Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA‑4327 regulates TGF‑β1 stimulation of matrix metalloproteinase‑13 expression via CREB‑binding protein‑mediated Runx2 acetylation in human osteoblasts

doi: 10.3892/etm.2024.12770

Figure Lengend Snippet: Runx2 acetylation and CBP protein expression were reduced by the overexpression of miR-4327 under TGF-β1 stimulation in human osteoblasts. Transiently transfected MG-63 cells with miR-4327 mimic or negative control miRNA (nc-miRNA) for 24 h were subjected to 5 ng/ml TGF-β1 treatment for 2 and 24 h. (A) The expression of miR-4327 was analyzed by reverse transcription-quantitative polymerase chain reaction. # P<0.05 vs. control. (B) Whole-cell lysates were subjected to IP (2 h of TGF-β1 treatment) followed by IB. Representative blots of IP and IB are depicted. Protein expression was quantified using IgG expression for normalization using ImageJ software. Levels of (C) acetylated-Runx2, (D) Runx2 and (E) CBP. (F) Western blot analysis of CBP levels. (G) Relative protein expression of CBP normalized with respect to α-Tubulin expression. Whole-cell lysates obtained 24 h after TGF-β1 treatment were analyzed by western blotting. (H) Representative blots of MMP-13 expression. (I) Relative expression of MMP-13 normalized with respect to α-Tubulin expression. * P<0.05 vs. control; # P<0.05 vs. negative control (n=3). Runx2, runt-related transcription factor 2; CBP, CREB-binding protein; miRNAs/miR, microRNAs; TGF-β1, transforming growth factor-β1; nc, negative control; IP, immunoprecipitation; IB, immunoblotting; MMP-13, matrix metalloproteinase-13.

Article Snippet: TGF-β1 was obtained from R&D Systems, Inc. Antibodies against CBP (cat. no. 7389; 1:1,000), acetylated-lysine (cat. no. 9441; 1:1,000) and α-Tubulin (cat. no. 2125; 1:1,000) were acquired from Cell Signaling Technology, Inc., and antibodies against Runx2 (cat. no. sc-390715; 1:100) and MMP-13 (cat. no. 18165-1-AP; 1:3,000) were purchased from Cell Signaling Technology, Inc., and Proteintech Group, Inc., respectively.

Techniques: Expressing, Over Expression, Transfection, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Software, Western Blot, Binding Assay, Immunoprecipitation