Study Of MicroRNA-93-5p And MicroRNA-20b-5p On The Relationship With VEGF Transcriptional Regulation And Deer Antler Cell Proliferation

Antler is the only organ which can be periodic shed and regenerated in mammal, it is secondary sexual characteristics of the male deer species in addition to reindeer. Current research involves different fields of pilose antler, mainly divided into the following several aspects: 1.The perspective of velvet antler of gene and cell activity to study the growth characteristics and reveals its origin. 2. Pharmacological drug research of sika deer antler. 3.The research of growth factors which plays an important role in the process of pilose antler growth. In recent years, on the growth of antler growth related factors in the process of research is a hotspot, which vascular endothelial growth factor(VEGF) in the growth process of pilose antler plays an important role. VEGF is a mainly exist in vascular cell specific mitogen and its role is mainly through binding to its receptor(VEGFR) to play in mediating microvascular proliferation and enhanced vascular permeability and other physiological functions. The miRNA was combined with 3 non coding region(3 ’UTR) to complement the effect of the target gene expression. The issue using RNA genomics technology, miRNA-mediated gene silencing VEGF antler cartilage cell proliferation inhibition studies were discussed to provide new ideas for the study of VEGF rapid growth of antler regeneration of molecular mechanisms.This experiment will be on the sequences sika deer antler VGRF 3’UTR gene mutants was cloned, using the previous system research group of preparation of cartilage and mesenchymal miRNA chips in antler tip and analysis the combined with bioinformatics software were used for preliminary screening. Applied of qPCR technology and Western blotting were further screening determine chose the miRNA which can binding the VEGF 3’UTR. The stability and quantify of miRNA mimics after transfect the cells was detected by qPCR, Construct the dual luciferase gene reporter vector, and detect the relative activity of luciferase and verified the VEGF miRNA binding sites. qPCR detection of miRNAs cells after transfection of pilose antler in its intracellular stability and relative expression levels. Changes of proliferation cartilage antler cells by MTT after transfection of miRNA. Changes of protein expression of VEGF in antler cells were detected by western blotting after transfection of mi RNA. Changes of proliferation cartilage of telomerase content antler cells by ELISA after transfection of miRNA. At the same time to explore the correlation between VEGF and cell factor bFGF and IGF.Experimental use of gene cloning technology successfullyobtained 356 bp of sika deer VEGF 3 ’wild type UTR partial sequence.By blast analysis and compare with bovine, human nucleotide, the sequence homology were 97.02% and 88.76%, The use of PCR site directed mutagenesis with wild type as a template successfully obtained mutant sequence was 336 bp in length, success will be the target GCACTTT sequence mutations into TGTAGCG. Initial screening using the bioin- formatics software and miRNA gene chip combined mode,screen out 8 significant difference of miRNAs expression. Through the SYBR Green real-time PCR and Western blotting techniques show further screened, miRNA-93-5p, miRNA-20b-5p can be combined with the VEGF 3’UTR sequence.After transfect miRNA-93-5p, miRNA-20b-5p,the expression level of VEGF protein inhibitory is most obvious. Dual luciferase report gene assay results showed that miRNA-93-5p, miRNA has the regulation and control function of VEGF gene, VEGF is a target gene of miRNA-93-5p,miRNA-20b-5p control.qPCR test results showed that compared with the control group, the high miRNA expression quantity was found in cells transfected group; Cells proliferation with MTT detection show that compared with the control group, miR-93-5p group and mi R-20b-5p transfection of pilose antler cells in vitro proliferation was inhibited; ELISA was used to measure the content of telomerase after transfection; compared with the control group, the content of telomerase gradually reduced with the extension of time; Western blotting detection after transfection, the expression level of VEGF protein in the cells decreased with the extension of time. After transfection, there is a certain correlation in the expression of VEGF in the cells and expression of bFGF and IGF respectively.