Study On Gga-miR-2954 Regulating Proliferation And Differentiation Of Chicken Myoblasts By YY1

Mi RNAs play an important role in the development of tissues and organs,disease occurrence,cell proliferation and apoptosis.In the previous studies,our results showed gga-mi R-2954 was universal male-biased expressed with tissues and YY1 was verified as its target gene in chicken fibroblasts by double-luciferase reporter gene assay.A number of studies in mammals have shown that YY1 inhibits myogenesis,this study intends to simulate the myogenesis process in vitro to investigate whether gga-mi R-2954 regulates the muscle development process through its target gene YY1.The research results are as follows:1.The expression profiles of gga-mi R-2954 in muscle tissues during the embryonic stages were conducted by quantitative PCR,results showed the male-biased expression pattern of gga-mi R-2954 was confirmed,and the higher expression was at E10-E18.5,then decreased significantly.2.The fluorescence in situ hybridization(FISH)results of the leg muscle tissue at E13.5 showed that gga-mi R-2954 was enriched in cytoplasm and expressed a little in nucleus.Myoblast differentiation was induced in vitro and quantitative PCR detection showed that the expression of gga-mi R-2954 increased with the increase of differentiation days,,while YY1 was the opposite.3.After overexpression and suppression of gga-mi R-2954,CCK-8,Ed U assay as well as the quantitative PCR detection of cycle related genes CCND1 and CDK6 were performed to measure effects.These results showed that gga-mi R-2954 inhibits myoblast proliferation.The expression of Myo G,My HC detected by q PCR and the result of My HC immunofluorescence staining showed that gga-mi R-2954 promotes the myoblast differentiation.4.The quantitative PCR and Western blot results showed that gga-mi R-2954 inhibit the expression of YY1 in proliferating and differentiated myoblasts.YY1 promotes myoblast proliferation but inhibits the myoblast differentiation.When myoblast co-transfected with gga-mi R-2954 mimics and pc DNA3.1-YY1,YY1 could rescue the decrease in the number of cells and inhibited the further differentiation due to the effect ofoverexpression of gga-mi R-2954.The above results reveal the important role of gga-mi R-2954 in chicken skeletal muscle development,and it is confirmed that gga-mi R-2954 could regulate embryonic myoblast proliferation and differentiation by targeting YY1 in chicken primary myoblast.The study of the regulatory mechanism further improves the molecular regulatory pathways involved in muscle development in chicken,and helps to reveal the regulatory process of muscle fiber growth and development in chicken embryos of different genders.This study not only further complements the molecular regulatory pathways involved in poultry skeletal muscle development,but also helps to reveal the molecular regulation process of sex difference on chicken skeletal muscle growth and development.