Effects Of PDLIM3 And PDLIM5 Genes On Proliferation And Differentiation Of Chicken Skeletal Muscle Satellite Cells

The PDZ-LIM superfamily member is present in the nucleus of many cells and consists of one PDZ domain at the N-terminus and one or more LIM domains at the C-terminus.The family includes 10 genes,of which PDLIM3 and PDLIM5 genes have been shown to promote mouse myoblast differentiation and participate in the regulation of skeletal muscle growth and development.To explore whether chicken PDLIM3 and PDLIM5 genes have the same function,this study explored the expression of PDLIM3 and PDLIM5 genes in the before and after birth of chicken,and after the chicken skeletal muscle satellite cells cultured in vitro interfered with the expression of these two genes,respectively.The proliferation and differentiation of satellite cells were finally analyzed by transcriptome sequencing.The molecular pathways involved in the regulation of cell proliferation and differentiation were analyzed,and the interaction of PDLIM3 and PDLIM5 genes on skeletal muscle development was further explored.The main results are as follows:(1)RT-PCR results showed that the expression levels of PDLIM3 and PDLIM5 genes in the skeletal muscle of broiler muscles from 10 embryos to 7 days after birth increased first and then decreased,and PDLIM5 gene was not expressed after shelling,the expression levels were highest at E18 and E12,respectively.(2)After interfering with PDLIM3 and PDLIM5 genes in chicken skeletal muscle satellite cells,the expression levels of cell proliferation-related genes CCND1,CCND2 and PCNA mRNA were significantly decreased(P<0.05).In addition,EDU and CCK8 test results also confirmed that skeletal muscle satellite cell proliferation was inhibited.(3)After the PDLIM3 and PDLIM5 genes were interfered in the chicken skeletal muscle satellite cells,the mRNA expression levels of myoblast differentiation markers MyHC,MyoG and MyoD were all decreased to different degrees;The protein abundance of MyHC and MyoG was decreased in different degrees after 24 h,48h and 72 h.The expression of MyHC fluorescence was detected in the samples transfected for 48 h,and the fluorescence expression in the interference group was significantly decreased.Therefore,the PDLIM3 and PDLIM5 genes were interfered with the inhibition of skeletal muscle satellite cell differentiation.(4)Transcriptome sequencing revealed that the differentially expressed genes of chicken skeletal muscle satellite cells interfering with PDLIM3 gene expression were mainly enriched in cGMP-PKG,Oxytocin,MAPK,Tight junction and other signaling pathways,and involved in cytoskeletal protein binding,actin binding,muscle tissue regeneration and development,adhesion and other functions.(5)Transcriptome sequencing revealed that compared with the control group,the differentially expressed genes of chicken skeletal muscle satellite cells that interfered with PDLIM5 gene expression were mainly enriched in signaling pathways such as Tight junction,Oxytocin,p53,MAPK,and involved in myocyte production and muscle contraction.Muscle cell differentiation,and participate in cytoskeletal protein binding,actin cytoskeleton formation,actin and actin binding.(6)PDLIM5 gene was significantly increased after interfering with PDLIM3 gene;PDLIM3 gene expression was significantly decreased after interfering with PDLIM5 gene.PDLIM3 gene and PDLIM5 gene were enriched into MAPK signaling pathway by transcriptome sequencing.In summary,the interference of PDLIM3 and PDLIM5 gene expression can inhibit the proliferation and differentiation of chicken skeletal muscle satellite cells.The PDLIM5 gene is capable of interacting with the PDLIM3 gene and may be involved in the regulation of satellite cell growth and development through the MAPK signaling pathway.