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Scope: Myogenesis involves a series of complex cellular and developmental processes regulated by many genes, transcription factors and non-coding RNAs. Recent studies have demonstrated the involvement of circular RNAs (circRNAs) in myogenesis. While previous studies have established a role for some circRNAs, the precise functions and mechanisms of circRNAs in skeletal muscle development are still not completely understood in chicken.Methods: To identify potential circRNAs during chicken embryonic skeletal muscle development, rRNA- libraries sequencing was performed in breast muscles from 12 broilers and 12 layers at four different embryonic points, embryonic day 10 (E10), E13, E16 and E19. Through circRNA differential expression analysis and target miRNA prediction, the circTMTC1 was predicted to participate in the embryonic muscle formation by sponging miRNA, which were verified in vitro experiments.Results: We identified 228 differentially expressed circRNAs between broilers and layers (fold change >2; p-value < 0.05), and 43 circRNAs were differentially expressed at multiple embryonic days. circTMTC1, a novel circRNA transcribed from the TMTC1 gene, was expressed significantly higher in layers than in broilers at E10, E13 and E16. Furthermore, circTMTC1 knockdown accelerated proliferation and differentiation in chicken skeletal muscle satellite cells (SMSCs), besides, circTMTC1-overexpressing cells showed opposite effects. circTMTC1 functioned as a miR-128-3p sponge at the differentiation stage of SMSCs, and circTMTC1 inhibited the expression of miR-128-3p. Furthermore, miR-128-3p promoted differentiation of chicken SMSCs, and circTMTC1 inhibited the promotion effect of miR-128-3p on chicken SMSC differentiation.Conclusion: Our study revealed that circRNAs are differentially expressed during chicken embryonic development between the two chicken models, and circTMTC1 inhibits chicken SMSC differentiation by sponging miR-128-3p.

microRNAs (miRNAs) are 20-24 nucleotide (nt) RNAs that regulate eukaryotic gene expression post-transcriptionally by the degradation or translational inhibition of their target messenger RNAs (mRNAs). To identify miRNA target genes will help a lot by understanding their biological functions. Sophisticated computational approaches for miRNA target prediction, and effective biological techniques for validating these targets now play a central role in elucidating their functions. Owing to the imperfect complementarity of animal miRNAs with their targets, it is difficult to judge the accuracy of the prediction. Complexity of regulation by miRNA-mediated targets at protein and mRNAs levels has made it more challenging to identify the targets. To date, only a few miRNAs targets are confirmed. In this article, we review the methods of miRNA target prediction and the experimental validation for their corresponding mRNA targets in animals.

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