Evolutionary conserved circular MEF2A RNAs regulate myogenic differentiation and skeletal muscle development
Fig 4
CircMEF2A1 activates PPP3CA/NFATC1 signaling to regulate myogenesis via targeting miR-30a-3p.
(A) Venn analysis revealed human, mouse, and chicken miR-30a-3p was predicted co-targets 76 genes, and only 8 genes were found reduced in the circMEF2A1 knockdown SMSCs among these 76 genes, further 3 myogenesis relative genes were identified among these 8 genes. (B) qRT-PCR analysis of these 3 myogenesis relative genes in the cDNA samples generated from miR-30a-3p mimic or mimic NC and ov-circMEF2A1 or ov-NC co-transfected SMSCs, n = 3. (C) qRT-PCR analysis of these 3 myogenesis-relative genes in the cDNA samples generated from si-circMEF2A1 and siRNA-NC transfected SMSCs, n = 3. (D) Partial sequence of PPP3CA 3’UTR containing wild-type (PPP3CA-WT) and mutant-type (PPP3CA-MT) of miR-30a-3p response element was subcloned into the dual-luciferase reporter vector. (E) Dual-luciferase report analysis of PPP3CA-WT and PPP3CA-MT in DF-1 cells which co-transfected with miR-30a-3p mimic or mimic NC, n = 3. (F) qRT-PCR analysis of myogenic genes in the cDNA samples generated from PPP3CA siRNA (si-PPP3CA) and siRNA-NC transfected SMSCs, n = 3. (G) Immunofluorescence of MyHC in si-PPP3CA and siRNA-NC transfected SMSCs. Scale bars: 200 μm. (H) The relative myotube area of si-PPP3CA and siRNA-NC transfected SMSCs was calculated by Image pro plus software, n = 9. (I) The proportion of MyHC+ cells of si-PPP3CA and siRNA-NC transfected SMSCs was calculated by Image pro plus software, n = 9. (J) Western blot analysis of myogenic proteins including MyoG and MyHC, and circMEF2A1/miR-30a-3p target proteins including PPP3CA, NFATC1 and phosphorylated NFATC1 (p-NFATC1), and housekeeper proteins including β-tubulin, GAPDH, and H3, in total proteins or nuclear proteins extracted from si-PPP3CA and siRNA-NC transfected SMSCs. (K) Western blot analysis of myogenic proteins, circMEF2A1/miR-30a-3p target proteins, and housekeeper proteins in total proteins or nuclear proteins extracted from miR-30a-3p mimic or mimic NC and ov-circMEF2A1 or ov-NC co-transfected SMSCs. (L) Western blot analysis of myogenic proteins, circMEF2A1/miR-30a-3p target proteins, and housekeeper proteins in total proteins or nuclear proteins extracted from si-PPP3CA or siRNA-NC and ov-circMEF2A1 or ov-NC co-transfected SMSCs. Data were displayed as mean ± SEM, independent sample t-test was used to analyze the statistical differences between each dataset, **P < 0.01 and *P < 0.05.