Functional Exploration Of Lrtm1 Gene In Myogenic Differentiation

PART ONE:CONSTRUCT C2C12-LRTM1 CELL LINESObjectiveThe mouse Lrtm1 lentiviral vector was constructed.Screened stable expression of the exogenous Lrtm 1 in C2C12 cell lines.MethodsThe expression of Myogenin,Myosin and Lrtm1 mRNA levels were detected during the process of differentiation.Hoechst staining was used to observe the structure of myotubes after C2C12 cell lines differentiation.Transient overexpressed GFP and Lrtm1 plasmid in C2C12 cell lines.Construction of recombinant vector pLJM1-Lrtm1-DDK:PCR fragments of target gene from p CMV6-Lrtm1-DDK plasmid.pLJM1-GFP vector cutted by the restricyion enzymes AgeI and EcoRI.In recombination,the target gene was linked with cleaved pLJM1-GFP vector.Lentivirus Package.C2C12 were infected by lentivirus and screened C2C12-Lrmt1 cell lines.Detected commercial Lrtm1 antibody titer.Detected the protein expression of mutant Lrtm1.ResultsThe expression of Lrtm1 and Myosin mRNA levels were detected during the process of differentiation.Myotubes had formed during its differentiation.Transient expression system was instable in C2C12 cell lines.Successfully amplificated the mouse Lrtm1 gene.The pLJM1-GFP vectors were cutted by AgeI and EcoRI restriction enzymes.Successfully selected the positive clones that were linked to the Lrtm1 gene.Sequencing analysis of p LJM1-Lrtm1-DDK blast the sequencing of Lrtm1 CDS and pCMV6-Lrtm1-DDK.The recombinant plasmid p LJM1-Lrtm1-DDK was expressed in HEK293 T cells.The expression of Lrtm1 mRNA in C2C12-Lrtm1 cell line was detected by Real-time PCR.The specific recognition of the commercial Lrtm1 antibody was no work.The protein expression of mutant Lrtm1 was detected.ConclusionSuccessfully constructed of Lentivirus expression vector pLJM1-Lrtm1-DDK.C2C12-Lrtm1-DDK cell lines were screened.PART TWO:FUNCTIONAL EXPLORATION OF LRTM1 GENE IN THE PROCESS OF MYOGENETIC DIFFERENTIATIONObjectiveConstructed a myogenic differentiation model.After inducting differentiated stable cell lines,observed the effect of Lrtm1 gene on myogenic differentiation to clarify the mechanism of regulation of Lrtm1 protein expression in C2C12 cells.MethodsThe stable cell lines were induced to differentiate.Real-time PCR and Western blot were used to detect the effect of Lrtm1 gene on myogenin and Myosin genes.In the stable cell lines,the expression of the mRNA levels,such as muscle regulation factor,PAX3 and PAX7,were detected and the mechanism of Lrtm1 gene affecting the process of muscle differentiation was explored.Detected the expression of Lrtm1 at protein levels afer treating C2C12-Lrm1,C2C12 and HEK293 T cell lines with MG132.ResultsThe expression of Lrtm1 gene in mRNA level and protein level has inhibitory effect on Myogenin and Myosin.Real-time PCR detected the expressionof Lrtm1 gene at mRNA level promoting the expression of myogenic differentiation genes PAX3,and Myo D.Lrtm1 protein in C2C12 cells was degraded by proteasome.ConclusionThe Lrtm1 gene was highly expressed during myogenic differentiation.Overexpression of Lrtm1 inhibited the expression of Myogenin and Myosin genes.Overexpression of Lrtm1 gene promoted PAX3 gene expression.The expression of Lrtm1 protein in C2C12 cells was regulated by the proteasomal degradation.