Study Of Let-7b Rugulating Alpaca Hair Growth Through Down-Regulating Target Gene EDA

Alpaca coat has good quality, the ear hair is mainly coarse wool and has a long growth cycle; the back hair is mainly fine wool and has a short growth cycle. Studies show that EDA signaling pathway has certain regulation effect in the development of skin and hair growth process.To study the effect of EDA in alpaca hair growth, while exploring the targeted relationship between let-7b and EDA, as well as the regulatory relationship of let-7b to EDA.In this experiment,the young male alpacas as the research object.To explore the effect of EDA in alpaca hair growth,the experiment was first to detect the EDA differential expression in alpaca ear and back skin by Real-time fluorescent quantitative PCR and Western blot technique；and to detect the location of EDA protein in alpaca ear and back skin by immunohistochemistry.Then to construct the dual-luciferase reporter vector to verify the targeting relationship let-7b and EDA. Finally, let-7b over eukaryotic expression vector was transfected alpaca skin fibroblasts to detect the EDA expression levels in transfected and untransfected cells,these aims to explore how let-7b regulate EDA. The experimental results are as follows:(1) The detection of EDA mRNA and protein levels in alpaca ear and back were showed that EDA mRNA and protein levels in alpaca ear skin relative expression were significantly higher than the back skin (P<0.01); and the Immunohistochemical results showed EDA protein positive reaction was obvious in hair root sheath portion of alpacas ear and back skin hair follicles, the hair matrix positive reaction was weak.Analysis of data obtained that the mean optical density value of ear skin was higher than the back skin, the difference was significant (P<0.01).(2) The dual-luciferase reporter vector showed that the experimental group luciferase expression was decreased, indicated that let-7b with EDA had obvious targeted relationship.(3) Let-7b eukaryotic expression vector was transfected alpaca skin fibroblasts and the real-time PCR and Western blot results showed that EDA mRNA expression of the transfected group was decreased(P<0.01) and the protein expression level also was decreased (P<0.05). While using cell immunohistochemical analysis EDA expression in transfected and untransfected group, the results showed: EDA protein was abundantly expressed in fibroblast cytoplasm, and in transfected cells positive reaction was weaker than the untransfected cells.All the results prompted that EDA differentially expressed in alpaca ear and back skin, suggested that EDA played a certain role in alpaca hair growth;EDA was the target gene of let-7b, and let-7b may down-regulate EDA mRNA and protein.