m6A Methylation of Precursor-miR-320/RUNX2 Controls Osteogenic Potential of Bone Marrow-Derived Mesenchymal Stem Cells

Abstract

Methyltransferase-like 3 (METTL3) is the main enzyme for N⁶-methyladenosine (m⁶A)-based methylation of RNAs and it has been implicated in many biological and pathophysiological processes. In this study, we aimed to explore the potential involvement of METTL3 in osteoblast differentiation and decipher the underlying cellular and molecular mechanisms. We demonstrated that METTL3 is downregulated in human osteoporosis and the ovariectomized (OVX) mouse model, as well as during the osteogenic differentiation. Silence of METTL3 by short interfering RNA (siRNA) decreased m⁶A methylation levels and inhibited osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and reduced bone mass, and similar effects were observed in METTL3^+/- knockout mice. In contrast, adenovirus-mediated overexpression of METTL3 produced the opposite effects. In addition, METTL3 enhanced, whereas METTL3 silence or knockout suppressed, the m⁶A methylations of runt-related transcription factor 2 (RUNX2; a key transcription factor for osteoblast differentiation and bone formation) and precursor (pre-)miR-320. Moreover, downregulation of mature miR-320 rescued the decreased bone mass caused by METTL3 silence or METTL3^+/- knockout. Therefore, METTL3-based m⁶A modification favors osteogenic differentiation of BMSCs through m⁶A-based direct and indirect regulation of RUNX2, and abnormal downregulation of METTL3 is likely one of the mechanisms underlying osteoporosis in patients and mice. Thus, METTL3 overexpression might be considered a new approach of replacement therapy for the treatment of human osteoporosis.

Keywords: BMSCs; METTL3; RUNX2; m(6)A RNA methylation; osteogenic differentiation; pre-miR-320.

Graphical abstract

Figure 1

Decreases in Global m⁶A Level and METTL3 Expression in Osteoporosis Bone Tissues (A and B) Decreases of global m⁶A levels in total RNAs isolated from bone tissues of patients with osteoporosis compared with those of healthy subjects. m⁶A modification of RNAs was determined by m⁶A dot blot analysis (A) and an m⁶A ELISA kit (B). n = 3. (C and D) Decrease of global m⁶A level in bone tissues of OVX mice with osteoporosis relative to that in sham-operated control counterparts. n = 4. (E and F) Expression downregulation of m⁶A methyltransferases METTL3 and METTL14 mRNAs in bone tissues from osteoporosis patients (E) and OVX mice (F) relative to those from healthy human subjects and sham control mice, respectively. In comparison, the transcript levels of demethylases FTO and ALKBH5 were not different between osteoporosis and controls. mRNA levels were determined by qRT-PCR. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. n = 4.

Figure 2

Overexpression of METTL3 Rescues Impaired BMSC Function in OVX Mice (A) Illustration of experimental protocols for the creation of osteoporosis mice by ovariectomy and administration of METTL3-carrying adenovirus (METTL3-OE) or the empty vector-carrying adenovirus (NC-OE) to OVX mice and sham-operated control counterparts. (B) Verification of the efficiency of METTL-OE to induce METTL3 overexpression at the mRNA (upper, n = 5) and protein (lower) level on day 7 after adenovirus injection in mice. (C) Representative μ-CT images of trabecular bone of the femoral metaphysis (top) and entire proximal femur (bottom) showing the rescuing effects of METTL3-OE on the impaired bone microstructure in OVX mice. Scale bars, 10 mm. (D) μ-CT analysis of femur from 4-month-old females showing the rescuing effects of METTL3-OE on bone volume/tissue volume ratio (BV/TV), bone mineral density (BMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). n = 5. (E) Representative images of H&E staining of mouse femurs showing the ameliorating effects of METTL3-OE on the reduction of bone formation in OVX mice relative to the sham-control counterparts. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. n = 5.

