Fig 1: miR-23a-5p regulates C2C12 myoblast proliferation and differentiation by interacting with lncDum. (A) RT-qPCR analysis of the expression levels of lncDum in myoblasts transfected with si-lncDum, si-NC, p-lncDum or pcDNA3.1+ empty vector during proliferation. (B) EdU assay was performed following 24 h of transfection. Scale bar, 100 µm. (C) Percentage of EdU-positive/DAPI-positive cells was quantified. (D) Cell count was measured via a CCK-8 assay in C2C12 myoblasts transfected with si-lncDum, si-NC, pcDNA3.1+ empty vector or p-lncDum. RT-qPCR analysis of the expression levels of (E) Cyclin E, Cyclin B, CCND1 and P21, and (F) lncDum in myoblasts transfected with si-lncDum, si-NC, pcDNA3.1+ empty vector or p-lncDum during differentiation. (G) Bright-field microscopy was used to observe the ability of C2C12 myoblasts to differentiate into myotubes at 6 days after transfecting with si-lncDum, si-NC, pcDNA3.1+ empty vector or p-lncDum. Scale bar, 100 µm. (H) RT-qPCR measured the expression levels of MyoG, MyoD, MRF4, Myf5 and MyHC. (I) Western blotting analysis of MyoG protein levels in C2C12 myoblasts transfected with si-lncDum, si-NC, p-lncDum or pcDNA3.1+ empty vector during differentiation. (J) lncDum contained a binding site for miR-23a-5p. RT-qPCR analysis of the expression levels of (K) lncDum and miR-23a-5p during myoblast proliferation and differentiation, and (L) lncDum in myoblasts transfected with miR-23a-5p mimics and p-lncDum, NC or p-lncDum during proliferation. (M) Luciferase assays assessed the effect of miR-23a-5p on the activity of lncDum. (N) Cell count was measured via a CCK-8 assay in C2C12 myoblasts transfected with p-lncDum, NC or p-lncDum and miR-23a-5p mimics. (O) RT-qPCR analysis of the expression levels of Cyclin E, Cyclin B, CCND1 and P21. (P) Bright-field was used to microscopy observe the ability of C2C12 myoblasts to differentiate into myotubes at 6 days after transfecting with p-lncDum, NC or p-lncDum and miR-23a-5p mimics. Scale bar, 100 µm. (Q) RT-qPCR analysis of the expression levels of MyoG, MyoD, MRF4, Myf5 and MyHC. (R) Western blotting analysis of MyoG protein levels in C2C12 myoblasts transfected with miR-23a-5p mimics and p-lncDum, NC or p-lncDum during differentiation. Data are presented as the mean ± SEM of three independent repeats. *P<0.05, **P<0.01. miR, microRNA; lnc, long non-coding RNA; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; NC, negative control; CCK-8, Cell Counting Kit-8; MyHC, myosin heavy chain; OD, optical density; NS, not significant.