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.

microRNA biogenesis, 7 and is dynamically regulated by functional interplay among the catalytic proteins of methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms tumour 1-associated protein (WTAP), KIAA1429, fat mass and obesity-associated protein (FTO), and alkB homology 5 (ALKBH5). The maturation process of miRNA is suppressed along with decreases in m6A levels. 8 Inhibition of miRNA expression affects the post-transcriptional regulation of mRNA. 9 The diverse roles of miRNAs in fracture healing have been well documented. 10,11 This led us to investigate the m6A modification status during facture healing and whether modified m6A levels affect the miRNA maturation process, which subsequently impacts fracture healing.
Here, we assessed the role of m6A modification in fracture healing. Further, we investigated the mechanisms underlying m6A modification during fracture healing.

| Femoral fracture model
A total of 120 C57BL/6J mice (age, 6 weeks) were purchased from the Center of Experimental Animals, Tongji Medical College, Huazhong University of Science and Technology, China. All animal experiments were approved by the Institutional Animal Care and Use Committee of the said university. After the mice were anesthetized with 10% chloral hydrate (0.3 mL/100 g body weight), the femoral fracture model was created. Briefly, a longitudinal incision was made on the skin and the muscles were separated to expose the femur. A transverse osteotomy was performed in the mid-diaphysis of the femur, and the bones were stabilized by inserting a 23-gauge intramedullary needle. Equal amounts (100 μL) of phosphate-buffered saline (PBS), plasmid METTL3 and agomiR-7212-5p (10 mg/ kg body weight) were locally injected into the femoral fracture site.
Local injection was administrated on days 0, 4 and 7. The mice were sacrificed at a designated time, and callus samples were collected for Western blot, qRT-PCR and microCT analysis.

| Radiographic analysis of callus formation
The entire femoral fracture site from each mouse was scanned ex vivo using the SkyScan 1176 scanner microCT system (BRUKER) and reconstructed as three-dimensional images. Bone volume (BV), total volume (TV), BV/TV and bone mineral density (BMD) were calculated to evaluate the degree of fracture healing. Bone radiographs were taken using an in vivo FX PRO imaging system (BRUKER).

| In vivo tracking
Cy3-labelled miR-7212-5p was locally injected into the femoral fracture site of C57BL/6J mice on days 0, 4 and 7. The mice were anesthetized and observed under a bioluminescence system (BRUKE) on the above-mentioned days, and fluorescence images for miR-7212-5p distribution were acquired under 740 nm excitation and 790 nm emission filters.

| RNA m6A quantification
Total RNA was isolated using TRIzol reagent (Invitrogen). Nanodrop (Thermo Scientific) was used to quantify the RNA concentration.
The m6A content in total RNA was measured using the m6A RNA methylation quantification kit (Abcam), according to the manufacturer's instructions.

| qRT-PCR
Total RNA (including miRNA) was extracted from cells or calluses using TRIzol reagent (Invitrogen). Concentration of the extracted RNA was quantified using Nanodrop (Thermo Scientific). miRNA quantification was performed using a TaqMan miRNA assay (Applied system), the iScript Select cDNA synthesis kit (Bio-Rad) and iQSupermix kits (Bio-Rad). GAPDH and U6 snRNA were used as controls for standardizing the mRNA and miRNA concentrations, respectively.
A real-time PCR system with a comparative Ct method (2 −ΔΔCt ) was used to quantify RNA expression. Sequences of the primers used to probe miRNA and mRNA are shown in Table S1.

| 3ʹ-Untranslated region (UTR) cloning and luciferase assay
Fragments of the 3ʹUTR of FGFR3 and the miR-26a-5p binding site were amplified using PCR and then subcloned into pGL3 vector (Promega). Binding-region mutations were obtained using the Quik Change Site-Directed Mutagenesis Kit (Stratagene). For the luciferase assay, MC3T3-E1 cells were seeded into a 96-well plate. Then, cells were cotransfected with WT-or mutant-type FGFR3 3ʹUTR-Luc reporter plasmid and miR-NC or miR-7212-5p. After 48 hours, the cells were lysed, the lysates were harvested, and luciferase activity was evaluated using the dual-luciferase reporter assay system (Promega). The activity of luciferase activity was normalized against that of firefly luciferase. For the m6A-pri-miRNA and DGCR8-pri-miRNA binding experiments, the overexpressed METTL3 cells were UV-cross-linked and lysed using the Magna RIP™ kit (Millipore). The extracts were immunoprecipitated with an anti-m6A antibody and DGCR8 antibody or IgG as a control. Beads were incubated with 200 µL of proteinase K (10 mg/mL), and RNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1). The extracted RNA was used to synthesize cDNA, which was subjected to qRT-PCR using specific pri-miRNA primers (normalized to input).

