Screening Of Body Weight Related MiRNAs And Functional Validation Of Mir-460b-5p In Jinghai Yellow Chicken

The meat production of broilers is crucial to economic benefits of broiler industries,while the slaughter performance of broilers is directly determined by skeletal muscle development.Hence,the broiler breeding for growth traits shows a great importance.As a kind of small noncoding RNA,miRNAs can regulate the expression of multiple genes and perform a wide range of regulation in organisms.Currently,more and more studies have confirmed that miRNAs are closely associated with skeletal muscle development of chickens.Jinghai yellow chicken is one of the high quality yellow-feathered broiler breeds in China,and molecular regulation theories related to its growth are also gradually improving.In this study,through transcriptome sequencing technology,the differential analysis of miRNA expression profiles in leg muscle from fast-and slow-growing groups of Jinghai yellow chickens were performed,and the miRNAs with close relevance to skeletal muscle development were screened out subsequently.Furthermore,functional studies and targeting relationship verifications of the key miRNA were conducted to provide a theoretical basis for molecular breeding of Jinghai yellow chicken.The research results are as follows:1.Through transcriptome sequencing analysis,12 differentially expressed miRNAs were screened(novel-miR-133,novel-miR-318,novel-miR-151,novel-miR-304,novel-miR-174,novel-miR-302,gga-miR-1416-5p,gga-miR-338-5p,gga-miR-100-5p,gga-miR-30a-3p,ggamiR-460b-5p and gga-miR-24-3p).The prediction analysis showed that a total of 150 potential target genes were targeted by 12 differentially expressed miRNAs.After the analysis of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG),150 predicted target genes were significantly enriched in 148 GO terms and 5 KEGG pathways(p<0.05),including positive regulation of BMP signaling pathway(GO:0030513),positive regulation of multicellular organismal process(GO:0051240),cell cycle(gga04110),NOTCH signaling pathway(gga04330)and other biological processes and signaling pathways related to growth and development.According to protein interaction network analysis of predicted target genes,20 hub genes with high "degree centrality" were further determined.Combined with above analysis,3 miRNAs(novel-miR-133,gga-miR-24-3p and gga-miR-460b-5p)may involved in the growth regulation of Jinghai yellow chicken were ultimately screened out.2.Based on the results of sequencing analysis,miR-460b-5p was selected for functional assays at the cellular level.At first,the expression pattern of miR-460b-5p was investigated in the proliferation and differentiation stages of chicken primary myoblasts.It was showed that the expression level of miR-460b-5p gradually decreased from the proliferation stage(GM 50%)to the lowest at 24 h of differentiation.As differentiation proceeded,miR-460b-5p expression increased significantly(p<0.05),reaching the highest and stabilizing at 72 h and 96 h of differentiation.Through mRNA quantitative analysis of proliferation marker genes,CCK-8 and EdU assays,miR-460b-5p was found to significantly facilitate the transition of myoblasts from G1 to S phase(p<0.01)and promote chicken myoblast proliferation.In addition,mRNA and protein quantitative analysis of differentiation marker genes,as well as the indirect immunofluorescence results of my otubes,revealed that miR-460b-5p significantly stimulated myotube development(p<0.05)and promote chicken myoblast differentiation3.The target relationship validation of miR-460b-5p was further performed according to the target gene prediction results.In the dual-luciferase reporter assay,the firefly luciferase activity of RBM19 wild-type plasmid was significantly inhibited(p<0.01)after transfection with miR-460b-5p.Quantitative analysis showed that the mRNA expression of RBM19 was significantly decreased(p<0.05)with overexpression of miR-460b-5p in myoblasts,while the contrary result was observed after interference with miR-460b-5p(p<0.05).In summary,miR460b-5p was able to regulate RBM19 expression by specifically binding to the 3’ UTR of RBM19.