Experimental Studies On Therapy Of Lung Cancer By VEGF-C SiRNA Mediated By Liposomes

Objectives: Lung cancer is one of respiratory system malignancies and occurs at a higher incidence in China. Lung cancer usually connects with changes in cell cycle and apoptosis mechanisms caused by some gene mutation. Recently many experiments have shown that VEGF-C plays a very important role in tumor occurring and progress. Our object is to evaluate the application of RNA interference in the study of VEGF-C gene in Lung cancer cell line A-549. Observe the effects of VEGF-C silencing on Lung cancer cell line A-549 growth suppression, apoptosis and expression of VEGF-C and VEGF-C mRNA.Methods:Designed the specific small interference RNA (siRNA) of the gene sequence of VEGF-C and transfected into A-549 lung cancer cells using liposomes LipofectamineTM2000. The efficiency of infection of A-549 cells transfected with VEGF-C siRNA in vitro was observed by the fluorescence microscope. The change of cell morphology was observed by invert microscope. The toxicity and inhibition effect of VEGF-C siRNA for A-549 cells proliferation was determined by MTT assay. The curve of growth. was drawn . mRNA and protein levels of VEGF-C were detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. Flow cytometry was used to study apoptosis after transfected VEGF-C siRNA. The ultrastructure changes of A549 cells transfected with VEGF-C siRNA was observed by the electron microscopy.Results: The specific siRNA of the gene sequence of VEGF-C can transfect A-549 cells efficiently. The density of siRNA and liposomes for experiments have no toxicity for A-549 cells. The invert light microscopy demonstrated that A-549 cells transfected with VEGF-C siRNA appeared inhibitory effects including float growth, volum lessen and high photonasty. The VEGF-C siRNA can suppress the VEGF-C mRNA and protein expression efficiently and stably. The results also revealed that down regulation of VEGF-C resulted in significantly inhibition growth and proliferation of A-549 cells in vitro. At 48h, after the transfection the rates of growth inhibitory(38.87%) and apoptotic cells[(19.31±2.21)% ]were significantly higher in specific VEGF-C siRNA group than those in the control. The electron microscopy demonstrated that A-549 cells transfected with VEGF-C siRNA appeared typical morphological characteristics of apoptosis including the compaction and margination of the chromosomes, nuclear fragments and formation of apoptotic bodies.Conclusion: These results provided solid testimony that VEGF-C gene was vulnerable to RNA interference and, selective inhibition of VEGF-C had anti-proliferation effect on A-549 cells. Inhibition of the expression of VEGF-C can increase apoptosis of A-549 cells. RNA interference could be a powerful tool in further investigations of VEGF-C and a novel therapeutic strategy for lung cancer patients.