Mutation in myostatin 3'UTR promotes C2C12 myoblast proliferation and differentiation by blocking the translation of MSTN.

The point mutation in myostatin (MSTN) can produce the Texel sheep double muscle phenotype. However, whether other species have the same mode of action as MSTN and whether breeding materials can be obtained through cross-species genetic editing remain unclear. The mutation in the mouse MSTN 3'UTR could create a target site for mmu-miR-1/206, as verified by the dual luciferase reporter system. A C2C12 cell model with the mutation in MSTN 3'UTR was constructed using CRISPR/Cas9 gene editing. Then, the mRNA and protein expression of MSTN was analyzed in the mutant C2C12 cell model. Results revealed that the mutation blocked the translational level of MSTN. By inhibiting mmu-mir-206, low expression of MSTN protein in mutant C2C12 cell can be rescued. Furthermore, the proliferation and differentiation abilities of the mutant C2C12 cell model were tested by RT-PCR, CCK8 analysis, EDU (5-ethynyl-2'-deoxyuridine) proliferation analysis, immunofluorescence analysis, Western blot, and myotube fusion statistics. This study may serve as a reference for elucidating the function and molecular mechanism of MSTN and as a foundation for accurate breeding improvement.

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