The relation between circMEF2As and MEF2A.

(A) qRT-PCR analysis of linear MEF2A, preMEF2A, circMEF2A1, and circMEF2A2 in the cDNA samples generated from MEF2A siRNA (si-MEF2A) and siRNA-NC transfected SMSCs, n = 3. (B) qRT-PCR analysis of linear MEF2A, preMEF2A, circMEF2A1, and circMEF2A2 in the cDNA samples generated from MEF2A-flag fusion protein overexpression vector (ov-MEF2A-flag) and ov-NC transfected SMSCs, n = 3. (C) Partial MEF2A gene promoter sequences containing MEF2A protein binding site, the chicken MEF2A promoter sequence segment containing wild-type (WT) and mutant-type (MT) of MEF2A protein binding site were subcloned into promoter activity analyze dual-luciferase reporter vector. (D) Dual-luciferase report analysis of empty vector, promoter WT and promoter MT transfected SMSCs, additionally, promoter WT and promoter MT also co-transfected with ov-MEF2A-flag independently, n = 3. (E) Cut & tag PCR analysis were performed to test MEF2A protein binding ability on MEF2A promoter in ov-MEF2A-flag transfected SMSCs; DNA marker: DL2000. (F) RNA level and protein level of MEF2A in si-circMEF2A1 and siRNA-NC transfected SMSCs, n = 3. (G) RNA level and protein level of MEF2A in ov-circMEF2A1 or ov-NC and miR-30a-3p mimic or mimic NC co-transfected SMSCs, n = 3. (H) RNA level and protein level of MEF2A in si-circMEF2A2 and siRNA-NC transfected SMSCs, n = 3. (I) RNA level and protein level of MEF2A in ov-circMEF2A2 or ov-NC and miR-148a-5p mimic or mimic NC co-transfected SMSCs, n = 3. (J) Dual-luciferase report analysis of MEF2A-WT and MEF2A-MT in DF-1 cells which co-transfected with miR-30a-3p mimic or mimic NC, n = 3. (K) Dual-luciferase report analysis of MEF2A-WT and MEF2A-MT in DF-1 cells which co-transfected with miR-148a-5p mimic or mimic NC, n = 3. (L) qRT-PCR analysis of myogenic genes in the cDNA samples generated from ov-circMEF2A1, ov-circMEF2A2, ov-NC, siRNA-NC, and si-MEF2A co-transfected SMSCs, n = 3. (M) Immunofluorescence of MyHC in ov-circMEF2A1, ov-circMEF2A2, ov-NC, siRNA-NC, and si-MEF2A co-transfected SMSCs. Scale bars: 200 μm. (N) The relative myotube area of ov-circMEF2A1, ov-circMEF2A2, ov-NC, siRNA-NC, and si-MEF2A co-transfected SMSCs was calculated by Image pro plus software, n = 9. (O) The proportion of MyHC+ cells of ov-circMEF2A1, ov-circMEF2A2, ov-NC, siRNA-NC, and si-MEF2A co-transfected SMSCs was calculated by Image pro plus software, n = 9. Data were displayed as mean ± SEM, independent sample t-test was used to analyze the statistical differences between each dataset, **P P < 0.05.</p

Differentially expressed gene analysis between siRNA-NC and si-circMEF2A1 transfected SMSCs (fold change > 1.2 and <i>P</i>-value < 0.05).

(A) The volcano plot of the differentially expressed genes between circMEF2A1 knockdown and negative control. (B) The heat map of myogenic marker genes and circMEF2A1/miR-30a-3p target genes in the differentially expressed genes. (TIF)</p

Detection of the knockdown efficiency of different siRNAs.

