Preliminary Function Analysis For Selenoprotein W In Chicken Skeletal Muscle And Its Response To Selenium

Selenium is an essential trace element for humans and animals,which plays an important biological functions in avian, including the growth and development, productivity, and involved in the immune function and disease control. Selenium may exert its biological function as the formulation of selenoproteins in the organism. Selenoprotein W (SelW) is one kind of selenoproteins that undetectable in skeletal muscle and cardiac muscle of animals suffered with selenium deficiency myopathies but abundant in that feed with common diets. Some evidences confirmed that there is a close relationship between the selenium status in organism and the content of SelW in skeletal muscles and speculated that SelW could influence the metabolism of muscle and diseases related. At present, the investigations of SelW are predominatly focused on mammals (including primates, rodents, sheep, pigs, etc), especially on the relationships of SelW with selenium deficiency diseases. Nonetheless, there are few studys published has ever reported on the function or/and the regulation of gene expression of SelW in poultry. In this study, a reliable primary culture of chicken embryo myoblasts was established with the application of cell culture techniques and the purity of cells prepared was identified by immunocytochemical technology. And then, the chicken skeletal muscles (swing muscle, pectoralis, thigh muscle) and chicken embryo myoblasts were obtained as models for investigating the effects of selenium on the expression levels of SelW mRNA in chicken muscle tissues and myoblasts by using real time quantitative PCR (qPCR) technology. Then, the expression levels of SelW and myogenic regulatory factors (Myf-5, Myogenin, MRF4) as well as P21, the distribution of cell cycles and the contents of total Glutathione (GSH) of myoblasts were detected respectively by using qPCR, flow cytometer, and Total GSH detection kit after gene knockdown of SelW in chicken embryo myoblasts was applied. The results as follows:1. Myoblasts was prepared after several processes such as the digestion with collagenase I, a further purification with Percoll separation medium. Myoblasts kept a good growth state in growth medium, and began to differentiate and fuse with each other to become multinucleated myotubes in differentiation medium. Immunocytochemistry for Desmin andα-Sacromeric Actin indicated that over 95% of myoblasts were positive for both markers, which suggest these myoblasts posess a high purity. Above all, it provided a good foundation for the subsequent experiments refered to selenium exposure and SelW gene knockdown.2. The content of selenium and the expression levels of SelW mRNA in muscle tissues of chickens during the different ages (1 d, 15 d, 25 d, 35 d) could be affected by the different levels of selenium in dietary (0.033 mg·kg^-1,0.150 mg·kg^-1,1.500 mg·kg^-1). Diverse sensitivities and responsive ability were observed in muscle tissues (wing muscle, pectoralis and thigh muscle) at same age of chickens. Meanwhile, the similar results were observed in myoblasts treated various concentrations of selenium in vitro that selenium could regulate the transcript level in them. These data suggest that SelW played an important role in the growth and development of chickens'skeletal muscles.3. In this study, a high transfection was observed in primary cultured chicken myoblasts 24 h after the Stealth RNA (S-RNA) transfection, and the optimal condition for transfection with S-RNA are: 30 nM S-RNA along with 3.0μL LipofectamineTM RNAiMAX for six-well plate. And a S-RNA sequence with highest gene knockdown efficiency for SelW mRNA (to 20% of control)in chicken was selected and S-RNA targeted in SECIS region instead of ORF also obtained a good knockdown efficiency.4. After RNAi for SelW in chicken myoblasts, the expression levels of Myogenin, MRF4, P21 and Myf-5mRNA were detected, the results showed that significant decreases of gene expression of Myogenin and MRF4 were observed 12 h and 36 h post transfection (P < 0.05), respectively. And P21 decreased at 24 h and 48 h (P < 0.05). The results suggest that SelW knockdown could involved in the regulation of the process of muscle differentiation. Meanwhile, Myf-5 transcription level was always lower than that of Control group at each time point indicated, Cell cycle distribution assay found that the proportion of myoblasts in G₀/G₁ phase increased, the proportion of cells in G₂/M phase and S phase decreased, which indicated that the decrease of SelW in myoblasts may also disrupt the cell proliferation.5. A significant decrease in the content of GSH in myoblasts was detected 24 h after SelW RNAi, which means SelW RNAi weaken the anti-oxidant capacity of myoblasts.