Silence Of Cbfa1 Gene By RNA Interference Inhibites Osteogenesis Of Antler Stem Cells

Objectives:In this study we constructed the recombinant lentiviral vector for RNAi of cbfal gene and established the micromass culture system to investigate the role of cbfal in antler enchondral ossification. The purpose of this study was three fold:1) provide the essential data for the research of antler ossification,2) provide the direction and ideal model for the research of ossification and osteoporosis in general,3) silence cbfal gene expression in antler stem cells by RNAi to develop a powerful approach for the improvement quality of the antler.Methods:Pedicle periosteum (PP) tissue explantes was enzymatically digested in this experiment to release antler stem cells, and the biological characteristics of the cells were invstigagted in vitro. Cbfal gene of sika deer was cloned by RT-PCR and RACE. Six RNAi target sequences of cbfal were selected following authoritative and general design rules. shRNA was ligated into the lentiviral carrier plasmid using T4 DNA ligase. This carrier plasmid and constructed plasmids were co-transfected into 293t cell line. The recombinant lentivirus was collected and concentrated from the supernatant, its titre was surveied, and subsequently used to infect antler regeneration stem cells. Then the stem cells were seeded at high dendity for micromass culture. Inhibition rate of cbfal mRNA and expression of collagen I were assayed after carrying out RNA interference using fluorescence quantitative PCR.Results:PP cells were successfully cultured. PP cells had short incubation period, after 24h seeding the cells finished adherence,3-7d entered its exponantial growth phase period, and the PP cells expressed cbfal gene. In this experiment the cds sequence and 3'UTR of cbfal gene were cloned, the cds sequence is 1599bp. The nucleotide had 96%,96%,94%,96%,95%,95%,91% homology with cattle, Equus caballus, Sus scrofa, Macaca mulatta, Homo sapiens, Pan troglodytes, Mus musculus respectively. The deduced amino acid had 84%,84%,83%,84%,84%,83%,80% homology with cattle, Equus caballus, Sus scrofa, Macaca mulatta, Homo sapiens, Pan troglodytes, Mus musculus respectively, and the phylogentic tree was constructed. The predicted protein molecular weight was 58.4KD, isoelectric point approximately was at pH 9.33. The analysis of secondary structure of protein indicated that the protein was composed of Alafa helix, turn, beta sheet and Coil (non-regular curl). Six shRNAs aiming to knock down cbfal gene were designed and successfully synthesized chemically. Each shRNA was inserted into a vector plasmid (pLVTHM) correctly and formed 6 recombinant vectors S1-S6. Large quantity of recombinant lentivirus was collected after three plasmids (vector plasmid, envelop and packaging plasmid) co-transfection carried out. Titre of the concentrated lentivirus was 106-108. Antler stem cell was infected successfully and pellet had been formed in micromass cultures. Real time fluorescence quantitative PCR showed that cbfalgene expression was inhibited and collagen I gene expression was significantly downregulated compared with negative control. Among these, S6 was the most effective, The highest inhibition ratio of cbfal was 88%, downregulation ratio of collagen I mRNA expression was 86.8%. The results from the present study showed that shRNA can silence cbfal gene efficiently and specifically in antler ossification system. Conclusion:We successfully established micromass culture system for inducing antler stem cell differentiation toward osteoblast lineage in vitro and using this system mimiced the in vivo process of antler endochondral ossification, hence solved the problems of long-term, costly and involve animal ethic issues in vivo experiments. These results provided the direction and ideal model for the research of ossifacation and osteoporosis. The cbfal gene of sika deer was cloned successfully, and the expression of cbfal gene had been significantly inhibited by RNAi. At the same time the expression of collagen I gene was also downregulated after cbfal gene silence. Therefore, the antler stem cell differentiation towards osteoblast was inhibited. The results have also filled the gap in this research area of ossifacation mechanism and provided a new method for improving antler quality.