METTL7A-mediated m6A modification of corin reverses bisphosphonates-impaired osteogenic differentiation of orofacial BMSCs

Abstract

Bisphosphonate-related osteonecrosis of jaw (BRONJ) is characterized by impaired osteogenic differentiation of orofacial bone marrow stromal cells (BMSCs). Corin has recently been demonstrated to act as a key regulator in bone development and orthopedic disorders. However, the role of corin in BRONJ-related BMSCs dysfunction remains unclarified. A m6A epitranscriptomic microarray study from our group shows that the CORIN gene is significantly upregulated and m6A hypermethylated during orofacial BMSCs osteogenic differentiation. Corin knockdown inhibits BMSCs osteogenic differentiation, whereas corin overexpression or soluble corin (sCorin) exerts a promotion effect. Furthermore, corin expression is negatively regulated by bisphosphonates (BPs). Corin overexpression or sCorin reverses BPs-impaired BMSCs differentiation ability. Mechanistically, we find altered expression of phos-ERK in corin knockdown/overexpression BMSCs and BMSCs under sCorin stimulation. PD98059 (a selective ERK inhibitor) blocks the corin-mediated promotion effect. With regard to the high methylation level of corin during osteogenic differentiation, we apply a non-selective m6A methylase inhibitor, Cycloleucine, which also blocks the corin-mediated promotion effect. Furthermore, we demonstrate that METTL7A modulates corin m6A modification and reverses BPs-impaired BMSCs function, indicating that METTL7A regulates corin expression and thus contributes to orofacial BMSCs differentiation ability. To conclude, our study reveals that corin reverses BPs-induced BMSCs dysfunction, and METTL7A-mediated corin m6A modification underlies corin promotion of osteogenic differentiation via the ERK pathway. We hope this brings new insights into future clinical treatments for BRONJ.

Fig. 1

m6A-epitranscriptomic microarray results illustrated the changes in mRNA methylation levels during orofacial BMSCs osteogenic differentiation. a Differential m6A-methylated sites in orofacial BMSCs underwent 7-day osteogenic differentiation are displayed in hierarchical clustering. b Scatter plot shows differentially methylated genes associated with alterations in gene expression. c qRT-PCR analyses of Corin, MYOCD, and ANLN. d MeRIP-qPCR results for Corin, MYOCD, and ANLN. e KEGG analyses of the differentially expressed m6A hypermethylated genes. f KEGG analyses of the differentially expressed m6A hypomethylated genes. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, 0 d vs. 7 d

Fig. 2

Corin promoted orofacial BMSCs osteogenic differentiation in vitro. a qRT-PCR analyses of the corin mRNA expression at 0d, 3d, 7d, 14d after osteogenic differentiation. b MeRIP-qPCR results show the m6A modification levels of corin mRNA. c Representative immunoblotting shows the protein expression of corin. d ELISA results show the sCorin levels at different time points during osteogenic differentiation process. e, f The mRNA and protein expression of corin from sh-NC/ sh-CORIN Group. g, h Representative images and quantitative analyses of ALP and ARS staining of BMSCs from sh-NC/ sh-CORIN Group. i Representative immunoblotting shows the protein expression of Runx2/OCN from sh-NC/sh-CORIN Group. j, k The mRNA and protein expression of corin from Vector/oe-CORIN Group. l, m Representative images and quantitative analyses of ALP and ARS staining of BMSCs from Vector/oe-CORIN Group. n Representative immunoblotting shows the protein expression of Runx2/OCN from Vector/oe-CORIN Group. o The sCorin levels at different time points from Vector/oe-CORIN Group. p, q Representative images and quantitative analyses of ALP and ARS staining of BMSCs from Vector/oe-CORIN supernate Group. r Representative immunoblotting shows the protein expression of Runx2/OCN from Vector/ oe-CORIN supernate Group. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, sh-NC or Vector vs. sh-CORIN or oe-CORIN

Fig. 3

Corin promoted orofacial BMSCs osteogenesis in vivo. a, b HE staining results show bone-like tissue generation. c, d Immunohistochemical staining of Runx2 and quantification of its expression. e, f Immunohistochemical staining of OCN and quantification of its expression. Scale bar: 100 μm. Five-pointed star: bone-like tissue; black arrow: Runx2/OCN-positive BMSCs; HA hydroxyapatite tricalcium carrier, CT connective tissue. Data are expressed as mean ± s.e.m. **P < 0.01, ***P < 0.001, Vector vs. oe-CORIN

