The Effects Of CDC10 Gene And Transcriptional Factor ELK1 On The Proliferation Of Bovine Myoblasts

The cell divison cycle 10(CDC10,also known as SEPT7)gene is a member of the septins family.Septins are GTP-binding and membrane-interacting proteins with a highly conserved domain structure involved in various cellular processes,such as,cytoskeleton organization,cytokinesis and membrane dynamics.In beef cattle,the CDC10 gene is located in the quantitative trait locus(QTL)of growth traits and the expression level of the CDC10 gene is positively correlated with the growth traits in several breeds.Studies have shown that one single nucleotide polymorphism(SNP)is located at-323 bp upstream of the transcription initiation site in the promoter of CDC10 gene,which the SNP was associated with growth-related traits in Japanese Black cattle.However,the molecular mechanism of CDC10-323G>C SNP to regulate the expression of CDC10 gene and muscle growth is still unclear.In this study,PCR-RFLP,RNAi,cell culture,Real-time PCR,Western blot and dul luciferase reporter gene system were applied to analyse the allele frequency of the CDC10-323G>C SNP in different cattle breeds and the expressions of CDC10 gene in different tissues and organs.In order to study the effect of CDC10 on the proliferation of myoblasts,the interference and overexpression vectors constructed,and the WST1 and EdU assay were used to detect the proliferation of the myoblasts.Luciferase reporter gene system was used to detect the effect of G and C alleles on CDC10 promoter activity.In addition,a transcription factor,ELK1,was predicted by bioinformatics,which may combine with the-323G>C mutation region and regulate CDC10 gene expression.The interference and overexpression of the ELK1,as well as dul luciferase reporter gene system and WST1/EdU assay were applied to reveal the molecular mechanism of-323G>C regulating the expression of CDC10 and its effect on the proliferation of myoblasts.The major results are as followings: 1)The CDC10 gene expression profile showed that CDC10 gene expression was significantly higher in the subcutaneous fat,lungs and testis than in other tested tissues in adult simmental cattle;2)The promoter activity of C allele at CDC10-323 SNP site was significantly stonger than G allele(P<0.05);3)The proliferation rate of myoblasts were significantly increased after overexpression of CDC10 by WST1(P<0.05)and EdU assay(P<0.05);4)After interference of CDC10,the proliferation rate of myoblasts was significantly decreased by WST1(P<0.05)and was nearly significantly decreased by EdU assay(P=0.052);5)The CDC10 expression was down-regulated after interference of ELK1(P<0.01)and CDC10 was up-regulated after overexpression of ELK1(P<0.01);6)There was no significant difference in the proliferation rate of myoblasts after overexpression of ELK1 by WST1 and EdU assay(P>0.05)as well as after interference of ELK1 by WST1(P>0.05),however,after interference of ELK1,the proliferation rate of myoblasts was down-regulated significantly by EdU(P<0.05);7)The luciferase reporter gene system showed that the promoter activity of wild-type G allele was significantly reduced when the ELK1 expression vector was co-transformed(P<0.01).The results showed that the CDC10 gene significantly affected the proliferation rate of myoblasts,but the ELK1 had no significant effect through CDC10-323G>C SNP site to regulate proliferation of myoblasts.Whether the other transcription factors interaction with-323G>C mutation affects the proliferation of myoblasts remains to be further studied.