| Duroc pigs are characterized by fast growth and high lean meat percentage,but their growth and meat production performance gradually decreased after they reach 110 kg body weight.Therefore,it is not only the development trend of modern pig breeding,but also the key problem to be solved in this study to determine and breed the individuals who can continue to maintain good growth and meet production performance during the 110-130 kg body weight stage.In this study,the performance of Duroc pigs was measured,four individuals with high ADG(Average daily gain)(774.89 ± 39.03 g)(H group)and low ADG(658.77 ± 36.20 g)(L group)were selected from Duroc pig population with ADG of 110-130 kg body weight,and the longissimus dorsi muscle(LDM)was analyzed by whole transcriptome sequencing.Through ncRNA-mRNA association analysis,the relationship network of differentially expressed gene miR-874/MEOX1 related to skeletal muscle growth and development was screened out,and the effects and mechanisms of miR-874/MEOX1 on proliferation and differentiation were investigated in this study.1.The gene expression profiling associated with different ADG in Duroc pigs was analyzed through whole transcriptome sequencing(1)A total of 703 DE(differentially expressed)mRNAs were identified,including MEOX1,MYO1 F,SH2B3,PIK3R5,PLIN2,ACSL5 and MEF2 C,which were related to skeletal muscle growth and development.The DE mRNAs were enriched in GO terms including cell proliferation,positive regulation of MAPK cascade,positive regulation of ERK1 and ERK2 cascade,binging to myosin heavy chain and cell differentiation.KEGG analysis showed DE mRNAs were enriched in PPAR,Gn RH and production of steroids.(2)A total of 74 DE lncRNAs were identified.Functional enrichment analysis showed that lncRNAs mainly regulated mRNAs expression in a Trans mode of action,and the GO terms of their target genes mainly enriched included cellular process,biological regulation and metabolic process;and KEGG analysis showed that target genes were enriched in pathways including complement and coagulation cascades,interactions between viral proteins and cytokines and cytokine receptors and lysosomes.(3)A total of 296 DE circRNAs were identified,which were enriched in GO terms including cellular process,metabolic process,biological regulation and catalytic activity,and KEGG analysis showed that circRNAs were enriched in thyroid hormone signaling pathway,cell aging,Fox O signaling pathway,prolactin signaling pathway and growth hormone synthesis,secretion and action.(4)A total of 51 DE miRNAs were identified,including ssc-miR-874,ssc-miR-375,sscmiR-141,ssc-miR-216,ssc-miR-146a-5p and ssc-miR-21-3p.GO analysis showed that the target genes were enriched in GO terms including cellular processes,metabolic processes,biological regulation,cells,cell components and catalytic activity.The significantly enriched pathways included MAPK,Fo XO,m TOR,actin cytoskeleton regulation,phosphatidylinositol signaling system,and sphingolipid signaling pathways.(5)Through ncRNA-mRNA association analysis,the target relationship pairs such as ssc-miR-874/MEOX1,ssc-miR-874/MYO1 F,ssc-miR-4331-3p/MAP2K6,ssc-miR-21-3p/ETAA1,ssc-miR-216/IGFBP4 and ssc-miR-10386/ARRB2 were found.Among them,the expression level of miR-874 was the highest,and the difference was the most significant.MEOX1 gene could be mapped to carcass length,body weight,backfat thickness and marbling and other QTLs related to growth and meat quality traits.Therefore,miR-874/MEOX1 was taken as the main targets for exploring the mechanisms of skeletal muscle growth and development.2.The functions and mechanisms of miR-874/MEOX1 in cell proliferation and myogenic differentiation of C2C12 cellsIn this study,C2C12 cells were used as the experimental object.C2C12 cells were transfected with miR-874 or MEOX1 to detect the expression of cell activity,cell cycle,proliferation marker genes(PCNA,CDK2,CDK4 and Cyclin D1)and myoblast marker genes(Myo D,Myo G and MEF2C).The results were as follows:(1)It was predicted that MEOX1 was a target gene of miR-874 by Target Scan and Miranda.Dual-luciferase assay showed that miR-874 mimics significantly reduced the luciferase activity of the GP-miRGLO-MEOX1-Wt reporter gene,confirming the targeting relationship between the two genes.