Effect Of MicroRNA 140 On Regulation Of BMP2 Gene In Zebrafish Skeletal Development

BMP2(Bone Morphogenetic Protein 2)is a member of transforming growth factor-?(TGF-?)superfamily,it is a gene of the BMPs family that has been widely researched.Study showed that the BMP2 gene not only plays a promoting role in the process of cartilage and bone development,but also is involved in neural precursor cells differentiation and apoptosis,and it plays an important role during the development of the nervous system.In this study,through the software of bioinformatics analysis we found that:there is a microRNA-140's binding site in the 3 'untranslated region(3' UTR)of zebrafish and human and some other vertebrates'BMP2 gene.In order to verify whether the microRNA-140 combined with zebrafish BMP2 gene in vivo and influenced the expression of BMP2 gene,we constructed the wild type zebrafish BMP2 gene 3 'UTR and mutant plasmid into pmirGLO dual luciferase reporter vector,wild-type and mutant plasmids were constructed with Negative Control microRNA mimics and dre-mir-140-5p microRNA mimics were co-transfected into Hek293 cells.24h after transfection,we collected protein and detected the luciferase activity.The results showed that,dre-mir-140-5p microRNA mimics can reduce the wild type zebrafish BMP2 gene 3 'UTR luciferase expression,that is,dre-mir-140-5p microRNA mimics could bind to zabrafish BMP2 gene 3' UTR.In order to study the role of BMP2 gene in zebrafish embryonic development,we used the dre-mir-140-5p microRNA mimics to inject zebrafish embryo at 1-2 cell stage,cultured and observed the effects on early embryo development of zebrafish.The study proved,injection of dre-mir-140-5p microRNA mimics embryos of vertebral caused craniofacial bone deformities.Further zebrafish skeletal staining showed that the injection of dre-mir-140-5p microRNA mimics could affect zebrafish cartilage growth and ossification process,the experimental group Meckel's cartilage of zebrafish embryo appeared deformity and loss of ceratobranchial.Alizarin red staining result showed:notochord and parasphenoid deletion,some severe congenital malformation or absence of otolith.