Effect Of MiR-144 On Proliferation And Differentiation Of Cattle Skeletal Muscle Cells And Screening Of Its Target Ene

Skeletal muscle is the main component of livestock and poultry weight.The speed of its growth and development directly affects the meat production of livestock and poultry and the economic benefits of farms.Therefore,the exploration of skeletal muscle development mechanism is of great significance to livestock and poultry production.miR-144 is one of the differentially expressed miRNAs in high and low residual feed intake(RFI)of beef cattle screened by the research group through full transcriptome sequencing in the early stage,and previous studies have shown that miR-144 is highly expressed in muscle tissue.Therefore,this experiment takes bovine miR-144 as the research object to explore the expression mode of miR-144 in different tissues and different muscle types Analyze its regulatory effect on proliferation,differentiation and apoptosis of bovine skeletal muscle cells and its effect on mitochondrial synthesis and autophagy in skeletal muscle cells,and finally screen the potential target gene of miR-144.The results will provide theoretical basis and molecular materials for the molecular regulation mechanism of miR-144 in bovine muscle development.The main results are as follows:(1)Molecular characteristics and tissue expression analysis of cattle miR-144miR-144 was highly conserved in different species,and the target genes were significantly enriched in MAPK,TNF,FoxO and other signal pathways related to muscle development(P<0.05).The expression of miR-144 was the highest in cattle liver and the lowest in subcutaneous fat.The expression of miR-144 in liver was significantly higher than that in leg muscle and heart tissue(P<0.05);The expression in longissimus dorsi muscle was significantly higher than that in rumen and subcutaneous fat(P<0.05),but there was no significant difference between rumen and subcutaneous fat tissue(P>0.05);The expression of miR-144 in heart and longissimus dorsi muscle was significantly higher than that in subcutaneous fat(P<0.05),but the expression of miR-144 in the same tissue at different stages was not significant(P>0.05).(2)Effect of miR-144 on proliferation and differentiation of cattle skeletal muscle cellsRT-qPCR was used to detect the expression pattern of miR-144 in cattle skeletal muscle cells during proliferation and differentiation.The results showed that the expression of miR-144 decreased significantly in the proliferation stage and increased in the differentiation stage.At the same time,the mRNA expression levels of differentiation marker genes MYOD1,MYOG and MYH3 were up-regulated,indicating that the cattle skeletal muscle cell model was successfully constructed;miR-144 simulant,inhibitor and corresponding negative control were transfected to detect the expression of miR-144 and proliferation and differentiation related genes.The results showed that the expression of miR-144 was significantly up-regulated(P<0.01)and the expression of CyclinD1,CDK2,CDK4 and PCNA were significantly down regulated(P<0.05);When miR-144 was overexpressed during differentiation,the expression of differentiation marker genes MYOG,MYOD1,MYH3 and MYF6 was significantly upregulated(P<0.01),while it was significantly down regulated after inhibiting miR-144(P<0.01);The above results preliminarily showed that miR-144 inhibited the proliferation and promoted the differentiation of cattle skeletal muscle cells.(3)Effects of miR-144 on apoptosis and mitochondrial synthesis of cattle skeletal muscle cellsRT-qPCR was used to detect the effects of miR-144 on mitochondrial biosynthesis,autophagy and apoptosis in cattle skeletal muscle cells.The results showed that after overexpression of miR-144,the mitochondrial biosynthesis gene PGC-1α、The mRNA expressions of Nrf1,TFAM,division genes Drp1,MTP18,mitochondrial autophagy gene LC3B and apoptosis genes Bax and P53 were up-regulated,while the mRNA expressions of fusion genes Mfn2 and Opa1 were down regulated;The results were opposite after inhibition of expression,which preliminarily showed that miR-144 participated in mitochondrial biosynthesis by promoting mitochondrial division and inhibiting fusion;At the same time,miR-144 promotes mitochondrial autophagy and cattle skeletal muscle cell apoptosis.(4)Mining potential target genes of cattle miR-144After overexpression of miR-144,476 differentially expressed genes were screened by transcriptome sequencing,of which 310 were up-regulated and 166 were down regulated.The key genes were MAFK,EGR2,TFAP4,ERG,PLCB1,EGR3,FGF18 and so on;Differential genes were significantly enriched in MAPK,PI3K-AKT,cGMP-PKG and other signal pathways related to muscle development.