Fat mass and obesity-associated protein in mesenchymal stem cells inhibits osteoclastogenesis via lnc NORAD/miR-4284 axis in ankylosing spondylitis

Figure 1 Fat mass and obesity-associated protein expression was upregulated in mesenchymal stem cells from individuals with ankylosing spondylitis compared with mesenchymal stem cells from healthy donors. A: Protein levels of N6-methyladenosine-related methyltransferase and demethylases in mesenchymal stem cells from individuals with ankylosing spondylitis and mesenchymal stem cells from healthy donors were determined by western blot; B: Quantitative data of protein levels of fat mass and obesity-associated, alkB homolog 5, methyltransferase 3, methyltransferase 14, and WT1 associated protein are shown; C: Immunofluorescence staining (scale bar = 100 mm) showed fat mass and obesity-associated expression in the osteocalcin positive mesenchymal stem cells in healthy donors group and ankylosing spondylitis group. The statistical data are represented as the means ± SDs, n = 3 in each group. NS: No significant difference, ^aP < 0.05, ^bP < 0.01. AS-MSCs: Mesenchymal stem cells from individuals with ankylosing spondylitis; HD-MSCs: Mesenchymal stem cells from healthy donors; ALKBH5: AlkB homolog 5; METTL3: Methyltransferase 3; METTL14: Methyltransferase 14; WTAP: WT1 associated protein; OCN: Osteocalcin; FTO: Fat mass and obesity-associated; AS: Ankylosing spondylitis; HD: Healthy donors.

Figure 2 Fat mass and obesity-associated positively regulates the ability of mesenchymal stem cells to inhibit osteoclast formation. CD14⁺ monocytes were cultured with mesenchymal stem cells from healthy donors or mesenchymal stem cells from individuals with ankylosing spondylitis after knocking down fat mass and obesity-associated in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor κB ligand. A: Representative images of tartrate resistant acid phosphatase (TRAP) staining (× 100); B: Quantitative data of TRAP⁺ osteoclasts number in each well is shown; C: Representative images of F-actin assays (× 200); D: Western blot analysis was performed to detect protein levels of TRAP, nuclear factor of activated T cells 1 (NFATc1) and cathepsin K (CTSK); E: Quantitative data for NFATc1, TRAP, and CTSK protein levels determined by western blot analyses are shown; F: Representative images of TRAP staining (× 100) and F-actin assays (× 200); G: Quantitative data of TRAP⁺ osteoclasts number in each well is shown; H: Western blot analysis was performed to detect protein levels of NFATc1, TRAP, and CTSK. The statistical data are presented as the means ± SDs; n = 3 in each group. NS: No significant difference, ^bP < 0.01, ^cP < 0.005, ^dP < 0.001. AS: Ankylosing spondylitis; HD: Healthy donors; si-NC: Small interfering RNA of negative control; si-FTO: Small interfering RNA targeting fat mass and obesity-associated; OE-NC: Overexpression of negative control; OE-FTO: Overexpression of fat mass and obesity-associated; TRAP: Tartrate resistant acid phosphatase; NFATc1: Nuclear factor of activated T cells 1; CTSK: Cathepsin K.

Figure 3 Fat mass and obesity-associated modulates the expression of microRNA-4284 by targeting ong non-coding RNA activated by DNA damage stability. A: Relative expression of fat mass and obesity-associated (FTO) and miR-4284 were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in mesenchymal stem cells (MSCs) after small interfering RNA (siRNA) transfection; B: Volcano plots of long noncoding RNA (lncRNA) high-throughput sequencing results for MSCs compared between control group and FTO knockdown group; C: The possible binding sites between long non-coding RNA activated by DNA damage (NORAD) and miR-4284 are predicted by DIANA TOOLS; D: The expression of FTO and lnc NORAD were quantified by qRT-PCR in MSCs after siRNA transfection; E: N6-methyladenosine modification level of lncRNA NORAD in si-NC or si-FTO treated MSCs (n = 3); F: Degradation curves of lncRNA NORAD in si-NC or si-FTO treated MSCs (n = 3); G: The possible binding sites between lncRNA NORAD and miR-4284 are shown, and a mutant lnc NORAD site was constructed; H: Luciferase reporter assay using empty luciferase vector (luci-control), wild type reporter with binding site (luci-NORAD wild type), and the vector containing mutated binding sequence (luci-NORAD mutant) in the presence of miR-4284 mimic (n = 3); I: The expression of lnc NORAD and miR-4284 were quantified by qRT-PCR in MSCs after siRNA transfection; J and K: RNA immunoprecipitation assays were used to detect the interaction between lnc NORAD and miR-4284. The statistical data are presented as the means ± SDs; n = 3 in each group. NS: No significant difference; ^aP < 0.05, ^bP < 0.01, ^cP < 0.005. FTO: Fat mass and obesity-associated; NC: Negative control; si-NC: Small interfering RNA of negative control; si-FTO: Small interfering RNA targeting fat mass and obesity-associated; IgG: Immunoglobulin G; m6A: N6-methyladenosine; WT: Wild type; MUT: Mutant; Ago2: Argonaute 2; NORAD: Non-coding RNA activated by DNA damage.