(2)The effects of miR-874 on cell proliferation and myogenic differentiation of C2C12cells:(1)During cell proliferation process,CCK-8 and Ed U assay showed that miR-874 mimics significantly inhibited cell activity and Ed U-positive cells,flow cytometry results showed that the number of cells in S phase was decreased,and the mRNA expression of proliferative marker genes was significantly decreased(P < 0.05).In addition,miR-874 inhibitor promoted cell activity and EDU-positive cells,and the number of S phase and mRNA expression of proliferative marker genes was significantly increased(P < 0.05).These results strongly suggested that miR-874 can inhibit the proliferation of C2C12 cells.(2)During myogenic differentiation process,the expression of miR-874 was first down-regulated and then up-regulated,while the expression of MEOX1 was first up-regulated and then downregulated,and reached the highest value on d 6,and the mRNA expression of Myo D and Myo G also gradually up-regulated,as did the cell fusion index,and myotube formation was evident.On d 0 and d 6,miR-874 mimics can significantly down-regulate the mRNA expression of myogenic marker genes and MEOX1;in addition,the expression of myogenic marker proteins and MEOX1 protein were significantly down-regulated(P < 0.05).After miR-874 was interfered,the results were opposite,the mRNA and protein expression of myogenic marker genes were significantly up-regulated(P < 0.05).These results indicated that miR-874 inhibited myogenic differentiation by targeting MEOX1.(3)The effects of MEOX1 on cell proliferation and myogenic differentiation of C2C12cells:(1)During cell proliferation process,CCK-8 and Ed U assay showed that pc DNA3.1-MEOX1 can significantly increase cell activity and Ed U-positive cells,the cell count of S phase was significantly increased,and the mRNA expression of proliferative marker genes was significantly up-regulated(P < 0.05).On the contrary,si-MEOX1 can significantly inhibited the cell activity and Ed U-positive cells,the cell count of S phase and the mRNA expression of proliferative marker genes was significantly down-regulated(P < 0.05).These results indicated that MEOX1 played a positive regulatory role in cell proliferation.(2)During myogenic differentiation process,the expression of myogenic marker genes and their proteins were significantly up-regulated in pc DNA3.1-MEOX1 group(P < 0.05).After MEOX1 interference,mRNA and protein expression of myogenic marker genes were significantly down-regulated.These results suggested that MEOX1 can promote cell differentiation.(4)In the co-transfection test,C2C12 cells were transfected with miR-874 mimics and pc DNA3.1-MEOX1,the mRNA expression of myogenic marker genes and proteins were detected in rescue assay.It was found that on d 0,the mRNA and protein expression of myogenic marker genes were significantly up-regulated(P < 0.05),but on d 6,there was no significant difference in My HC protein.These results showed that overexpression of MEOX1 reversed the negative regulatory effect of miR-874 on cell differentiation.In conclusion,whole transcriptome sequencing analysis was performed on longissimus dorsi muscle of Duroc pigs with different ADG,and it was found that DE genes(MEOX1,MYO1 F,SH2B3,PIK3R5,PLIN2,ACSL5 and MEF2C)and the target relationship pairs(sscmiR-874/MEOX1,ssc-miR-874/MYO1 F,ssc-miR-4331-3p/MAP2K6,ssc-miR-21-3p/ETAA1,ssc-miR-216/IGFBP4 and ssc-miR-10386/ARRB2)were related to the skeletal muscle growth and development or lipid metabolism.In addition,miR-874 can mediate MEOX1 to regulate cell proliferation and differentiation,thereby regulating skeletal muscle growth and development. |
