Methyltransferase-like 3-mediated N6-methyladenosine modification of miR-7212-5p drives osteoblast differentiation and fracture healing

Abstract

N6-methyladenosine (m6A) modification has been reported in various diseases and implicated in increasing numbers of biological processes. However, previous studies have not focused on the role of m6A modification in fracture healing. Here, we demonstrated that m6A modifications are decreased during fracture healing and that methyltransferase-like 3 (METTL3) is the main factor involved in the abnormal changes in m6A modifications. Down-regulation of METTL3 promotes osteogenic processes both in vitro and in vivo, and this effect is recapitulated by the suppression of miR-7212-5p maturation. Further studies have shown that miR-7212-5p inhibits osteoblast differentiation in MC3T3-E1 cells by targeting FGFR3. The present study demonstrated an important role of the METTL3/miR-7212-5p/FGFR3 axis and provided new insights on m6A modification in fracture healing.

Keywords: METTL3; fracture; m6A; miRNA.

FIGURE 1

Decreased METTL3 expression contributes to lower levels of m6A methylation during fracture healing. A, Percentage of m6A in total RNAs in callus samples during fracture healing (n = 5); B, mRNA levels of m6A modification‐associated genes in calluses during fracture healing (n = 5); C, percentage of m6A in total RNAs in METTL3 overexpression or knockdown in MC3T3‐E1 cells. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < .0001

FIGURE 2

Overexpression of METTL3 delays femoral fracture healing in mice. A, Radiological changes in the femur of mice on days 7, 14 and 21 post‐fracture. B, μCT images of the fractured femur on days 14 and 21. C‐F, BV, TV, BV/TV and BMD of calluses on days 14 and 21. G‐J, Expression levels of Runx2 and BMP2 from mice calluses on days 14 and 21 were quantified using qRT‐PCR and Western blot. K, METTL3 level in the calluses on day 21 was evaluated using qRT‐PCR. L, Percentage of m6A in total RNAs in the calluses on day 21. n = 5. Data are expressed as mean ± SD. ****P < .0001

FIGURE 3

METTL3 inhibits osteoblast differentiation and matrix mineralization. A, Relative levels of METTL3 in MC3T3‐E1 cells after transfection with PBS, plasmid METTL3, plasmid‐NC, si‐NC and si‐METTL3 were detected using qRT‐PCR. B, Western blot detected the expression of METTL3 in MC3T3‐E1 cells after transfection with PBS, plasmid‐METTL3, plasmid‐NC, si‐NC and si‐METTL3. C, Quantification of the ratio of METTL3/GAPDH in (B) groups. D, mRNA level of collagen I, ALP, OCN and Runx2 was detected using qRT‐PCR. E, ALP staining of MC3T3‐E1 cells after transfection with PBS, plasmid‐NC, plasmid METTL3, si‐NC and si‐METTL3. F, Quantification of the absorbance at 405 nm in (E) groups. G, Alizarin red staining of MC3T3‐E1 cells after transfection with PBS, plasmid‐NC, plasmid METTL3, si‐NC and si‐METTL3. H, Quantification of the absorbance at 570 nm in (G) groups. Data are expressed as mean ± SD. Scale bar = 50 μm. All experiments were performed in triplicates. ****P < .0001

FIGURE 4

METTL3‐dependent m6A methylation mediates miR‐7212‐5p maturation via DGCR8. A, Co‐expression of down‐regulated miRNAs during fracture healing. B, Level of miR‐7212‐5p during fracture healing was detected using qRT‐PCR. N = 5. C, miR‐7212‐5p and pri‐miR‐7212‐5p levels were detected in METTL3‐overexpressing cells and METTL3‐knockdown cells using qRT‐PCR. D, Coimmunoprecipitation of the METTL3‐interacting protein DGCR8. E, Immunoprecipitation of DGCR8, METTL3, and associated RNAs from control and METTL3‐knockdown cells. (F) Immunoprecipitation of m6A‐modified RNAs in control or METTL3‐overexpressing cells to detect the levels of pri‐miR‐7212‐5PM6A modification. G, Immunoprecipitation of DGCR8‐associated RNAs in control or METTL3‐overexpressing cells to assess the pri‐miR‐7212‐5p binding with DGCR8. Pri‐let‐7e was used as a positive control. *P < .05, ***P < .001 and ****P < .0001. Day 0: intact control sample. Day 3: post‐day 3 fracture sample. Day 5: post‐day 5 fracture sample. Day 7: post‐day 7 fracture healing. Day 11: post‐day 11 fracture healing. Day 14: post‐day 14 fracture healing. All cell experiments were performed in triplicates

