The Effect Of The Lentivirus-mediated RNA Interference Targeting Aurora-A Gene On The Growth Of Human Lung Adenocarcinoma Cell Line In Vitro And In Vivo

Objective: We aim at studying the effect of RNA interference targeting Aurora-Agene on the biological behaviors of H1299cells and exploring the relevant mechanism soas to build an experimental basis for its research in the clinical treatment of lung cancer.Methods: Lentiviral vectors (Lv-shRNA) were constructed to deliver small hairpinRNA (shRNA) targeting Aurora-A into H1299cells. The control group was the H1299cells transfected with negative vector. The transfection efficiency was determined bydetecting the GFP expression with the fluorescent microscopy. The expression level ofAurora-A mRNA was examined by reversed transcript polymerase chain reaction (RT-PCR)and real-time quantitative polymerase chain reaction (RT-qPCR). The expression level ofAurora-A protein was finally examined by Western blotting for identifing the inhibitoryefficiency of RNAi. In order to analyze the effects of silencing Aurora-A gene on theexpression of Caspase-3,Caspase-9,Bcl-2and Bax, as well as cell cycle, apoptosis andproliferation, the methods mentioned above was applied to detect the changes inexpression of Caspase-3,Caspase-9,Bcl-2and Bax on mRNA and/or protein levelrespectively. For the same purpose, cell counting Cellomics method and flow cytometrywere performed to examine the changes in cell cycle, apoptosis and proliferation afterRNAi targeting Aurora-A. The growth of subcutaneously transplanted tumors wasobserved in nude mice after inoculated with H1299cells with Aurora-A stably silenced andthe control H1299cells. The expression of Aurora-A protein was examined byimmunohistochemistry.Results: RNAi lentivirus expression vectors targeting to four various sites ofAurora-A gene were produced, and the sequence and correct site of ds oligos inserted were confirmed by PCR and sequencing assay; the lentivirus were packaged in293T, cells withhigh titer; Small hairpin RNA (shRNA) targeting Aurora-A was transfected to lungcarcinoma cells H1299by recombinants of lentiviral vectors and the transfection efficiencycould reach more than70%; The expression of Aurora-A gene was inhibited by lentiviralrecombinants of LV-Aurora-A. The inhibitory efficiency of LV-Aurora-A could reachover50%; Silencing Aurora-A gene by RNAi could up-regulate the expression ofCaspase-3,Caspase-9and Bax protein and down-regulate the expression of Bcl-2protein;Inhibiting the expression of Aurora-A gene had the influence on the processes of H1299cell cycle thus induced G2/M arrest; Down-regulating the expression of Aurora-A geneinhibited proliferation of H1299cell and slowed down its growth velocity; Obviously,effect on the apoptosis of H1299cell was observed by inhibiting the expression ofAurora-A gene. The transplanted tumor of nude in the group of silencing Aurora-A gene byRNAi was observed apoptotic bodies under a microscope (HE). The Aurora-A proteinexpression was reduced significantly by immunohitochemistry under a microscope. Theresults showed that the growth of transplanted tumor was restrained.Conclusions: The H1299cell line with surviving gene stable interference wassuccessfully constructed through lentivirus vector; The apoptosis level of H1299cellincreased and cell growth was inhibited in vitro when the survivin gene interfered by thelentivirus vector; The growth of the H1299transplanted tumor was significantly inhibitedby the RNAi targeting Aurora-A gene, induce apoptosis of transplanted tumor cells.Aurora-A might be a potential adjuvant gene therapeutic target for human lung carcinoma.