Dysregulated circARF3/miR-27b-3p/YAP1 contributes to impaired osteogenic differentiation of periodontal ligament stem cells in periodontitis

Figure 1 Circular RNA ADP-ribosylation factor 3 expression is correlated with periodontitis. A and B: Expression levels of periodontal ligament stem cell surface markers including CD90, CD105, CD45, and CD34 in periodontal ligament stem cell from periodontitis patients (A) and healthy donors (B) identified by flow cytometry; C: Circular RNA ADP-ribosylation factor 3 expression in periodontitis tissues and heathy samples. ^bP < 0.01, compared with healthy group; D: Level of circular RNA ADP-ribosylation factor 3 at 0 hour, 24 hours, 7 days, 14 days, and 21 days after osteogenic induction. ^aP < 0.05, compared with 0 hour; ^bP < 0.01, compared with 0 hour. pPDSLC: Periodontitis periodontal ligament stem cell; hPDSLC: Healthy periodontal ligament stem cell; ARF3: ADP-ribosylation factor 3.

Figure 2 Circular RNA ADP-ribosylation factor 3 modulates osteogenic differentiation of periodontal ligament stem cells. A: Level of circular RNA ADP-ribosylation factor 3 (circARF3) in periodontal ligament stem cells treated with circARF3 overexpressing plasmids and small hairpin RNAs that target circARF3. ^bP < 0.01, compared with shNC group or ovNC group; B and C: Alkaline phosphatase and alizarin red staining of periodontal ligament stem cells treated with circARF3-overexpressing plasmids and small hairpin RNAs that target circARF3. ^bP < 0.01, compared with ovNC group; ^cP < 0.01, compared with shNC group; D: The expression of osteoblast makers, Runx2, Osterix, Osteocalcin and Col1a1, was determined by western blotting assay. ^bP < 0.01, compared with ovNC group; ^cP < 0.01, compared with shNC group. ARF3: ADP-ribosylation factor 3; ARS: Alizarin red staining; ALP: Alkaline phosphatase.

Figure 3 Circular RNA ADP-ribosylation factor 3 acts as sponge for miR-27b-3p. A: Potential binding site of miR-27b-3p on circular RNA ADP-ribosylation factor 3 (circARF3) predicted by ENCORI website; B: Level of miR-27b-3p determined by quantitative real-time polymerase chain reaction. ^bP < 0.01, compared with mimic NC group; C: Luciferase activity of wild-type (WT) and mutated (MUT) circARF3 promoter gene vectors. ^bP < 0.01, compared with mimic NC group; D: RNA pulldown assay to check interaction between biotin-labeled WT and MUT miR-27b-3p and circARF3. ^bP < 0.01, compared with Bio-NC group; ^cP < 0.01, compared with Bio-miR-27b-3pWT group; E: RNA immunoprecipitation assay to check interaction between miR-27b-3p and circARF3. ^bP < 0.01, compared with IgG group; F: Level of miR-27b-3p in periodontal ligament stem cells after transfection with circARF3-overexpressing vectors determined by quantitative real-time polymerase chain reaction. ^bP < 0.01, compared with ovNC group. ARF3: ADP-ribosylation factor 3; WT: Wild-type; MUT: Mutated.

Figure 4 Circular RNA ADP-ribosylation factor 3 regulates periodontal ligament stem cell osteogenic differentiation via miR-27b-3p. A: Level of miR-27b-3p in periodontal ligament stem cells transfected with miR-27b-3p mimics or inhibitors. ^bP < 0.01, compared with inhibitor NC group; ^cP < 0.01, compared with mimics NC group; B and C: The expression of osteoblast markers Runx2, Osterix, Osteocalcin and Col1a1, was determined by western blotting assay. ^bP < 0.01, compared with inhibitor NC group; ^cP < 0.01, compared with mimics NC group; D: Alkaline phosphatase and alizarin red staining of periodontal ligament stem cells treated with miR-27b-3p mimics or inhibitors. ^bP < 0.01, compared with inhibitor NC group; ^cP < 0.01, compared with mimics NC group; E: Level of miR-27b-3p in periodontitis tissues and heathy samples. ^bP < 0.01, compared with healthy group. ARS: Alizarin red staining; ALP: Alkaline phosphatase.

Figure 5 miR-27b-3p targets the Yes associated protein 1 3’ untranslated region. A: Potential binding site of miR-27b-3p on Yes associated protein 1 (YAP1) 3’ untranslated region was predicted by ENCORI website; B: Luciferase activity of wild-type (WT) and mutated (MUT) YAP1 promoter gene vectors. ^bP < 0.01, compared with mimics NC group; C: Level of YAP1 in periodontal ligament stem cells after transfection with miR-27b-3p mimics was determined by quantitative real-time polymerase chain reaction. ^bP < 0.01, compared with mimics NC group; D: Level of YAP1 in periodontitis tissues and heathy samples. ^bP < 0.01, compared with healthy group; E and F: RNA and protein levels of YAP1 in periodontal ligament stem cells were determined by quantitative real-time polymerase chain reaction and western blotting assay, respectively. ^bP < 0.01, compared with shNC group; ^cP < 0.01, compared with shARF3 group. YAP1: Yes associated protein 1.

Figure 6 Circular RNA ADP-ribosylation factor 3 affects periodontal ligament stem cell osteogenic differentiation via miR-27b-3p/Yes associated protein 1 axis. A and B: Alkaline phosphatase and alizarin red staining of periodontal ligament stem cells; C and D: Expression of osteoblast makers, Runx2, Osterix, Osteocalcin and Col1a1 was determined by western blotting assay. ^bP < 0.01, compared with ovNC group; ^cP < 0.01, compared with ovARF3 group. ARF3: ADP-ribosylation factor 3; ARS: Alizarin red staining; ALP: Alkaline phosphatase.