FIGURE 5

miR‐7212‐5p inhibits fracture healing in vivo. A, Fluorescent images of C57BL/6J mice before and after injection of Cy3‐labelled miR‐7212‐5p. B, Level of miR‐7212‐5p in calluses on days 4, 7, 14 and 21 after injection of PBS or agomiR‐7212‐5p. n = 5. C, X‐ray showed the fracture site of the control group and agomiR‐7212‐5p group. D, μCT images of the fractured femur on days 14 and 21. n = 5. E‐H, BV, TV, BV/TV and BMD of the calluses on post‐fracture days 14 and 21. n = 5. I‐L, Levels of Runx2 and BMP2 in mice calluses on days 14 and 21 were quantified by qRT‐PCR and Western blot. n = 5. Data are expressed as mean ± SD. **P < 0.01, ****P < .0001

FIGURE 6

miR‐7212‐5p suppresses osteoblast differentiation. A, Relative expression of miR‐7212‐5p in MC3T3‐E1 cells after transfection with PBS, antagomiR‐NC, antagomiR‐7212‐5p, agomiR‐NC or agomiR‐7212‐5p. B, Relative levels of Collagen I, ALP, OCN and Runx2 in MC3T3‐E1 cells were quantified using qRT‐PCR. C, ALP staining of MC3T3‐E1 cells after transfection with either PBS, antagomiR‐NC, antagomiR‐7212‐5p, agomiR‐NC or agomiR‐7212‐5p. D, Quantification of the absorbance at 405 nm in (C) groups. E, Alizarin red staining of MC3T3‐E1 cells after 21 d following transfection with PBS, antagomiR‐NC, antagomiR‐7212‐5p, agomiR‐NC or agomiR‐7212‐5p. F, Quantification of the absorbance at 570 nm in (E) groups. The data are expressed as mean ± SD. Scale bar = 50 μm. All experiments were performed in triplicates. ****P < .001

FIGURE 7

miR‐7212‐5p targets FGFR3 to inhibit osteoblast differentiation. A, Venn diagram showing that miR‐7212‐5p targets FGFR3. B, Expression level of FGFR3 during fracture healing was detected using qRT‐PCR. C, Binding site of miR‐7212‐5p with the 3ʹ‐UTR region of FGFR3. D, Luciferase reporter assay of miR‐7212‐5p with wild‐type FGFR3‐3’UTR (3’UTR‐wt) or the mutated FGFR3‐3’UTR (3’UTR‐mut). E‐G, Expression level of FGFR3 after the fracture site was injected with agomiR‐7212‐5p detected using PCR and Western blot. H and I, Western blot analysis revealed decreased FGFR3 expression after the cells were transfected with agomiR‐7212‐5p. J, qRT‐PCR was used to assess the level of FGFR3 after transfection with PBS, si‐NC, si‐FGFR3, antagomiR‐7212‐5p+si‐NC or antagomiR‐7212‐5p+si‐FGFR3. K, The levels of Col1a1, ALP, OCN and Runx2 in MC3T3‐E1 cells after they were transfected with PBS, siRNA‐NC, si‐FGFR3, antagomiR‐7212‐5p+si‐NC or antagomiR‐7212‐5p+si‐FGFR3 were quantified with qRT‐PCR. L, ALP staining of MC3T3‐E1 cells after transfection with PBS, si‐NC, si‐FGFR3, antagomiR‐7212‐5p+si‐NC and antagomiR‐7212‐5p+si‐FGFR3 for 48 h. M, Quantification of the absorbance at 405 nm in (L) groups. N, Alizarin red staining of MC3T3‐E1 cells after 21 d following transfection with PBS, si‐NC, si‐FGFR3, antagomiR‐7212‐5p+si‐NC or antagomiR‐7212‐5p+si‐FGFR3. O, Quantification of the absorbance at 570 nm in (N) groups. The data are expressed as mean ± SD. Scale bar = 50 μm. All experiments were performed in triplicates. *P < .05, **P < .01 and ***P < .001