Fig. 4

Corin reversed BPs-impaired orofacial BMSCs osteogenic differentiation in vitro and osteogenesis in vivo. a, b Representative images and quantitative analyses of ALP and ARS staining of BMSCs under dose-dependent zoledronic acid stimulation. c Representative immunoblotting shows the protein expression of Runx2/OCN from BMSCs under dose-dependent zoledronic acid stimulation. d Pre-treated with 5 µmol/L zoledronic acid for 3 days, qRT-PCR analyses of corin mRNA expression at 0 d, 3 d, 7 d, 14 d after osteogenic differentiation. e Pre-treated with 5 µmol/L zoledronic acid for 3 days, ELISA results show the sCorin levels at different time points. f, g Pre-treated with 5 µmol/L zoledronic acid for 3 days, representative images and quantitative analyses of ALP and ARS staining of BMSCs from Vector/oe-CORIN Group. h Representative immunoblotting shows the protein expression of Runx2/OCN from Vector/oe-CORIN Group. i, j Representative images and quantitative analyses of ALP and ARS staining of BMSCs from Vector/oe-CORIN supernate Group. k Representative immunoblotting shows the protein expression of Runx2/OCN from Vector/oe-CORIN supernate Group. l, n, p HE staining results, as well as immunohistochemical staining of Runx2 and OCN. m, o, q Quantitative analyses of HE staining, Runx2 and OCN. Scale bar: 100 μm. Five-pointed star: bone-like tissue; black arrow: Runx2/OCN-positive BMSCs; HA hydroxyapatite tricalcium carrier, CT connective tissue. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, NC or Vector vs. BP or oe-CORIN. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, Vector+BP vs oe-CORIN + BP

Fig. 5

ERK inhibitor blocked corin-mediated promotion of orofacial BMSCs osteogenic differentiation. a Representative immunoblotting shows protein expression of p-ERK from sh-NC/ sh-CORIN Group. b Protein expression of p-ERK from Vector/oe-CORIN Group. c Protein expression of p-ERK from BMSCs under sCorin stimulation. d, e Treated with 20 µmol/L PD98058, representative images and quantitative analyses of ALP and ARS staining of BMSCs from Vector/oe-CORIN Group. f Representative immunoblotting shows the protein expression of Runx2/OCN from Vector/oe-CORIN Group. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, Vector vs. oe-CORIN. ^###P < 0.001, oe-CORIN vs oe-CORIN + PD98059

Fig. 6

METTL7A promoted corin m6A methylation level. a, b Treated with 500 µg/mL Cycloleucine, representative images and quantitative analyses of ALP and ARS staining of BMSCs from Vector/oe-CORIN Group. c Representative immunoblotting shows the protein expression of Runx2/OCN from Vector/oe-CORIN Group. d, e qRT-PCR analyses, as well as representative immunoblotting show corin expression from sh-NC/sh-METTL7A Group. f MeRIP-qPCR results show m6A modification of corin mRNA from sh-NC/sh-METTL7A Group. g Comparation of corin mRNA stability from sh-NC/sh-METTL7A Group. h, i Corin expression from Vector/oe-METTL7A Group. j MeRIP-qPCR results show m6A modification on corin mRNA from Vector/oe-METTL7A Group. k Comparation of corin mRNA stability from Vector/oe-METTL7A Group. l Construct of Wildtype and mutant 3’UTR CORIN plasmids. m METTL7A binding to the CORIN 3′UTR is verified by the dual-luciferase reporter assay. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, sh-NC or Vector vs. sh-METTL7A or oe-METTL7A. ^###P < 0.001, oe-CORIN vs oe-CORIN+Cycloleucine

Fig. 7

METTL7A reversed BPs-impaired orofacial BMSCs osteogenic differentiation in vitro and osteogenesis in vivo. a, b Pre-treated with 5 µmol/L zoledronic acid for 3 days, representative images and quantitative analyses of ALP and ARS staining of BMSCs from Vector/oe-METTL7A Group. c Representative immunoblotting shows the protein expression of Runx2/OCN from Vector/oe-METTL7A Group. d, e HE staining results show bone-like tissue generation. f, g Immunohistochemical staining of Runx2 and quantification of its expression. h, i Immunohistochemical staining of OCN and quantification of its expression. Scale bar: 100 μm. Five-pointed star: bone-like tissue; black arrow: Runx2/OCN-positive BMSCs; HA hydroxyapatite tricalcium carrier, CT connective tissue. Data are expressed as mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001, Vector vs. METTL7A. ^#P < 0.05, ^##P < 0.01, Vector+BP vs. oe-METTL7A + BP