Figure 3

Negative Impact of METTL3 Knockout on Bone Mass and Density (A) Schematic illustration of generation of METTL3^+/− knockout (KO) mice (C57BL/6N) by CRISPR/Cas9-mediated genome engineering strategy. Eleven exons were identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 11. Exons 2–6 were selected as the target site. Cas9 and guide RNA (gRNA) were co-injected into fertilized eggs for mouse production. (B) Verification of the efficiency of CRISPR/Cas9-mediated deletion of METTL3 at the mRNA (left) and protein (right) level on day 7 after adenovirus injection in mice. (C) μ-CT images of trabecular bone of the femoral metaphysis (upper panels) and entire proximal femur (lower panels) showing the impairment of bone microstructure in METTL3^+/− mice without ovariectomy and the enhanced impairment of bone microstructure in METTL3^+/− mice with ovariectomy. n = 5. (D) μ-CT analysis showing the deceases in BV/TV, BMD, Tb.N and Tb.Th and an increase in Tb.Sp in OVX METTL3^+/− mice. n = 5. (E) H&E staining depicting the declined bone formation in METTL3^+/− mice with or without ovariectomy compared to the wild-type mice. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01. n = 5.

Figure 4

Overexpression of METTL3 Promotes Osteogenic Differentiation of BMSCs (A) METTL3 overexpression increased m⁶A content in total RNAs of BMSCs as revealed by m⁶A dot blot assay. n = 3. (B) Verification of METTL3 overexpression at the mRNA level in BMSCs transfected with METTL3-OE, as detected by qPCR. n = 4. (C) qRT-PCR results showing METTL3 mRNA level on day 14 after transfection with METTL3 plasmid in BMSCs, which cultured with normal complete medium. (D and E) Enhancing effects of METTL3 overexpression on the expression levels of RUNX2, BGLAP, and ALP mRNAs in BMSCs on day 7 (D) and day 14 (E) after transfection of METTL3-OE relative to NC-OE. n = 3. (F) Typical examples of ALP staining (top and middle panels) showing the promoting effect of METTL3 overexpression by METTL-OE on osteogenic differentiation of BMSCs cultured in osteogenic medium. Statistical data (bottom panel) show the increased ALP activities by METTL3-OE relative to NC-OE, as indicated by the elevated percentage of stained area. Scale bar, 150 μm, n = 6. (G) Typical examples of ARS staining (top and middle panels) showing the facilitating action of METTL3 on osteogenic differentiation of BMSCs cultured in osteogenic medium as indicated by enhanced extracellular matrix mineralization (EMM). Statistical data (lower panel) show the increased EMM by METTL3-OE relative to NC-OE, as indicated by the elevated percentage of the stained area. Scale bar, 150 μm, n = 6. (H) Representative H&E staining showing the favorable effects of adenovirus carrying METTL3 gene (METTL3-OE) on bone formation of BMSCs implanted into the back of nude mice, as compared with empty vector (NC-OE) for NC. Scale bars, 50 μm. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. n = 3.

Figure 5

Silence of METTL3 Mitigates Osteogenic Differentiation of BMSCs (A) m⁶A dot blot analysis showing the time-dependent increase of m⁶A content in total RNAs in BMSCs during osteogenic differentiation. n = 3. (B) qRT-PCR results showing the time-dependent upregulation of METTL3 transcripts in BMSCs 7 and 14 days after osteogenic induction. n = 8. (C) Verification of the efficiency of siMETTL3 in silencing endogenous METTL3 expression at mRNA (left panel) and protein (right panel) levels by qPCR and western blot analysis, respectively. n = 4. (D) m⁶A dot blot assay showing the inhibitory effects of siMETTL3 on m⁶A content in BMSCs. n = 3. (E) qRT-PCR results showing METTL3 mRNA level on day 14 after transfection with METTL3 siRNA in BMSCs, which cultured with normal complete medium. (F) The effects of METTL3 silence on the expression of key osteogenesis genes, including RUNX2, BGLAP, and ALP after 7 days (upper panels) and 14 days (lower panels) of osteogenic induction. n = 3. (G) Representative images of ALP staining (top and middle panels) showing the suppressing effect of METTL3 silencing by siMETTL3 on osteogenic differentiation of BMSCs cultured in osteogenic medium. Statistical data (bottom panel) show the decreased ALP activities by siMETTL3 relative to negative control (siRNA), as indicated by the declined percentage of the stained area. Scale bar, 150 μm, n = 6. (H) Representative images of ARS staining (top and middle panels) showing the inhibitory action of siMETTL3 on osteogenic differentiation of BMSCs cultured in osteogenic medium as indicated by diminished EMM. Statistical data (lower panel) show the increased EMM by siMETTL3 relative to siNC, as indicated by the decreased percentage of stained area. Scale bar, 150 μm, n = 6. (I) Representative H&E staining depicting the inhibitory effect of the adenovirus carrying METTL3 shRNA (siMETTL3) on bone formation of BMSCs implanted into the back of nude mice, as compared with siNC. Scale bar, 50 μm. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. n = 3.

