Construction Of Musclin Eukaryotic Expressive Vector And Regulation Of Musclin, Smad3and MEF2Gene Expression By Progesterone In Mice

Musclin gene is an active polypeptide derived from skeletal muscle, which was found recently. In skeletal muscle, the expression of musclin gene is regulated by a variety of factors such as the types of muscle, nutritional conditions, hormones and cytokines. Musclin was involved in the metabolism of glucose and fat by autocrine and paracrine pathway of skeletal muscle, and it may be closely related to diabetes and insulin resistance diseases. Therefore, the profound study on the biological function and regulation of musclin gene expression is very important.The primers were designed according to the sequences published in NCBI(NM198112.2), and the sequences of mouse musclin CDS was amplified by PCR and, ligated to T vetor. The sequence was confirmed by sequencing. Then, the mice’s pcDNA3.1(+)-Musclin eukaryotic expressive vector was constructed. This study laid a foundation for further studying the function and mechanism of musclin.Besides,24female mice about20g were selected and were randomly devided into four groups: control group (physiological saline),low-dose progesterone group (2mg/kg body weight), mid-dose progesterone group (4mg/kg body weight), high-dose progesterone group(8mg/kg body weight).0.1mL corresponding solution was injected to each mouse, once each day and the injection lasted for a continuous week. Then the samples of skeletal muscle were collected, and the expression of musclin mRNA and protein were examined. The mRNA expressions of some genes related to muscle such as Smad3, MEF2A, MEF2C, and MEF2D were also examined. The results showed that:1. Compared with the control group, the body weights of mice in different doses of progesterone groups were increased.2. The results of QRT-PCR showed:the expression of musclin mRNA was down-regulated respectively by low-dose and high-dose progesterone, but was up-regulated strikingly by mid-dose progesterone; The results of Western blotting showed:the expression of musclin protein in groups of low-dose and high-dose progesterone were slightly lower than that in the control group, while that in mid-dose progesterone group was significantly higher than that in control group.3. The results of QRT-PCR showed:the expression of Smad3mRNA was up-regulated by every dose of progesterone and the effect was siginificant in high-dose progesterone group; the expression of MEF2A mRNA was down-regulated by every dose of progesterone and the effect was significant in mid-or high-dose progesterone group; the expression of MEF2C mRNA was down-regulated by both mid-dose and high-dose progesterone, and the effect was significant in the mid-dose progesterone group; the expression of MEF2D mRNA was down-regulated by both mid-dose and high-dose progesterone, and the effect was obvious in mid-or high-dose progesterone groupIn conclusion, the expression of musclin and Smad3, MEF2A, MEF2C, MEF2D gene were increased or decreased by different doses of progesterone, and the expression of musclin gene was related to the expression of Smad3, MEF2gene. The study offered some theoretic evidences for the biological function and the expressional regulation of musclin gene, and laid a theoretic foundation for the further study on the expression/function mechanisms of musclin.