Expression Pattern And Target Gene Screening Of MiR-27b In Goat Muscle

The stock of goats and sheep is equal in China.Compared with sheep,goats generally grow more slowly and produce less meat.In order to improve the individual goat meat yield through molecular breeding,study on the regulation mechanism of goat muscle growth and development is one of the hotspots in goat breeding.Muscle satellite cells,as the main components of skeletal muscle in animals,are of great significance to the formation and regeneration of skeletal muscle,and are the basic model for studying the gene regulation mechanism of skeletal muscle growth and development in vitro.A large of studies have found that some mi RNAs have specific or high expression patterns in skeletal muscle cells,which can participate in the regulation of myogenesis and play a key role in skeletal muscle development.Specificially,miR-27 b is an important muscle regulatory factor in model animals such as mice,which can inhibit the proliferation of skeletal muscle satellite cells and promote differentiation.However,these studies only focused on mice,humans or cattle,and few studies have described the regulatory mechanism of mi R-27 b in skeletal muscle satellite cells of goats.Therefore,this study took DaZu black goat as the research object,to study the expression of miR-27 b in the tissues at different growth stages and its effect on the differentiation of skeletal muscle satellite cells in vivo and in vitro.To explore its regulatory function in the growth and development of goat skeletal muscle and its possible mechanism,so as to provide basic data for molecular breeding of meat goats.The main results of this study are as follows:1.Study on the expression of mi R-27 b in goat tissues.We detected miR-27 b in three months of embryonic stage,0d after birth,and 24 months of goat tissues,respectively.Using qRT-PCR,we found that miR-27 b was expressed in myocardium,liver,spleen,lung,kidney,rumen,small intestine,longissimus dorsi,leg muscles,pectoral muscle and gluteus maximus in the Dazu black goats at different growth stages.Among them,mi R-27 b was highly expressed in liver.Further study found that following the increasing of goat age,the expression of mi R-27 b expression was gradually decreased in myocardium,dorsal longest muscle,leg muscle,pectoral muscle,and gluteus maximus.2.Subcellular localization of miR-27 b in goat skeletal muscle satellite cells.The localization of miR-27 b was studied by fluorescence in situ hybridization.When the probe bound to the miR-27 b in the cell to form a hybrid strand,the green fluorescence excited by the probe was mostly present in the cytoplasm,indicated that miR-27 b was indeed present in the goat skeletal muscle satellite cells and was abundantly expressed in the cytoplasm and only a small amount was expressed in the nucleus.3.Effects of mi R-27 b on differentiation of skeletal muscle satellite cells in goats.The morphological changes of goat skeletal muscle satellite cells at different stages of differentiation can be observed by differentiation induction culture.It was found that muscle tube could be formed,and three myoblast marker genes(MyoD,MyoG and MyHc)were expressed stably at each stage,indicating that a stable satellite cell system of goat skeletal muscle was established in this study.Further studies found that the expression of miR-27 b continued to increase with the extension of the differentiation period of satellite cells.The expression of mi R-27 b at 2d of differentiation was significantly higher than that of undifferentiated satellite cells(P<0.05),and the expression of miR-27 b at 4d of differentiation was significantly higher than that at 0(P<0.01).The results showed that mi R-27 b can effectively promote the differentiation of goat skeletal muscle satellite cells.4.Screening of mi R-27 b target genes.In this study,the over-expression and interfering expression vectors of miR-27 b gene were successfully constructed.In addition,293 T cells were used to prepare mi R-27 b overexpression and interference lentivirus(viral titer was 6×10^8TU/mL),and the optimal transfection concentration was found to establish a skeletal muscle cell model of mi R-27 b overexpression and interference in goats.The TargetScan7.1 software was applied to predict target gene of miR-27 b and found that MDFI gene may be a target gene of miR-27 b and overexpression of mi R-27 b could inhibit the expression of MDFI gene with a significant difference(P<0.01).Moreover,55 differentially expressed genes were screened by transcriptome sequencing analysis of mi R-27 b overexpression and untreated skeletal muscle cell lines.A large number of candidate genes that may have potential targeting relationships with miR-27 b were further explored.The conclusions of this study are as follows:1.mi R-27 b was detected in multiple tissues of goats,and the expression level in muscle tissues gradually decreases with increasing of goat age.2.miR-27 b may participate in goat myogenesis by targrting to MDFI gene to promote skeletal muscle satellite cell differentiation process.