(A) Knockdown efficiency of three siRNAs against circMEF2A1 analyzed by qRT-PCR, and siRNA-3 was chosen for further analysis and named si-circMEF2A1 in the main documents, n = 3. (B) Knockdown efficiency of three siRNAs against PPP3CA analyzed by qRT-PCR, siRNA-2 was chosen for further analysis and named si-PPP3CA in the main documents, n = 3. (C) Knockdown efficiency of three siRNAs against circMEF2A2 analyzed by qRT-PCR, siRNA-2 was chosen for further analysis and named si-circMEF2A2 in the main documents, n = 3. (D) Knockdown efficiency of three siRNAs against SLIT3 analyzed by qRT-PCR, siRNA-3 was chosen for further analysis and named si-SLIT3 in the main documents, n = 3. (E) Knockdown efficiency of three siRNAs against ROBO2 analyzed by qRT-PCR, siRNA-3 was chosen for further analysis and named si-ROBO2 in the main documents, n = 3. (F) Knockdown efficiency of three siRNAs against MEF2A analyzed by qRT-PCR, siRNA-2 was chosen for further analysis and named si-MEF2A in the main documents, n = 3. (G) Knockdown efficiency of two siRNAs against mmu-circMef2a1 analyzed by qRT-PCR, siRNA-2 was chosen for further analysis and named si-mmu-circMef2a1 in the main documents, n = 3. (H) Knockdown efficiency of two siRNAs against mmu-circMef2a2 analyzed by qRT-PCR, siRNA-1 was chosen for further analysis and named si-mmu-circMef2a2 in the main documents, n = 3. Data were displayed as mean ± SEM, independent sample t-test was used to analyze the statistical differences between each dataset, **P P (TIF)</p

CircMEF2A2 promotes myogenesis in vitro and in vivo.

(A, B) qRT-PCR analysis of circMEF2A2 in the cDNA samples generated from circMEF2A2 siRNA (si-circMEF2A2), siRNA-NC, overexpression vector (ov-circMEF2A2), and ov-NC transfected SMSCs, n = 3. (C, D) qRT-PCR analysis of myogenic genes in the cDNA samples generated from si-circMEF2A2, siRNA-NC, ov-circMEF2A2, and ov-NC transfected SMSCs, n = 3. (E) Immunofluorescence of MyHC in si-circMEF2A2, siRNA-NC, ov-circMEF2A2, and ov-NC transfected SMSCs. Scale bars: 200 μm. (F, G) The relative myotube area of si-circMEF2A2, siRNA-NC, ov-circMEF2A2, and ov-NC transfected SMSCs was calculated by Image pro plus software, n = 9. (H, I) The proportion of MyHC+ cells of si-circMEF2A2, siRNA-NC, ov-circMEF2A2, and ov-NC transfected SMSCs was calculated by Image pro plus software, n = 9. (J, K) qRT-PCR analysis of myogenic genes in the cDNA samples generated from lentivirus packaged circMEF2A2 shRNA (LV-si-circMEF2A2), LV-si-NC, overexpression vector (LV-ov-circMEF2A2), and LV-ov-NC infected breast muscles of Tianfu chicks, n = 3. (L, M) The breast muscle rate of the LV-si-circMEF2A2, LV-si-NC, LV-ov-circMEF2A2, and LV-ov-NC infected Tianfu chicks, n = 6. (N, O) Representative photographs of the unilateral breast muscles of the LV-si-circMEF2A2, LV-si-NC, LV-ov-circMEF2A2, and LV-ov-NC infected Tianfu chicks. (P, Q) Hematoxylin and eosin (H&E) staining of the cross-section of LV-si-circMEF2A2, LV-si-NC, LV-ov-circMEF2A2, and LV-ov-NC infected chicks’ breast muscle. Scale bars: 200 μm. (R, S) The myofiber cross-sectional area of LV-si-circMEF2A2, LV-si-NC, LV-ov-circMEF2A2, and LV-ov-NC infected chicks’ breast muscles calculated by image J software, n = 9. Data were displayed as mean ± SEM, independent sample t-test was used to analyze the statistical differences between each dataset, **P P < 0.05.</p

Conservative target analysis of miR-30a-3p or miR-148a-5p on MEF2A.

(A) The interaction site of miR-30a-3p on MEF2A mRNA in humans, mice, and chickens. (B) The target site of miR-30a-3p on MEF2A 5’UTR is conserved in humans, mice, and chickens. (C) The interaction site of miR-148a-5p on MEF2A mRNA in humans, mice, and chickens. (D) The target site of miR-148a-5p on the MEF2A coding sequence is conserved in humans, mice, and chickens. (TIF)</p

Evolutionary analysis of circMEF2As across multiple species.

