The Study On The Functions And Mechanisms Of PLAG1 Regulating Skeletal Muscle Development In Cattle

Skeletal muscle is an important tissue involved in voluntary movement,energy metabolism,and secretion and is the primary product of livestock.The growth and development of skeletal muscles are key determinants of muscle mass and livestock meat quality,and have an important impact on the economic benefits of animal husbandry.In addition to the key factor named MRF,many factors,including multiple genes,non-coding RNAs,and transcription factors,play critical roles in regulating muscle growth and development.Pleomorphic adenoma gene 1(PLAG1)encodes a zinc-finger transcription factor that plays an essential role in affecting biological growth and development.However,research on whether PLAG1 is involved in muscle development is very limited,and the function and molecular mechanism remain unclear.This study investigated the function and regulatory mechanisms of PLAG1 in Qinchuan cattle with molecular and bioinformatics methods.The results are as follows:1.A novel polymorphism of the PLAG1 gene and its association with growth traits in cattleThis study investigated an SNP(g.48308C>T)of bovine PLAG1 in 646 cattle from Qinchuan cattle(QC),Jiaxian cattle(JX),Xianan cattle(XN),Pinan cattle(PN),and Ji’an cattle(JA)using the PCR-RFLP method.The results of association analysis between the SNP(g.48308C>T)of the PLAG1 gene and growth traits of beef cattle showed that g.48308C>T locus had a significant effect on cattle body height in QC,JX and XN.In these three breeds,TT type individuals had the lowest body height(P<0.05),and these was no significant difference of body height between TC type individuals and CC type individuals(P>0.05).This locus also had a significant effect on the heart girth of PN,QC and JX cattle and individuals of TC type have larger heart girth than that of TT type individuals(P<0.05).The mutation was also significantly correlated with the body length of PN,QC,and JX,and TT type individuals had lower body length than those of TC type individuals and CC type individuals(P<0.05).The height at hip cross of PN,QC and XN cattle was also affected by this SNP,and TT type individuals had the lowest height at hip cross(P<0.05).These results indicated that the SNP may be associated with the body height,body height,heart girth,and the height at the hip cross to affect cattle growth traits.2.Detection of different transcripts and the tissue expression profiles of PLAG1 gene in Qinchuan cattleThis study identified four transcripts of PLAG1 and explored the expression manners across multiple tissues and developmental stages.The results indicated that the expression of PLAG1 was more active in the fetal stage,and had relatively higher expression levels in fetal heart,kidney,and skeletal muscle;and had a relatively higher expression levels in adult heart,spleen,and skeletal muscle.The expression results of 4 types of PLAG1 transcripts in muscle tissue suggested that the expression patterns of PLAG1-X3 and-X4 were more similar to PLAG1,and the expression of PLAG1-X4 in fetal skeletal muscle tissue was significantly higher than those in adult bovine skeletal muscle tissue(P<0.05).Moreover,the PLAG1-X2 and-X5 had higher expression levels in adult stage than those in fetal stage and the expression of PLAG1-X2 in adult bovine skeletal muscle was significantly higher than that in fetal muscle tissue(P<0.05).In addition,the amino acid sequence of the PLAG1 gene had the closest evolutionary distance among ruminants,and was highly conserved in many species such as humans,mice,and cattle.Twenty-two muscle development-related proteins,including NCAPG,LCORL,MSTN,CHCHD7,HMGA2,and IGF2,were predicted to have strong interactions with PLAG1,and the m RNA expression trends were the same.These results suggest that PLAG1 may be involved in the regulation of bovine skeletal muscle development.3.The effects of PLAG1 on regulating myoblast proliferation,apoptosis and muscle repair after injuryThe overexpression vector of PLAG1 or siRNA was