Function And Regulation Of Gga-miR-205a In Chicken Primary Myoblast

MicroRNA is a kind of endogenous non-coding RNA,which is located in the non-coding region of the genome or the intron of the gene.They usually modulate gene expression post-transcriptionally through base pairing with the 3'-untranslated region(UTR)of target mRNA to inhibit translation of their specific targets.Previous study on miRNA sequencing of the two-tail samples(highest and lowest body weight)of breast muscle tissues from Recessive White Rock and Xinghua Chickens found that miR-205 a was one of the differentially up-regulated miRNAs.MiR-205 a and CDH11 gene were also differentially expressed in the high-throughput sequencing of leg muscles from female Xinghua Chicken at three developmental time points,11 embryo age,16 embryo age and 1 day post hatch.Therefore,miR-205 a and CDH11 gene are selected as candidates to explore their roles in myoblasts,and the molecular mechanism of the regulation of miR-205 a.Flow cytometry and EdU cell proliferation detection demonstrated that miR-205 a can inhibit the proliferation of both QM-7 cells and chicken primary myoblasts,MyHC immunofluorescence staining results show that miR-205 a can promote the differentiation of primary myoblast and the expression of MyHC gene.CDH11 is a potential target gene of miR-205 a predicted by bioinformatics.Dual-luciferase reporter assay showed that miR-205 a could inhibit the expression of CDH11 by combining with its 3'UTR,qRT-PCR further identified that miR-205 a could negatively regulate the expression of CDH11 at a post-transcriptional level.The result of flow cytometry and EdU cell proliferation detection showed that CDH11 could promote the proliferation of QM-7 cells and chicken primary myoblasts,which was exactly the opposite of miR-205 a.At the meantime,the results of MyHC immunofluorescence staining,the results of the statistical results of myotube area,and the qRT-PCR results showed that CDH11 could inhibit the differentiation of myoblasts and the expression of MyoD,MyoG,MyHC genes.We constructed a series of 5' deletion promoter PGL3-Basic vector on the upstream sequence of pre-miR-205 a and found the potential promoter active region by dualluciferase reporter assay.In the potential active region we predicted two possible binding transcription factors HNF-1 and MyoG/NF-1 through bioinformatics.We detected the activity of the potential promoter active region and found that the mutation of the transcription factors binding site could remarkably change the relative expression of the activity region.In conclusion,this study reveals the function of miR-205 a in myoblast and its regulation mechanism,which provides new data for miRNA regulate skeletal muscle proliferation and differentiation.