Effects Of Ssc-miR-21-5p On Myogenic Cell Proliferation,Differentiation And Intramuscular Lipid Deposition By Targeting FBXO11

The quality of pork is related to the development of skeletal muscle and intramuscular lipid deposition in pigs,and is influenced by genetic and other factors.The Bama minipig,which is unique to China,is the fatty pig.Its growth rate and meat quality are significantly different from those of Landrace pig,which is the lean pigs.Comparative studies between them can help reveal the formation mechanisms of skeletal muscle and intramuscular fat.As the key post-transcriptional regulators,miRNAs are involved in the regulation of skeletal muscle development and intramuscular lipid deposition in pigs.Therefore,resolving the differentially expressed miRNAs and their effects in skeletal muscle and intramuscular lipids of different meat-quality pigs will provide the theoretical basis for improving pork quality and other production performance.In this study,the transcriptomic data of muscle and adipose tissues of different breeds of pigs were analyzed comprehensively,and the differentially expressed miRNAs were identified by RT-q PCR in the longissimus dorsi muscle and intramuscular adipose tissues of Landrace pigs and Bama minipigs to screen candidate miRNAs which may affect skeletal muscle development and intramuscular lipid deposition in different meat-quality pigs.Then,skeletal muscle satellite cells were isolated and purified from the longissimus dorsi muscle of Landrace pigs and Bama minipigs,and the expression of Pax7 was detected by immunofluorescence for cell identification.The m RNA expression of candidate miRNAs,cell cycle-related factors,myogenic-related factors and adipogenic factors in skeletal muscle satellite cells of two pigs was detected by RT-q PCR,and the proliferation of both cells was detected by CCK-8.The results showed that the five candidate miRNAs that were screened(ssc-miR-133 b,ssc-miR-21-5p,ssc-miR-128-3p,ssc-miR-99b-5p and sscmiR-30d-5p)were differentially expressed between skeletal muscle satellite cells of two pigs(P<0.05),and two kinds of porcine skeletal muscle satellite cells exhibited different proliferative ability and differentiation potential: Compared with Bama minipigs,the skeletal muscle satellite cells from Landrace pigs showed higher proliferative ability and higher myogenic differentiation potential and lower adipogenic differentiation potential.The target genes of the five candidate miRNAs were predicted by using the miRNA target gene prediction tools,and screened by integrating the binding site of target gene 3’UTR to candidate miRNAs and homology analysis in pigs and mice.The candidate target genes were further screened by RT-q PCR to detect the m RNA expression of the screened target genes at the tissue level and cellular level between Landrace pigs and Bama minipigs.Then,candidate miRNAs mimics were transfected into porcine skeletal muscle satellite cells,and the m RNA expression changes of candidate target genes were detected by RT-q PCR.The results showed that among the candidate target genes whose expression was down-regulated,ssc-miR-21-5p mimic had the most significant inhibitory effect on FBXO11(P<0.01).Therefore,sscmiR-21-5p-FBXO11 was identified as the study target and the bioinformatics analysis of ssc-miR-21-5p was performed.The temporal expression of ssc-miR-21-5p during the differentiation of C2C12 and 3T3-L1 was detected by RT-q PCR.The results showed that the expression of ssc-miR-21-5p showed an overall increasing trend during myogenic differentiation of C2C12,while an overall decreasing trend during adipogenic differentiation of 3T3-L1.To further verify the targeting relationship between ssc-miR-21-5p and FBXO11,the dual-luciferase reporter vectors were constructed.ssc-miR-21-5p mimic and inhibitor were transfected into C2C12,and the expression of FBXO11 was detected by RT-q PCR and Western blotting.The results showed that ssc-miR-21-5p targeted FBXO11 3’UTR and inhibited its m RNA and protein expression(P<0.05).To further determine the role played by ssc-miR-21-5p-FBXO11 in skeletal muscle development and intramuscular lipid deposition,the proliferating state,adult muscle differentiation state,and post-differentiation oleic acid-treated C2C12 cells were transfected with ssc-miR-21-5p mimic,ssc-miR-21-5p inhibitor and si-FBXO11.Cell proliferation was detected by CCK-8,m RNA expression levels of cell cyclerelated factors,myogenic-related factors and adipogenic-related factors were detected by RT-q PCR,the protein expression levels of Myo D were detected by Western blotting,MYHC immunofluorescence staining was observed to calculate the cell fusion index of myotubes,and Oil red O staining was performed to observe the number of lipid droplets deposited in myotubes.The results showed that overexpression of ssc-miR-21-5p and silencing of FBXO11 both significantly promoted the proliferation of C2C12 and the expression of cell cycle-related factors and myogenesis-related factors(P<0.05)and increased the formation of C2C12 myotubes,but inhibited oleic acid-induced lipid accumulation in myotubes of myogenic cells and significantly decreased the expression of adipogenic-related genes(P<0.05);The opposite result was obtained with inhibiting the expression of ssc-miR-21-5p.In conclusion,this study identified ssc-miR-21-5p as a key regulator of skeletal muscle development and intramyocellular lipid deposition in different skeletal pigs,which promoted the proliferation and differentiation of myogenic cells and inhibited the intramyocellular lipid deposition by targeting the 3’UTR of FBXO11.It provides new theoretical data to elucidate the mechanisms associated with the differences in skeletal muscle formation in different meat-quality pigs.