transfected into bovine primary myoblasts for gain-of-function and loss-of-function tests.The results of CCK-8 assay,Ed U proliferation assay,RT-q PCR,Western blot,and flow cytometry showed that PLAG1 could enhance cell viability,the percentage of proliferative cells,expression level of PCNA,and the proportion of S phage cells,which indicated that PLAG1 promotes myoblast proliferation.RT-q PCR,Western blot,and flow cytometry for Annexin V-FITC/PI as well as JC-1staining were performed to detect myoblasts apoptosis and the results suggested that PLAG1 could inhibit the apoptosis of bovine primary myoblasts.The muscle injury model was induced by cardiotoxin,and then PLAG1 was overexpressed to detect its effects on muscle repair.It was found that PLAG1 could upregulate the expressions of PAX3,PAX7,Cyclin D1 and CDK2,indicating that PLAG1 played a role in promoting muscle repair after injury.4.PLAG1 promotes proliferation and inhibits apoptosis of bovine primary myoblasts through the PI3K-AKT signaling pathwayChromatin immunoprecipitation sequencing(Ch IP-seq)was performed to identify the target genes of PLAG1 protein,and the results showed that the annotated genes can be enriched in PI3K-AKT signaling pathway,MAPK signaling pathway,c AMP signaling pathway,etc.,among which the number of genes enriched in PI3K-AKT signaling pathway was the most(reached to 122 genes).Western blot showed that PLAG1 could upregulate p-PI3 K,PI3K,p-AKT,AKT,Cyclin D1 and CDK2 and inhibit the expression of p21 and p27 to enhance myoblast proliferation,and inhibit the protein level of BAD to release Bcl-2 and Bcl-x L to inhibit apoptosis.5.miR-1 mediates a potential positive feedback regulation between PLAG1 and the PI3K-AKT signaling pathwayThe results of RT-q PCR showed that there might be a positive feedback regulation of PLAG1 and PI3K-AKT signaling pathways with LY294002 or 740 Y-P treated primary bovine myoblasts.A large number of Cp G islands in the PLAG1 promoter region suggested that DNA methylation might be involved in mediating this positive feedback regulation,but bisulfite sequencing PCR confirmed that DNA methylation was not involved in this feedback regulation.The dual-luciferase reporter assay revealed that miR-1 could target PLAG1,and miR-1 acted as a feedback regulator between PLAG1 and PI3K-AKT signaling pathway.PLAG1 increased the phosphorylation of PI3 K and AKT proteins to activate the PI3K-AKT signaling pathway,thereby inhibiting the downstream gene-Fox O3.The transcription factor Fox O3 can regulate the transcription of miR-1,and the reduction of Fox O3 led to the reduction of miR-1.In turn,reduced miR-1 expression led to the release of PLAG1,which constituted a closed positive feedback regulatory loop.6.Exosomal miRNAs participates in regulating the expression of PLAG1The exosomes released from bovine primary myoblasts were identified by electron microscopy and particle size analysis.A total of 36 exosomal miRNAs were identified using miRNA transcriptomes analysis.Exo-bta-miR-10 a,exo-bta-miR-122,exo-bta-miR-451,and exo-bta-miR-486 were upregulated in exosomes,and another 32 exosomes were downregulated in exosomes.The dual-luciferase reporter assay suggested that miR-181 a not only targets PLAG1 in cells,but also may be transported into exosomes to regulate the expression of PLAG1 in recipient cells,which provided new insight into the regulatory mechanism of PLAG1.This study investigated the effects of PLAG1 on Qinchuan cattle,which included the influence of the SNP site in PLAG1 on cattle growth traits,identification of PLAG1 transcripts and the expression patterns,the effects of PLAG1 on the proliferation and apoptosis of Qinchuan cattle primary myoblasts,the effects on muscle repair,and the regulatory mechanism between PLAG1 and the PI3K-AKT signalling pathway.Moreover,this study explored the potential regulatory relationship between exosomal miRNA and PLAG1.Altogether,those findings deepened the understating of PLAG1 function on muscle development,which help provide candidate gene for cattle breeding in the future.