Figure 6

METTL3 Alters the m⁶A Methylation Status of pre-miR-320 and the Expression Levels of pre-miR-320 and miR-320 (A) Flowchart showing the procedures for identifying the METTL3 targets using m⁶A-RIP microarray analysis for RNA methylation. (B) Inhibitory effects of siMETTL3 to silence endogenous METTL3 on m⁶A methylation of pri-miRNAs (left panel) and pre-miRNAs (right) relative to those of siNC in BMSCs. n = 3. (C) Schematic diagram showing the procedure of m6A-RIP to measure the changes of m6A enrichment in RUNX2 mRNA upon silence of METTL3 in BMSCs. (D) Inhibitory effect of siMETTL3 on pre-miRNA methylation by m⁶A relative to that of siNC, as revealed by gene-specific m⁶A miR-320 assay in BMSCs. n = 4. (E) Upregulation of both pre-miR-320 and miR-320 levels by siMETTL3 as determined by qRT-PCR. n = 3. (F) Downregulation of both pre-miR-320 and miR-320 levels by METTL3 overexpression (METTL3-OE). n = 3. (G) siYTHDF2 decreased expression of pre-miR-320 after co-transfection of siMETTL3. n = 3. (H) miR-320 antisense inhibitor (AMO-320) reverses the inhibitory effects of METTL3 silence on the mRNA level of key osteogenesis genes, including RUNX2, BMP2, and SPP1, after 7 days of osteogenic induction. n = 3. (I) Counteracting effect of AMO-320 to the weakening of osteogenic ability caused by siMETTL3, as indicated by ALP staining. Scale bar, 150 μm. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. n = 3.

Figure 7

RUNX2 as a Target Gene of miR-320 and a Mediator of the Osteogenic Action of the METTL3/m⁶A Modification Axis (A) Sequence alignment showing the complementarity between miR-320 and RUNX2 gene with three potential binding sites (seed sites). The bases in red indicate the seed site, and the vertical lines represent the base pairing between miR-320 and RUNX2. (B and C) Lower expression levels of RUNX2 in bone tissues in osteoporosis patients than in healthy subjects (B; n = 6) and in OVX mice than in sham-control counterparts (C; n = 7). (D) Western blot results showing the repressive effect of miR-320 on RUNX2 protein level in BMSCs. n = 3. (E and F) Downregulation of RUNX2 at both mRNA (E) and protein (F) levels by silencing METTL3 with siMETTL3, as reported by qRT-PCR and western blot analysis. n = 3. (G) Potential sites and regions for m⁶A modification in the sequence of RUNX2 gene. (H) Gene-specific m⁶A-qPCR assay showing the reduction of m⁶A modification in specific regions of RUNX2 gene by siMETTL3 in BMSCs. (I) Changes of half-life (t_1/2) of RUNX2 mRNA in BMSCs with or without METTL3 depletion. (J) YTHDF1 siRNA (siYTHDF1) downregulates RUNX2 expression in the presence of METTL3-OE. (K) Alizarin red S staining results showing a slight recovery to osteogenic ability weakened by siMETTL3 in BMSCs. Scale bar, 150 μm. (L) RUNX2 overexpression exhibited a reversion effect on mRNA levels of BMP2 and SPP1, which was decreased by siMETTL3 after 7 days of osteogenic induction. n = 3. Data are expressed as mean ± SEM. **p < 0.01, ***p < 0.001.