(A) Source information and basic characteristics of circMEF2A1 in 6 species including humans, macaque, mice, rats, pigs, and chickens. (B) Source information and basic characteristics of circMEF2A2 in 6 species. (C-E) Neighbor-Joining tree analysis of MEF2A, circMEF2A1 and circMEF2A2 in 6 species performed by MEGA7 software; Rats and mice are clustered together, humans and macaques are clustered together, pigs are in a separate cluster, and chickens are also in a separate cluster but are the furthest away from the other five species. (F) Phylogenetic tree of the 6 species, data were downloaded from the NCBI database; the results of the cluster analysis are basically similar to (C-E). (G) Species differentiation time of chicken and mouse analyzed by TimeTree database. (TIF)</p

Information of antibodies.

Circular RNAs (circRNAs) have been recognized as critical regulators of skeletal muscle development. Myocyte enhancer factor 2A (MEF2A) is an evolutionarily conserved transcriptional factor that regulates myogenesis. However, it remains unclear whether MEF2A produces functional circRNAs. In this study, we identified two evolutionarily conserved circular MEF2A RNAs (circMEF2As), namely circMEF2A1 and circMEF2A2, in chicken and mouse muscle stem cells. Our findings revealed that circMEF2A1 promotes myogenesis by regulating the miR-30a-3p/PPP3CA/NFATC1 axis, whereas circMEF2A2 facilitates myogenic differentiation by targeting the miR-148a-5p/SLIT3/ROBO2/β-catenin signaling pathway. Furthermore, in vivo experiments demonstrated that circMEF2As both promote skeletal muscle growth. We also discovered that the linear MEF2A mRNA-derived MEF2A protein binds to its own promoter region, accelerating the transcription of MEF2A and upregulating the expression of both linear MEF2A and circMEF2As, forming a MEF2A autoregulated positive feedback loop. Moreover, circMEF2As positively regulate the expression of linear MEF2A by adsorbing miR-30a-3p and miR-148a-5p, which directly contribute to the MEF2A autoregulated feedback loop. Importantly, we found that mouse circMEF2As are essential for the myogenic differentiation of C2C12 cells. Collectively, our results demonstrated the evolution, function, and underlying mechanisms of circMEF2As in animal myogenesis, which may provide novel insight for both the farm animal meat industry and human medicine.</div

The retrieval of MEF2A-derived circRNAs.

(A) Myogenic regulate factors and myocyte enhancer factor 2 family produced circRNA scanning from the circAtlas database. (B) CircMEF2As were identified from the sequencing data in the SRA database with accession number PRJNA516545 (assembly to genome Gallus gallus-5.0). (C) CircMEF2As were identified from the sequencing data in the BIG Data Center with accession number CRA002573 (assembly to genome Gallus gallus-5.0). (D) CircMEF2As were identified from the sequencing data in the GEO database with accession number GSE89355 (assembly to genome Gallus gallus-4.0). Data were displayed as mean ± SEM. (TIF)</p

CircMEF2A2 targets and regulates miR-148a-5p.

