Exogenous Mmu-miR-505 Of Expression And Function In Mammalian Cells

Objective: miRNA is a class of about 22 nt conserved non-coding small RNA with the regulatory function, playing an important role in the regulation of developmental timing, cell proliferation and apoptosis as well as tumor development. At the same time, miRNA also involved in a variety of biological processes and signaling pathways, including nerve cell self-renewal, the development of vertebrates, the formation of heart, muscle and nervous system.The mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in almost every aspect of cell function. It with a variety of biological functions such as protein synthesis, immune, cell motility and metabolism, apoptosis and autophagy. By reading the previous literatures, we hypothesise that miRNA-505 may participate in the regulation of the mTOR pathway by affecting the expression of ASF/SF2.In order to study the biological function of miRNA-505, we use the plasmid pcDNA TM 6.2, Lentiviral vectors FUGW and Fsy, to construct recombinant vector which can express miRNA-505-3p,5p in mammalian cells, they are regulated by the promoter CMV, which derived from viruses (cytomegalovirus), endogenous mammalian cell promoter UBC (Ubiquitinenzyme) and neuron-specific promoter Syn (synapsin I), to get different efficiencies and tissue-specific expression.The recombinant vectors were transfected into HEK 293T cell line and primary cultured neurons by lipofectamineTM2000, the expression of EGFP and the transfection efficiency were examined by fluorescent microscope and flow cytometry after transfection, the expression of miR-505-3p/5p and ASF/SF2 were examined by RT-PCR and western blot.Methods: The research were about 173bp、175、171bp of the DNA encoding the miR-505 shRNA gene which were obtained from the pcDNATM6.2-GW/EmGFP-miR-505 vector by PCR, the restricted endonuclease digestion and T4 DNA ligase were used to construct the recombinant lentiviral expression vector:FUGW-miR-505-3p/5p/nc, Fsy-miR-5O5-3p/5p/nc by inserting miR-505-3p/5p/nc into FUGW and L26Fsy(1.1)GW. Lentivirus vector which has been constructed, and transfected into HEK293T cell line and primary cultured neurons by lipofectamine TM2000, the expression of EGFP and the transfection efficiency were examined under fluorescent microscope and flow Cytometry after transfection, the expression of miR-505-3p/5p and ASF/SF2 were examined by RT-PCR and western blot.Results:1.Successfully constructed four lentiviral vector:FUGW-miR-505-3p, FUGW-miR-505-5p, Fsy-miR-505-3p and Fsy-miR-505-5p.2. Lentiviral vector Fsy-miR-505 transfection efficiency was the best in primary cultured neuronal cells.However, FUGW-miR-505 transfection efficiency was the best in the other cell lines.3. Real-time PCR showed the expression of miR-505-3p in cells which transfected pcDNA-miR-505-3p,FUGW-miR-505-3p and Fsy-miR-505-3p were increased significantly; Meanwhile, the expression of miR-505-5p in cells which transfected pcDNA-miR-505-5p,FUGW-miR-505-5p and Fsy-miR-505-5p were increased significantly.4. Western blot analysis showed, ASF/SF2 was reduced in cell when co-transfected with miR-505-3p plasmid and ASF/SF2 plasmid.Conclusion:HEK 293T cells were transfected with three plasmids,Respectively, and their transfection efficiency and expression are not the same. miR-505-3p inhibits the expression of ASF/SF2 in hela cells.