CircRNA Profiling of Skeletal Muscle in Two Pig Breeds Reveals CircIGF1R Regulates Myoblast Differentiation via miR-16

Abstract

Muscle development is closely related to meat quality and production. CircRNAs, with a closed-ring structure, have been identified as a key regulator of muscle development. However, the roles and mechanisms of circRNAs in myogenesis are largely unknown. Hence, in order to unravel the functions of circRNAs in myogenesis, the present study explored circRNA profiling in skeletal muscle between Mashen and Large White pigs. The results showed that a total of 362 circRNAs, which included circIGF1R, were differentially expressed between the two pig breeds. Functional assays showed that circIGF1R promoted myoblast differentiation of porcine skeletal muscle satellite cells (SMSCs), while it had no effect on cell proliferation. In consideration of circRNA acting as a miRNA sponge, dual-luciferase reporter and RIP assays were performed and the results showed that circIGF1R could bind miR-16. Furthermore, the rescue experiments showed that circIGF1R could counteract the inhibitory effect of miR-16 on cell myoblast differentiation. Thus, circIGF1R may regulate myogenesis by acting as a miR-16 sponge. In conclusion, this study successfully screened candidate circRNAs involved in the regulation of porcine myogenesis and demonstrated that circIGF1R promotes myoblast differentiation via miR-16, which lays a theoretical foundation for understanding the role and mechanism of circRNAs in regulating porcine myoblast differentiation.

Keywords: circIGF1R; miR-16; myoblast differentiation; pig; skeletal muscle.

Figure 1

DEcircRNA analysis between two breeds. (A): Numner of DEcircRNAs. (B–D): Volcano plot of DEcircRNAs at 1, 90, and 180 days of age, respectively. (E): Venn diagram of DEcircRNAs. (F–H): Venn diagram showing the expression specificity of DEcircRNAs between the two pig species.

Figure 2

Functional enrichment results of the host genes of DEcircRNAs. (A,D): GO and KEGG analysis at 1 day of age; (B,E): 90 days of age; and (C,F): 180 days of age.

Figure 3

CeRNA network of DEcircRNAs. (A): 1 day of age; (B): 90 days of age; and (C): 180 days of age.

Figure 4

Effect of circIGF1R on myoblast differentiation of porcine SMSCs. (A,B): Cell transfection efficiency. (C,D): Myogenic factor expression at the mRNA level. (E,F): MyoD protein expression. (G,H): Cell immunofluorescence. Blue indicates nuclei stained with DAPI; red indicates MyHC protein. ** p < 0.01, * p < 0.05.

Figure 5

Effect of circIGF1R on the proliferation of porcine SMSCs. (A,B): Expression level changes of genes related to cell proliferation. (C,D): The results of CCK8. (E,F): The results of EdU.

Figure 6

CircIGF1R serves as a miR-16 sponge. (A,B): circIGF1R and miR-16 expression in muscle tissue at different stages. (C,D): circIGF1R and miR-16 expression during the myoblast differentiation of pig satellite cells. (E,F): miR-16 expression after overexpression/interference of circIGF1R. (G): RNhybrid was used to predict binding sites of circIGF1R and miR-16. (H): The sequence of wild and mutant type vectors. (I): Dual-luciferase reporter assay. (J): AGO2-RIP assay. Different lower case letters indicate p < 0.05. ** p < 0.01, * p < 0.05.

Figure 7

CircIGF1R promotes myoblast differentiation of SMSCs via miR-16. (A): Expression changes of key myogenic factors after transfection with OE-NC+mimics NC, OE-NC+miR-16 mimics, and OE-circIGF1R+miR-16 mimics. (B): Immunofluorescence staining results after transfection with OE-NC+mimics NC, OE-NC+miR-16 mimics, and OE-circIGF1R+miR-16 mimics. Blue indicates nuclei stained with DAPI; green indicates MyHC protein. ** p < 0.01, * p < 0.05.

Figure 8

Prediction of the translation ability of circIGF1R. (A,B): Translation ability and ORF prediction of circIGF1R. (C): IRES prediction of circIGF1R. (D): m6A site prediction of circIGF1R.

Figure 9

Mechanism of circIGF1R regulating myoblast differentiation through miR-16.