Screening Of Key MiRNA-mRNA For Muscle Development In Longlin Goat And Study On The Function Of MiR-495-3p

To reveal the molecular regulation mechanism of miRNA on goat muscle growth and development,the longissimus dorsi muscle samples of 1-month-old and 10-month-old Longlin goats were used as materials,and the differentially expressed miRNA and mRNA were screened byRNA-seq technique,bioinformatics analysis was performed on the differentially expressed miRNAs and mRNAs closely related to muscle traits.The predicted target genes of differentially expressed miRNAs and the mRNA screened by sequencing were enriched by GO and KEGG pathways,and the function of candidate miRNAs in goat skeletal muscle satellite cells was verified,The main results are as follows:1.The miRNA sequencing Library of the Longlin goat was constructed successfully.A total of 233 differentially expressed miRNAs(P<0.05)were screened by filtration quality control and other steps.Compared with 10-months group,103 up-regulated miRNAs and 130 down-regulated miRNAs were found in 1-month group.10 randomly selected differential miRNAs were verified by q PCR,and the results were the same as the sequencing results.Functional enrichment and pathway analysis of target genes of differentially expressed miRNA showed that the Notch signaling pathway and Wnt signaling pathway related to muscle development were enriched,and many target genes were related to the development and protein synthesis.2.Through high-throughput sequencing of longissimus dorsi muscle of1-month-old and 10-months-old goats,1999 differentially expressed genes were screened(P<0.05).Compared with 10 months old Longlin goats,938up-regulated differentially expressed genes and 1061 down-regulated differentially expressed genes were found in Longlin goats.Pax7,ACTC1,Myf5,IGF2,BMP1,MEF2 A genes affect muscle development.Functional and pathway enrichment analysis was performed for the differentially expressed genes showed that the differentially expressed genes were enriched in biological processes such as development,and were also annotated into the PPAR signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway and other signaling pathways related to muscle development.3.miR-495-3p was screened from the results of miRNA sequencing.Combined with transcriptome sequencing and software,it was predicted that the target gene of miR-495-3p might be the ACTC1 gene.The tissue expression profile analysis of 1-month-old Longlin goat showed that miR-495-3p was the highest expressed in spleen,and higher expressed in the dorsal muscle and leg muscle;ACTC1 was the highest expressed in the myocardium,followed by dorsal muscle and leg muscle.It is speculated that miR-495-3p may target ACTC1 to regulate muscle development.4.To explore the effect of miR-495-3p on the proliferation and differentiation of goat skeletal muscle satellite cells and the expression of ACTC1,we used the research methods of miR-495-3p overexpression and inhibition.CCK-8 test showed that overexpression and inhibition of miR-495-3p had no significant effect on the proliferation of goat skeletal muscle satellite cells.However,overexpression of miR-495-3p could promote the expression of Myo G,Myf5 and ACTC1,inhibition of miR-495-3p decreased the expression of Myo G,Myf5 and ACTC1.miR-495-3p can target ACTC1 and promote the differentiation of goat skeletal muscle satellite cells.5.The wild-type and mutated vectors of ACTC1 gene were successfully constructed,and the dual luciferase reporting test proved that miR-495-3p had a targeting relationship with the 3’UTR of ACTC1 gene.Based on the above results: miR-495-3p and its possible target gene ACTC1 were screened through miRNA sequencing and transcriptome sequencing.Functional verification showed that miR-495-3p could promote the differentiation of goat skeletal muscle satellite cells and bind to the 3’UTR of ACTC1 gene.