MiR-27b Regulates Myogenic Proliferation And Differentiation By Targeting Pax3 In Goat

MiRNA are a class of non-coding RNAs which represent approximately 18-22 nucleotides,of which the "seed sequence" in the 5' region usually will bind to 3'UTR of the target genes followed by degradation of mRNAs or inhibition of protein translation playing roles in regulating gene expression in life organisms.At present,the influence of miRNA on the growth and development of skeletal muscle was very important.Some miRNAs are expressed in a tissue specific manner.MiRNA which are exclusively or preferentially expressed in striated muscle are called myomiRs.The group currently includes that miR-1,miR-133,miR-206 and so on.This study will explore the function of miR-27b on the proliferation and differentiation of goat skeletal muscle satellite cells,in order to improve the regulation and control network in the process of muscle growth and development,and provide theoretical basis for molecular breeding of goats.(1)The muscle tissue was collected from adult Boer goat and Anhuai goats which were under the same feeding condition.Tissue samples including myocardium,chest muscle,leg muscle,longissimus tnuscle and kidney.With the qRT-PCR,we collected the satellite cells in F7,F10,F13,and we also collected the different cells during differentiation included Id,3d,5d,7d to detect the expression of miR-27b and Pax3.The result showed the expression of miR-27b in adult Boer goat was lower than Anhuai goat,but the expression of Pax3 showed a opposite result.During the proliferation and differentiation of goat skeletal muscle satellite cells,that the expression of miR-27b was reduced gradually,and the expression of Pax3 was increased gradually during proliferation.But the expression of miR-27b was increased during differentiation,the expression of Pax3 was opposite.From the above experimental results,we can deduce that miR-27b can promote the differentiation of goat skeletal muscle satellite cells,but inhibit its proliferation.(2)At first,we predicted the bind site of miR-27b on the Pax3 3'UTR by bioinformatics,we constructed two dual luciferase reporter vectors included wild type and mutant type.MiR-27b mimics and miR-27b NC were cotransfected into the goat skeletal muscle satellite cells with two dual luciferase reporter vectors.The result showed that there was a site in the 3'UTR of Pax3 gene,and miR-27b mimics can reduce the fluorescence values of wild type vector,but it cannot reduce the mutant type vector.So we can come to a conclusion that Pax3 was a target gene of miR-27b.(3)We have verified that Pax3 was a target gene of miR-27b.Over-expressed miR-27b by transfecting miR-27b mimics into cells,we detected the expression of Pax3 mRNA,the result showed that its expression had no significant difference compared with the negative control.This result indicated that miR-27b did not make the Pax3 mRNA degradation to regulate the expression of Pax3 gene.In order to observe the effect of miR-2-7b on the proliferation of skeletal muscle satellite cells,the EdU assay was used to detect the proliferation of satellite cells.The result showed that miR-27b can inhibit the satellite cells proliferation.Then for the sake of understanding the influence of miR-27b on satellite cells differentiation,after tansfecting miR-27b mimics,we detected the expression of MyoG in satellite cells by qRT-PCR.The result showed that miR-27b can promote differentiation of goat skeletal muscle satellite cells.(4)We also over-expressed miR-27b by transfecting the miR-27b mimics,and set up the negative control by transfecting the miR-27b NC.The result of immunofluorescence staining showed that the fluorescence values of miR-27b mimics group were lower than negative control.The result of Western Blot also showed that miR-27b can inhibit the expression of Pax3 protein.In Experiment ?,we have found that the expression of Pax3 mRNA was not changed evidently,but miR-27b can inhibit the expression of Pax3 protein,in conclusion,miR-27b can restrain the translation of Pax3 mRNA to affect the proliferation and differentiation of goat skeletal muscle satellite cells.In summary,in this study,detected the expression of miR-27b and Pax3 in Boer goat and Anhuai goat muscle tissue and goat skeletal muscle satellite cells during proliferation and differentiation,and found that miR-27b can promote the differentiation and inhibit the proliferation,which provided a foundation for further clarify the molecule mechanism of miR-27b in goat skeletal muscle growth and development.