A: The expression of Runx2, OCN and ALP in hBMSCs of Control, SpA, SpA + METTL3-kd and SpA + METTL3-kd + miR-320a mimic groups was measured by Western blot. B-C: Calcium mineralization in hBMSCs of Control, SpA, SpA + METTL3-kd and SpA + METTL3-kd + miR-320a mimic groups was assessed by ARS staining (scale bars, 50 μm). D: The protein levels of SOD, CAT and GSH in hBMSCs of Control, SpA, SpA + METTL3-kd and SpA + METTL3-kd + miR-320a mimic groups were measured by ELISA. E: The expression of IL-1β, IL-6 and TNF-α in Control, SpA, SpA + METTL3-kd and SpA + METTL3-kd + miR-320a mimic groups of hBMSCs was assessed by ELISA. “ME-kd” is for “METTL3-kd” and “miR mimic” stands for miR mimic “miR-320a mimic”. *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 5
Fig. 5
PIK3CA is a target gene of miR-320a . A: The potential binding site between miR-320a and PIK3CA. B: The dual-luciferase reporter gene assay was conducted to confirm the direct binding relationship between miR-320a and PIK3CA. C: The mRNA expression of PIK3CA in SpA-treated hBMSCs was detected by RT-qPCR. D: The levels of PIK3CA in hBMSCs treatment with NC mimic or miR-320a mimic were measured by RT-qPCR. E: The expression of PIK3CA in NC mimic or miR-320a mimic-treated hBMSCs was detected by Western blot. *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 6
Fig. 6
miR-320a exacerbates osteomyelitis progression by inhibiting PIK3CA. A: The transfection efficiency of overexpressing PIK3CA in hBMSCs was examined by RT-qPCR. B: The expression of Runx2, OCN and ALP in hBMSCs of Control, SpA, SpA + miR-320a mimic and SpA + miR-320a mimic + oe-PIK3CA groups was measured by Western blot. C-D: ARS staining analysis of Control, SpA, SpA + miR-320a mimic and SpA + miR-320a mimic + oe-PIK3CA groups of mineralized nodules in hBMSCs (scale bars, 50 μm). E: The protein levels of SOD, CAT and GSH in hBMSCs of Control, SpA, SpA + miR-320a mimic and SpA + miR-320a mimic + oe-PIK3CA groups were measured by ELISA. F: The expression of IL-1β, IL-6 and TNF-α in Control, SpA, SpA + miR-320a mimic and SpA + miR-320a mimic + oe-PIK3CA groups of hBMSCs was assessed by ELISA. “oe-PIK3” is for “oe-PIK3CA” and “miR mimic” stands for “miR-320a mimic”. *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 7
Fig. 7
METTL3 promotes osteomyelitis progression by regulating the miR-320a/PIK3CA axis. A: The transfection efficiency of inhibiting PIK3CA in hBMSCs was examined by RT-qPCR. B: The expression of miR-320a in Control, SpA, SpA + METTL3-kd and SpA + METTL3-kd + PIK3CA-kd groups of hBMSCs. C: The expression of Runx2, BGP and ALP of Control, SpA, SpA + METTL3-kd and SpA + METTL3-kd + PIK3CA-kd groups was measured by Western blot. D: The protein levels of SOD, CAT and GSH of Control, SpA, SpA + METTL3-kd and SpA + METTL3-kd + PIK3CA-kd groups were measured by ELISA. E: The expression of IL-1β, IL-6 and TNF-α in Control, SpA, SpA + METTL3-kd and SpA + METTL3-kd + PIK3CA-kd groups of rats was assessed by ELISA. “ME-kd” is for “METTL3-kd” and “PIK3-kd” stands for “PIK3CA-kd”. *p < 0.05, **p < 0.01, ***p < 0.005
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