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

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.