(A) Venn analysis of human, mouse, and chicken circMEF2A2 targeted miRNAs which predicted by RNAhybrid software, three co-targeted miRNAs including miR-148a-5p, miR-34b-3p, and miR-34c-3p were observed. (B, C) qRT-PCR analysis of these three co-targeted miRNAs in the cDNA samples generated from si-circMEF2A2, siRNA-NC, ov-circMEF2A2, and ov-NC transfected SMSCs, n = 3. (D) RNA FISH analysis revealed the subcellular localization of circMEF2A2 and miR-148a-5p in normal growing SMSCs. Scale bars: 20 μm. (E) The image of circMEF2A2 and miR-148a-5p hybridization which was predicted by RNAhybrid software. (F) Partial sequence of circMEF2A2 containing wild-type (circMEF2A2-WT) and mutant-type (circMEF2A2-MT) of miR-148a-5p response element were subcloned into the dual-luciferase reporter vector. (G) Dual-luciferase report analysis of circMEF2A2-WT and circMEF2A2-MT in DF-1 cells which co-transfected with miR-148a-5p mimic or mimic NC, n = 3. (H, I) qRT-PCR analysis of miR-148a-5p in cDNA samples generated from miR-148a-5p inhibitor, inhibitor NC, miR-148a-5p mimic and mimic NC transfected SMSCs, n = 3. (J, K) qRT-PCR analysis of myogenic genes in the cDNA samples generated from miR-148a-5p inhibitor, inhibitor NC, miR-148a-5p mimic and mimic NC transfected SMSCs, n = 3. (L) Immunofluorescence of MyHC in miR-148a-5p inhibitor, inhibitor NC, miR-148a-5p mimic and mimic NC transfected SMSCs. Scale bars: 200 μm. (M, N) The relative myotube area of miR-148a-5p inhibitor, inhibitor NC, miR-148a-5p mimic, and mimic NC transfected SMSCs was calculated by Image pro plus software, n = 9. (O, P) The proportion of MyHC+ cells of miR-148a-5p inhibitor, inhibitor NC, miR-148a-5p mimic and mimic NC transfected SMSCs was calculated by Image pro plus software, n = 9. Data were displayed as mean ± SEM, independent sample t-test was used to analyze the statistical differences between each dataset, **P P < 0.05.</p

CircMEF2A1 promotes myogenesis in vitro and in vivo.

(A, B) qRT-PCR analysis of circMEF2A1 in the cDNA samples generated from circMEF2A1 siRNA (si-circMEF2A1), negative control siRNA (siRNA-NC), overexpression vector (ov-circMEF2A1), and negative control vector (ov-NC) transfected SMSCs, n = 3. (C, D) qRT-PCR analysis of myogenic genes including MyoD1, MyoG, MyF5, and MyHC in the cDNA samples generated from si-circMEF2A1, siRNA-NC, ov-circMEF2A1, and ov-NC transfected SMSCs, n = 3. (E) Immunofluorescence of MyHC in si-circMEF2A1, siRNA-NC, ov-circMEF2A1, and ov-NC transfected SMSCs. Scale bars: 200 μm. (F, G) The relative myotube area of si-circMEF2A1, siRNA-NC, ov-circMEF2A1, and ov-NC transfected SMSCs was calculated by Image pro plus software, n = 9. (H, I) The proportion of MyHC+ cells of si-circMEF2A1, siRNA-NC, ov-circMEF2A1, and ov-NC transfected SMSCs was calculated by Image pro plus software, n = 9. (J, K) qRT-PCR analysis of myogenic genes in the cDNA samples generated from lentivirus packaged circMEF2A1 shRNA (LV-si-circMEF2A1), negative control shRNA (LV-si-NC), overexpression vector (LV-ov-circMEF2A1), and negative control vector (LV-ov-NC) infected breast muscles of Tianfu chicks, n = 3. (L, M) The breast muscle rate of the LV-si-circMEF2A1, LV-si-NC, LV-ov-circMEF2A1, and LV-ov-NC infected Tianfu chicks, n = 6. (N, O) Representative photographs of the unilateral breast muscles of the LV-si-circMEF2A1, LV-si-NC, LV-ov-circMEF2A1, and LV-ov-NC infected Tianfu chicks. (P, Q) Hematoxylin and eosin (H&E) staining of the cross-section of LV-si-circMEF2A1, LV-si-NC, LV-ov-circMEF2A1, and LV-ov-NC infected chicks’ breast muscle. Scale bars: 200 μm. (R, S) The myofiber cross-sectional area of LV-si-circMEF2A1, LV-si-NC, LV-ov-circMEF2A1, and LV-ov-NC infected chicks’ breast muscles was calculated by Image J software, n = 9. Data were displayed as mean ± SEM, independent sample t-test was used to analyze the statistical differences between each dataset, **P P < 0.05.</p