Effects Of HuaiXiang Chicken GHR-AS On Primary Myoblasts Differentiation

Muscle is one of the important economic traits in poultry production,and its production is influenced by the development of skeletal muscle during the embryonic stage.Chicken GHR gene can transcribe in double directions,possessing not only GHR sense transcript(GHR-S)but also GHR antisense transcript(GHR-AS).The two kinds of transcripts are partially complemented in sequences and can interact with each other.Until now,the function of GHR-AS in muscle development is still not clear.In this study,firstly,we analyzed the GHR-AS and GHR-S expression patterns in muscle tissue.Then we isolated and cultured the primary myoblast from leg muscle to analyze the functions of GHR-AS in myoblast differentiation.The obtained results were as follows:(1)the GHR-AS expression appeared downtrend with chicken muscle development,and it was highly expressed in embryo than post hatching.E12 to E17 was the key stage for myofiber formation,indicating that GHR-AS might participate in skeletal development.The relation between the GHR-AS and GHR-S expression showed a positive significant.The GHR-AS level was significantly lower than that of GHR-S(P<0.05).(2)We induced cell from differentiation to myofiber by using DMEM culture medium containing 2% horse serum.Sub-cellular localization and half-life assays indicated that most GHR-AS are located in nuclear,and it is less stable than GHR-S in cell proliferation and differentiation stage.(3)The expression of GHR-AS appeared to be uptrend during primary chicken myoblast differentiation process,further showing that it may be involved in the differentiation progress.After transfecting the overexpression vector(p GHR-AS),the differentiation biomarker gene,MyoD and MyHC,significantly increased.(4)We added chicken GH protein and 5-aza during cell culture.The expression of GHR-AS,IGF1 and MyHC were increased after adding GH protein.Also,the myoblast differentiation was significantly increased.This study indicates that GHR-AS is involved in skeletal muscle development progress.The results of this study provide theoretical foundation for precision regulation of animal muscle development and for uncovering biomarker in poultry breeding.