Figure 4 Long non-coding RNA activated by DNA damage in mesenchymal stem cells inhibits osteoclastogenesis. A: Representative images of tartrate resistant acid phosphatase (TRAP) staining of osteoclasts co-cultured with mesenchymal stem cells on day 9 after knocking down long non-coding RNA activated by DNA damage (× 100); B: Quantitative data of TRAP⁺ osteoclasts number in each well is shown; C: Representative images of osteoclasts stained with F-actin assays (× 200); D: Western blot analysis was performed to detect protein levels of nuclear factor of activated T cells 1 (NFATc1), TRAP, and cathepsin K (CTSK); E: Quantitative data for NFATc1, TRAP, and CTSK protein levels determined by western blot analyses are shown; F: Representative images of TRAP staining (× 100) and F-actin assays (× 200); G: Quantitative data of TRAP⁺ osteoclasts number in each well is shown; H: Western blot analysis was performed to detect protein levels of NFATc1, TRAP, and CTSK. The statistical data are presented as the means ± SDs; n = 3 in each group. NS: No significant difference; ^aP < 0.05, ^bP < 0.01, ^cP < 0.005, ^dP < 0.001. AS: Ankylosing spondylitis; HD: Healthy donors; si-NC: Small interfering RNA of negative control; TRAP: Tartrate resistant acid phosphatase; NFATc1: Nuclear factor of activated T cells 1; CTSK: Cathepsin K; OE-NC: Overexpression of negative control; OE-NORAD: Overexpression of Non-coding RNA activated by DNA damage.

Figure 5 Fat mass and obesity-associated in mesenchymal stem cells inhibits osteoclastogenesis through the long non-coding RNA activated by DNA damage/miR-4284 axis. A: Representative images of tartrate resistant acid phosphatase (TRAP) staining (× 100) and F-actin assays (× 200); B: Quantitative data of TRAP⁺ osteoclasts number in each well is shown; C: Western blot analysis was performed to detect protein levels of nuclear factor of activated T cells 1 (NFATc1), TRAP, and cathepsin K (CTSK); D: Quantitative data for NFATc1, TRAP, and CTSK protein levels determined by western blot analyses are shown; E: Representative images of TRAP staining (× 100) and F-actin assays (× 200); F: Quantitative data of TRAP⁺ osteoclasts number in each well is shown; G: Western blot analysis was performed to detect protein levels of NFATc1, TRAP, and CTSK; H: Quantitative data for NFATc1, TRAP, and CTSK protein levels determined by western blot analyses are shown. The statistical data are presented as the means ± SDs; n = 3 in each group. ^aP < 0.05, ^bP < 0.01, ^cP < 0.005, ^dP < 0.001. si-NC: Small interfering RNA of negative control; si-FTO: Small interfering RNA targeting fat mass and obesity-associated; OE-NC: Overexpression of negative control; OE-FTO: Overexpression of fat mass and obesity-associated; TRAP: Tartrate resistant acid phosphatase; NFATc1: Nuclear factor of activated T cells 1; CTSK: Cathepsin K; NORAD: Non-coding RNA activated by DNA damage.

Figure 6 Targeting fat mass and obesity-associated alleviates enthesitis-related new bone formation in ankylosing spondylitis animal models. A: Histological analysis of bone formation in the enthesis was performed using hematoxylin and eosin (HE) staining and Safranin O. Scale bar = 500 μm (upper) or 250 μm (lower) (n = 5); B: The proposed model shows upregulation of fat mass and obesity-associated in mesenchymal stem cells leads to defects in osteoclastogenesis in ankylosing spondylitis via the long non-coding RNA activated by DNA damage/miR-4284 axis. HE: Hematoxylin and eosin; FTO: Fat mass and obesity-associated; Av-NC: Adeno-associated virus of negative control; Av-shFTO: Adeno-associated virus of short hairpin RNA targeting fat mass and obesity-associated; NORAD: Non-coding RNA activated by DNA damage.