Effect Of Porcine Akirin2 On Myogenic Differentiation Genes Expression In C2C12

Genetic analysis has revealed an important function in myogenesis for Myostatin(MSTN),a member of the TGF-? super-family.Akirin is one of the cascades of genes that responds to MSTN signals to regulate myogenesis,including Akirinl and Akirin2.In order to further study the function of the porcine Akirin2,in this study we cloned the porcine Akirin2 gene;detected the expression change of relative genes after transfected over-expression vector pEGFP-N1-Akirin2 or silencing vector Akirin2-pshRNA1 during the C2C12 differentiation and cloned the porcine Akirin2 promoter.1)In this study,a porcine Akirin2 gene was cloned from porcine muscle tissue.The nucleotide sequence of the cloned porcine Akirin2 gene indicated that the open reading frame consisted of 612 bp(GenBank accession no.KC140110.1),encoding a protein of 203 amino acids residues with an estimated molecular weight of 50 kDa.The theoretical isoelectric point(pI)of the porcine Akirin2 is 5.12.Further BLAST analysis of the protein revealed that porcine Akirin2 had high homology with the Akirin2 of Ovis aries(96%),Bos Taurus(96%),human(95%),Mus musculus(91%),Rattus norvegicus(89%).A 10-peptide nuclear localization signal"19-PASPKRRRCA-28"was found with high probability.The prediction of protein sequence secondary structure showed that the porcine Akirin2 protein contained two alpha-helix and random coils.To evaluate the evolutionary relationships of porcine Akirin2 with other species,we constructed a phylogenetic tree.Akirin2 proteins from different species were divided into three subgroups.This result suggested that the porcine Akirin2 protein had closer genetic relationship with the Akirin2 protein of human,Bos Taurus and Ovis aries.To characterize the Akirin2 gene further,RT-PCR was conducted to determine its expression in various tissues.The Akirin2 gene was obvious differentially expressed in many tissues and there were no signs of gene expression in some tissue,with the strongest expression in the brain;low expression in the lymph and testis;no signals in the heart,duodena,rectum,skin,thymus.2)To explore the function of porcine Akirin2 in the differentiation of myoblasts,in this study we successfully constructed the over-expression vector of pEGFP-N1-Akirin2.The pEGFP-N1-Akirin2 plasmid was transfected into C2C12 cells,the we detected the related genes of myoblasts differentiation by real-time PCR.The expression of myogenic regulator factor Myogenin and MyoD were significantly up-regulated in C2C12 cells during differentiation compared with control(P<0.05).The mRNA expression levels of IGF II and MHC were significantly up-regulated in C2C12 cells during differentiation compared with control(P<0.05).The expression of CyclinD was down-regulated and the mRNA level of P21 was increased during differentiation.At the same time,reverse transcription followed by quantitative PCR analysis confirmed the increased in IL-6 expression.The above data suggested that Akirin2 might play important role similar to Akirinl(mighty)promoted the differentiation of C2C12 myoblasts during the C2C12 myogeneis.3)To study the relationship between Akirin2 gene and C2C12 myogeneis through Akirin2 gene knockdown.According to Akirin2 gene sequences,a target sequence was designed based on shRNA design principles,and the DNA oligo was cloned into psiRNA-h7SKGFPzeo vector.The interference effect of Akirin2 on C2C12 cells was detected by real-time PCR.The recombination vector Akirin2-pshRNA1 mediated by turbo fect was transfected to the C2C12 cells.The Akirin2-pshRNA1 vector was successfully constructed.Akirin2 gene expression in C2C12 was decreased by 60.62%at mRNA level.The expression of myogenic regulator factor Myogenin was significantly down-regulated in C2Cl2 cells during differentiation compared with control(P<0.05).The mRNA expression level of IGF ?was down-regulated(P<0.05).The expression of myogenic regulator factor MyoD showed no significant difference between two test groups.The mRNA expression of MHC was up-regulated,but there was no significant difference compared with control group.Our data suggested that the differentiation of C2C12 was not inhibited with Akirin2 gene knockdown.The expression of CyclinD was obviously down-regulated and the mRNA level of P21 was obviously up-regulated in transfected C2C12 cells during differentiation.These data showed that the proliferation of C2C12 was significantly inhibited by effectively silencing of Akirin2 compared with control group.Akirin2 was involved in the regulation of cell proliferation of C2C12 cells.The expression of IL-6 was measured by real-time PCR.The result showed that the expression of IL-6 was obviously inhibited in transfected C2C12 cells with Akirin2-pshRNA1.4)Akirin is a recently discovered nuclear factor that plays an important role in skeletal myogenesis.However,its precise mechanisms are still lacking.To investigate the transcriptional regulation of Akirin2,the promoter of porcine Akirin2 was amplified.The potential transcriptional binding sites were analyzed by bioinformatics method.Four pairs of primers were designed and 4 fragments(about 200,400,700,1000bp)of promoter of porcine Akirin2 were cloned by PCR.The four different fragments were cloned into luciferase reporter vector pGL3-basic,and constructed recombinant vectors pGL3-F1.0?pGL3-F10.7?pGL3-F0.4 and pGL3-F0.2.The recombinant vectors were transfected into Hela cells.In 24h after transfection,the cells were lysed and luciferase activity was measured.The cloned porcine Akirin2 promoter sequences were correct by DNA sequencing.The promoter activity of pGL3-F1.0?pGL3-F0.7?pGL3-F0.4 and pGL3-F0.2 ranged from 2.694±0.282 to 8.372+0.306.The minimal functional promoter was located in the-180?+34,the region of-180?+34 plays an important role in the initiation of transcription.