The Effection On Myoblasts By MiR-340-5p Through Targetting Bai3, ROCK1, DMD

micro RNAs(mi RNAs),one kind of small non-coding RNA, which are containing 20-25 nucleotides in length, play key roles in myogenesis. Bai3 is a key gene forthe myoblasts fusion. DMD is related to a muscular disease, Duchenne. ROCK1 could promote theproliferation and migration of cells. The Dual-Luciferaseassay was performed to detect whether Bai3, DMD and ROCK1 were target genes of mi R-340-5p. Then, the expression levels of Bai3, DMD, and ROCK1 were detected by q RT-PCR and Western Blottingwith the overexpression and knockdown of mi R-340-5p. Finally, the function of mi R-340-5p for myoblasts fusion, proliferation and migrationwas identified through the methods such as immunofluorescence, x CELLigence system and wound-healing migration assay. The results of this study are as follows:(1) The sequence of mi R-340-5p was highly conservation in different species, including human, mouse, rat,pig and etc.(2) In order to investigate whether mi R-340-5p targets the 3’-UTR of Bai3, the Dual-Luciferase report system recombinant plasmid, and mutant recombinant plasmid of Bai33’UTR was constructed and co-transfected with mi R-340-5p mimics into BHK cells respectively. The luciferase of wild groups was significantly down-regulated, whereas the mutant groups was not, which confirmedthat mi R-340-5p targets 3’UTR of Bai3. Byobserving the expression of Bai3 in different periods of differentiation in C2C12, we found that Bai3 expression got to the peak value at the 5th day. The protein levels of Bai3 were down-regulatedwith the transfection with mi R-340-5p mimics atthe 5d after stimulating to myoblasts differentiation. Hence Bai3 should be a target of mi R-340-5p. Furthermore,myoblasts fusion could be suppressed by mi R-340-5p,which confirmed mi R-340-5p could inhibit Bai3 to repress myoblasts fusion.(3) In order to investigate whether miR-340-5p targets the 3’-UTR of ROCK1, the Dual-Luciferase report system recombinant plasmid, and mutant recombinant plasmid of ROCK13’UTR was constructed and co-transfected with mi R-340-5p mimics into BHK cells respectively. The luciferase of wild groups was significantly down-regulated, whereas the mutant groups was not, which confirmedthat mi R-340-5p targets 3’UTR of ROCK1. Byobserving the expression of ROCK1 in different periods of differentiation in C2C12, we found that ROCK1 expression got to the peak value at the 5th day. The protein levels of ROCK1 were down-regulatedwith the transfection with mi R-340-5p mimics atthe 5d after stimulating to myoblasts differentiation. Hence ROCK1 should be a target of mi R-340-5p. Furthermore,myoblasts fusion could be suppressed by mi R-340-5p,which confirmed mi R-340-5p could inhibit ROCK1 to repress myoblasts fusion.(4) Byobserving the expression of DMD in different periods of differentiation in C2C12, we found that DMD expression was increased accompanied bydifferentiation proceeding. The protein levels of DMD were down-regulatedwith the transfection with mi R-340-5p mimics atthe 5d after stimulating to myoblasts differentiation.Therefore, we demonstrate that DMD should be a target of mi R-340-5p.