| Identification of differentially expressed miRNAs
Microarray data (GSE76197) were obtained from the gene expression omnibus (GEO) database. GSE76197 is a free microarray series that evaluates the miRNAs expression profiles of mice femoral fracture callus on days 0, 3, 5, 7, 10 and 14. The down-regulated, differentially expressed miRNAs were identified using the parameters P ≤ .05 and log2FC ≤ −2.

| Alkaline phosphatase (ALP) staining
ALP staining was assessed using the BCIP/NBT alkaline phosphatase colour development kit (Beyotime). Briefly, cells were washed with PBS and then fixed with 4% paraformaldehyde. The cells were then incubated in BCIP/NBT liquid substrate for 24 hours. The entire procedure was performed in the dark.

| Alizarin red staining
Cells were cultured in 6-well plates in osteogenic medium (Cyagen Biosciences) to induce osteoblast mineralization for 21 days. After that, the cells were washed with PBS and fixed with 4% paraformaldehyde. The cells were then incubated with 500 μL 0.5% alizarin red stain for 15 minutes and rinsed with ddH 2 O on an orbital shaker for 5 minutes.

| Statistical analysis
All data have been presented as mean ± standard deviation for three independent experiments. One-way analysis of variance with a post hoc test was performed to test between-group differences.
Student's t test was employed to analyse statistical differences between two groups. All statistical analyses were performed using SPSS version 22.0. P ＜ .05 was considered statistically significant.

| m6A level was suppressed during fracture healing
To elucidate the potential role of m6A modification in fracture healing, we first examined the total levels of m6A at days 0, 3, 7, 10, 14 and 21. Our data revealed that the m6A levels were noticeably decreased during the first 7 days of fracture and then gradually increased with fracture healing ( Figure 1A). To identify the catalytic proteins that were mainly modified, we measured the  Figure 1C). Taken together, these data suggested that down-regulated METTL3 was the main factor determining the modified m6A expression during fracture healing.

| Local administration of METTL3 suppressed fracture healing in vivo
To verify the function of METTL3 in vivo, we created a femoral fracture in C57BL/6J mice. Equal volumes of PBS and plasmid METTL3 were injected locally into the fracture site on days 0, 4 and Furthermore, the METTL3 level was significantly increased after the mice were treated with plasmid METTL3, which also up-regulated the content of m6A in the calluses. Thus, up-regulation of METTL3 delays fracture healing.

| METTL3-dependent m6A methylation mediates miR-7212-5p maturation via DGCR8
Previous studies have reported that METTL3 suppresses the levels of miRNAs in an m6A-dependent pri-miRNA-process manner. To screen for candidate miRNAs during fracture healing, we selected microarray data concerning fracture healing from the Gene Expression Omnibus database (GSE76197). The following six miRNAs were markedly downregulated: miR-701-3p, miR-7223-5p, miR-7025-5p, miR-6929-5p, miR-7212-5p and miR-6979-5p ( Figure 4A). Next, we measured the expression levels of these miRNAs during femoral fracture healing in mice using callus samples. The levels of these miRNAs were found to show a similar trend as that of METTL3 levels ( Figure 4B), suggesting that changes in the levels of these miRNAs during fracture healing are mediated by METTL3. We then examined if these miRNAs were regulated by METTL3 in vitro. By measuring the pri-miRNAs and miRNAs in plasmid METTL3-and si-METTL3-transfected cells, we found that miR-
We then tested if METTL3 promotes pri-miRNA processing by regulating the binding of DGCR8 with pri-miRNA. Immunoprecipitation F I G U R E 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 assay showed that METTL3 coprecipitates with DGCR8 and that RNase treatment abates this interaction, indicating that the binding of DGCR8 with METTL3 is mediated by RNAs ( Figure 4D). By knocking down METTL3 in the MC3T3-E1 cells, the level of methylated RNA bound by DGCR8 is significantly decreased ( Figure 4E). Further, our data showed increased binding of pri-miR-7212-5p to DGCR8 in the METTL3-overexpressing cells when DGCR8 or m6A was immunoprecipitated in the control and overexpressed METTL3 cells ( Figure 4F, G).
The results obtained were consistent with those of previous studies that showed that METTL3 mediated the pri-miRNA process by recognizing DGCR8 and binding it with pri-miRNAs.

