Over-expression And RNAi Of Muclin And Its Effect On Muscle Related Gene Expression

Musclin is a kind of biological activity factors which secreted by skeletal muscle. Preliminary studies suggested that the expression of the gene was influenced by many factors, and it had a function of regulating muscle nutrition metabolism. The research about Musclin gene is fewer and its mechanism of action has not yet been elucidated till now. Therefore, it has important scientific significance to investigate the function and mechanism of musclin gene using the technology of over-expression and RNAi silencing.In the first experiment:The mouse C2C12cells were incubated and divided into four groups: including normal cells (control group), transfecting the pcDNA3.1-Musclin(mouse) eukaryotic expression vector (over-express group), transfecting musclin siRNA vector (RNAi group), co-transfecting the vectors of over-expression and siRNA(co-transfection group).We tested the changes of the expression of musclin mRNA after transient transfection and further tested the mRNA expression of some genes related to muscle such as Smad3and Myostatin gene. The fluorescence quantitative PCR results showed that compared with the musclin mRNA expression in the control group, it was significantly higher in the over-expression group(p<0.01), while it was lower in the RNAi group significantly,and it was significantly raised in the co-transfection group (p<0.01) but between the over-expression group and RNAi group.For the mRNA expression of Smad3and myostatin gene, compared with the control group, the expression of Smad3decreased obviously in the co-transfection group, and tended to increase in the RNAi group, but changed little in the over-expression group; The expression of myostatin mRNA, dropped significantly in the RNAi group and tended to increase in the co-expression group, but it is almost same in the over-expression group as in the control group. In conclusion, this experiment successfully realized the over-expression and silence of musclin gene, and also suggested that the expression of musclin gene affected the expression of Smad3and myostatin gene. The functional pathways of musclin genes regulating muscle growth might be related to inhibiting the expression of Smad3gene and promoting the expression of myostatin gene. This study laid a foundation for the further researches about the function and mechanism of musclin gene in the skeletal muscle growth and nutrition metabolism. In the second experiment:To deeply study the function and mechanisms of musclin gene in the nutrition metabolism and growing development of skeletal muscle,the musclin gBlocks DNA fragments was synthesized according to the sequence of musclin cds in sheep which was assembled using bioinformatics technology by my own laboratory, then cloned to T-vector.The sequence was confirmed by sequencing. After the above, the sheep pcDNA3.1(+)-Musclin eularyotic expressive vector was constructed. The experiments laid a foundation for the further study.