Expression Of Gga-miR-206 In Different Stages Of Skeletal Muscle Development In Jinmao Black Chicken Embryos And Its Targets Identification

miRNAs have recently been found important epigenetic factors,which play an essential role in regulating gene expression at the post-transcriptional level and are widely involved in various life processes of the organism.The embryonic development of the muscle determines the total number of muscle fibers,which will have a crucial impact on the muscle production,growth rate and adult weight of livestock and poultry.In order to explore the role of miRNAs in embryonic skeletal muscle development and identify their new targets,so as to reveal the molecular mechanisms of regulation for muscle development,based on the previous results of up-regulated miRNA:down-regulated mRNA predicted by miRANDA screened in chicken myoblasts from proliferation to differentiation by high-throughput sequencing,one of the key miRNAs gga-miR-206 was selected as the entry point of the study.Combined with results predicted by other databases,FUBP1(far upstream element binding protein 1),TMSB4X(Thymosin Beta 4 X-Linked)and YWHAB(tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta)were eventually chosen as candidate target genes in the study.The expression levels of miRNA and each candidate gene were investigated by quantitative real-time PCR and the correlationships between their expression were respectively analyzed by SPSS 19.0 software;the potential target interaction was further identified by dual luciferase reporter assay system between gga-miR-206 and YWHAB/FUBP1 gene,respectively.Each part was conducted with three independent experiments involved in at least two replicates.The main results were as follows:1.gga-miR-206 and candidate targets developmental expression profiles and the correlation analysis.The relative expression of gga-miR-206 and candidate targets in different stages of skeletal muscle development in chicken embryo was analyzed by qRT-PCR and the correlation was further conducted with pearson method by SPSS 19.0.The results showed that the expression of gga-miR-206 in the leg muscles increased first and then decreased at the whole level,which reached its peak at 16th embryonic age(E16)(P<0.05),and rised a little back in late foetal and post-natal stages.On the other hand,the expression in the pectoral muscle performed a downward trend as a whole,which performed the highest level at 10th embryonic age(E10)(P<0.05),followed by a small peak appearing on 16th embryonic age(E16).Correlation analysis revealed that in the development of embryonic skeletal muscles,the expression of YWHAB was negatively correlated with that of gga-miR-206(P<0.05).Likewise,TMSB4X was also found negatively expressed compared with gga-miR-206,significantly(P<0.05).However,opposite to this,expression of FUBP1 performed a strong positive correlation with that of gga-miR-206(P<0.05).2.Construction of candidate targets tissue expression profiles.qRT-PCR was used to detect the relative expression of three candidate genes in 8 tissues of 300-day-old Jinmao black chickens in further for preliminary functional identification.The results showed that FUBP1 highly expressed in the liver and leg muscles,significantly different from the other tissues(P<0.01),a little in cardiac muscle and little in breast muscle and ovary.In addition,YWHAB significantly high expressed in skeletal muscle tissues compared with the others(P<0.01),with a little in cardiac muscle and lowest in spleen and ovary;TMSB4X existed in all tissues to different degree.By comparison,the expression in the cardiac muscle and breast muscle was the highest,which was very significantly higher than that in the kidney and ovary(P<0.01)and significantly higher than that in the liver,spleen,lung and leg muscles(P<0.05).3.Verification of the target relationship between gga-miR-206 and YWHAB/FUBP1 gene.In view of the above findings,the target relationship identification was further carried out between gga-miR-206 and YWHAB/FUBP1,respectively.Both the wild-type and mutant of YWHAB/FUBP1 3' UTR over-expression pmirGLO vectors were respectively constructed,then were co-transfected into human embryo kidney cells(293T)together with miR-206 mimics and negative control(mimic NC),respectively.As a result,the dual luciferase reporter assay system indicated that the group with wild-type 3' UTR of YWHAB and miR-206 mimics significantly decreased the relative activity of luciferase compared with the negative control(P<0.05),while no significant differences existed in the other cases(P>0.05),indicating that YWHAB performed target binding properties with gga-miR-206.In addition,the key target loci were further confirmed to be 1429-1435th bases of the gene mRNA 3' UTR.On the contrary,FUBP1 showed no targeting interaction to gga-miR-206.