miR-29 is anti-fibrogenic in C2C12 cells.

<p>(A) C2C12 cells were differentiated (DM) for 0, 1, 2 or 4 days, at which times total RNAs were isolated for qRT-PCR measurement of the expressions of Col 1A1, Col 1A2, Col 3A1, α-SMA or VIM as well as MyHC, α-Actin, Troponin and MyoG. Expression folds are shown with respect to 0 hr cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (B and C) C2C12 cells were transfected with negative control (NC) or miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Myogenin, Troponin, α-Actin or MyHC as well as Col 1A1, Col 1A2, Col 3A1, α-SMA or VIM. Expression folds are shown with respect to NC cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (D) C2C12 cells were transfected with negative control (Anti-NC) or Anti-miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Col 1A1, Col 1A2, and Col 3A1. Expression folds are shown with respect to Anti-NC cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (E) Wild type (WT) or Mutant Col 1A1, Col 1A2, or Col 3A1-3′UTR luciferase reporter constructs were transfected into C2C12 cells with indicated miRNA or negative control (NC) oligos. Luciferase activities were determined at 48 h post-transfection and normalized to β-Galactosidase protein. Relative luciferase unit (RLU) is shown with respect to NC cells where normalized luciferase values were set to 1. The data represent the average of three independent experiments ± S.D. (F) A schematic illustration of base pairing between mmu-miR-29c with 3078–3099 region on 3′UTR of mouse Lims1. (G) Lims1 protein expression was measured in NC or miR-29 stable C2C12 cells by Western blotting using GAPDH as a loading control. (H) NC or miR-29 stable cells were differentiated and Lims1 expression levels were measured at the indicated time points. (I) C2C12 cells were transfected with negative control (Anti-NC) or Anti-miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Lims1. Expression folds are shown with respect to Anti-NC cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (J) Wild type (WT) or Mutant Lims1-3′UTR luciferase reporter constructs were transfected into C2C12 cells with miR-29 or negative control (NC) oligos. Luciferase activities were determined at 48 h post-transfection and normalized to β-Galactosidase protein.