| miRNA-7212-5p inhibits fracture healing in vivo
To evaluate the duration of effect of miR-7212-5p at the fracture site, we first injected Cy3-labelled miR-7212-5p into the fracture site of in the agomiR-7212-5p group. However, the control group exhibited a larger callus on the fracture site ( Figure 5C). In addition, the BV, TV and BMD of the callus were significantly higher in the control group than in the agomiR-7212-5p group, both on post-fracture days 14 and 21 ( Figure 5D-H). The expression levels of Runx2 and BMP2 were also decreased in the agomiR-7212-5p group ( Figure 5I-L), suggesting that miR-7212-5p delays fracture healing.

| miR-7212-5p mediates osteoblast differentiation by targeting FGFR3
To search for the potential target genes of miR-7212-5p, we compiled all the target scan-predicted genes, miRbase-predicted genes, and fracture-related genes and performed Venn analysis. Our results showed that FGFR3 may be a target of miR-7212-5p ( Figure 7A). Subsequently, we found that FGFR3 level was significantly elevated during the first 7 days following the fracture ( Figure 7B). However, these results were contradictory with those for the miR-7212-5p levels, suggesting that FGFR3 is the main target gene of miR-7212-5p during fracture healing.

| D ISCUSS I ON
m6A modifications in eukaryotic RNA play vital roles in various biological processes, and dysregulation of the m6A level contributes to diseases such as tumour and malignant hematopoiesis. 12,13 However, the functions of m6A in fracture healing remain elusive.
In the present study, we demonstrated that METTL3 inhibits osteoblast differentiation by targeting osteoblast-related miR-7212-5p in an m6A-dependent, pri-miRNA-processing manner. In addition, we found that miR-7212-5p suppressed osteoblast differentiation by targeting FGFR3 both in vitro and in vivo.  miRNAs play important roles in many biological processes. 14,24 The relationship between miRNAs and fracture healing has aroused great concern. Previous studies indicated that miRNAs mediate fracture healing through regulating of their target genes. 25,26 Fibroblast growth factor (FGF) signalling is reportedly involved in skeletal development. 27 Bone regeneration is accelerated by the activation of the FGF receptor signalling pathway, 28 whereas disruption of FGFR3 results in decreased endochondral ossification, thereby prolonging the growth period of long bones. 29 In the present study, we demonstrated that agomiR-7212-5p suppresses fracture healing by inhibiting FGFR3. The effective timing for miRNA administration in mouse models for fracture healing has not been well established till date, and the methods of injection vary from study to study. 30,31 Because blood flow to the bones is not as abundant as that to the heart or liver, we injected miRNA directly into the fracture site. The results from the animal experiments showed that the miR-7212-5p level was gradually increased at day 7 post-fracture. Given these results, we demonstrated that the injection of miR-7212-5p directly into the fracture site on post-fracture days 0, 4 and 7 is advisable.
It is impossible to obtain calluses from patients during fracture healing process. To study the relative roles of METTL3, miR-7212-5p and FGFR3 during fracture healing, we harvested calluses from mice with femoral fractures. Fracture healing is a complex process, and further studies are warranted to verify the effects of m6A methylation in fracture healing.
In summary, our results indicate that METTL3 regulates miRNA maturation during fracture healing. By suppressing miR-7212-5p expression, METTL3 silencing promotes osteoblast differentiation both in vitro and in vivo, suggesting the crucial role of METTL3 silencing in fracture healing. Overall, these results provide new insights towards studies on fracture healing in the future.

ACK N OWLED G EM ENT
This study was supported by the National Science Foundation of

DATA AVA I L A B I L I T Y S TAT E M E N T
All relevant data are available from the corresponding authors upon reasonable request.