Construction of the lentiviral vector containing the interfering RNA targeting against human AURKA gene andits effect on the proliferation of A549 cells

Objective: To construct and package the recombination lentiviral vector containing the interfering RNA gene targeting against human AURKA gene,and preliminary investigate the AURKA gene function.Methods: The recombination pGCLV-GFP lentiviral vector plasmid containing the interfering RNA gene targeting against human AURKA gene was transfected into the 293T cells,then which was packaged.The viral supernatant was collected to transfect A549 cells.The transfection efficiency was determined by detecting the GFP expression under the fluorescent microscopy.The expression level of AURKA protein was examined by Western blot for identifying the inhibitory efficiency of RNAi.The effect of AURKA silencd by RNAi on the proliferation of A549 cell was analyzed by BrdU assay.Colony formation assay was used to explore the sensitivity of the transfected cells to Vincristine.Results: The recombination lentiviral vector containing the interfering RNA gene targeting against human AURKA gene was successfully constructed,and the sequence and insert site of the interference segment were confirmed by PCR and DNA sequencing.The lentivirus was packaged in 293T cells,and the high titer virus supernatant was obtained.The transfection efficiency of small hairpin RNA(shRNA) targeting AURKA into A459 cells was 100%.The result of Western blot showed that the LV-AURKA could significantly inhibit the expression of AURKA protein,the result of BrdU showed down-regulating of AURKA gene could inhibit the proliferation of A549 cells,and the colony formation assay showed that the expression of AURKA gene could enhance the sensitivity of A549 cells to vincristine.Conclusions: The interfering RNA gene targeting against human AURKA mediated by lentiviral vector can effectively inhibit the AURKA gene expression,decreases the proliferation ability of A549 cells,and enhance the sensitivity of A549 cells to vincristine.