1. Expression Of Myostatin In Atrophic Gastrocnemius Of Mouse After Denervation 2. Construction And Identification Of Myostatin RNA Interference Plasmids

Objective To analysis and approach Myostatin, a negative regulator of skeletal muscle mass, expression pattern and its potential role in denervation- induced muscle atrophy.Methods We examined the time-dependent changes in Myostatin mRNA and protein expression levels in the denevated gastrocnemius muscle of mice after sciatic neurectomy by using quantitative real-time RT-PCR and Western blot respectively. We also conducted morphometric analyses to measure the wet weight ratio of the denervated muscle and the cross sectional area of muscle fibers and to observe muscle morphology.Results The experimental results showed that in the early stage of denervation, the level of Myostatin mRNA and protein in the denervated gastrocnemius muscle increased instantly, reaching a peak at day 3 or day 7 after sciatic neurectomy respectively, when compared to the normal value. In addition, the phospho-Smad2 protein was observed to have a similar expression profile to that of the Myostatin mRNA, the significant decrease of the ratio of muscle wet weight and CSA were observed.Conclusions The present study perhaps opens a new window into Myostatin modulation in muscle atrophy due to denervation and furthermore propagates the signal through TGF-β/Smad signaling pathway. Objective To evaluate the role of RNA interference (RNAi) in silencing Myostatin expression in mouse C2C12 cells (myoblast).Methods Three target sequences and a negative sequence were selected according to Myostatin mRNA sequence, the complementary DNA contained both sense and antisense oligonueleotides were designed and synthesized. After phosphorylation and annealing, the double strands DNA were cloned to the pSilencer2.0-U6 siRNA expression vector. Three positive RNAi plasmids Ml/pSilencer,M2/pSilencer,M3/pSilencer, and an nagetive plasmid Mc/pSilencer were transfected to RNAi plasminds for silencing the Myostatin expression was constructed. The plasmids were then transfected into the C2C12 cells, in which the silencing effect on Myostatin expression was investigated by Western blot.Results It was confirmed by digestion and sequencing that the structures of four RNAi plasmids structure were correct. Among three positive RNAi plasmids, plasmid M1/pSilencer could reduce Myostatin expression in C2C12 cells, and Myostatin expression was decreased nearly 40%, which has statistical significance versus negative control plasmid.Conclusion The positive RNAi plasmid M1/pSilencer constructed in this way can inhibit Myostatin expression in C2C12cells.