The Effects And Mechanism Of ShRNA Silencing VEGF Gene In Lung Cancer

Bacground&Objective Angiogenesis is associated with several pathological disorders. Tumor growth and metastasis are dependent on angiogenesis. Components of vascular basement membrane are involved in regulating angiogenesis. The binding of VEGF to VEGFR expressing on the endothelial cells membrane, which triggered the signaling transduction, resulted in the mitosis and proliferation of endothelial cells, and promoted angiogenesis. The VEGF/VEGFR signaling pathway could enhance specially the penetration of blood vessel and protect the damaged neural cells, which plays an important role in the physiological and pathological process, including the embryonic development, inflammation and tumor. Previous studies showed that most solid tumor cloud secret the VEGF. What's more, the VEGF expression was related with the malignant tumor development. Therefore, VEGF or VEGFR was the most potent target against tumor angiogenesis. RNA interference (RNAi), which could silence the mRNA transcription specially, was a mechanism of post-transcription gene silence induced by the double stranded RNA. RNAi is not only a technology in study of genome function, but also an effective method to treat disease, especially, in gene therapy of the tumor. Studies demonstrated that the proliferation and invadation in many solid tumors could be inhibited with shRNA silencing the VEGF expression.In this study, to investigate the effects and mechanism of shRNA silencing VEGF gene expression in lung cancer.Materials&Methods An effective and stable pSilencer-VEGF-shRNA plasmid to silence VEGF expression was screened and constructed successfully. A549 cells were transfected with pSilencer-VEGF-2 to observe its expression of VEGF, proliferation of the non small cell lung cancer and tubule formation of the endothelial cells in vitro. Finally the A549 cells and pSilencer-VEGF-A549 cells were injected subcutaneously into nude mice to observe the tumor growth and survival of the mice in vivo. Identify the changes of p53, Bcl-2 and Bax in VEGF interference A549 cells which interacted with HUVEC by RT-PCR and Western blot.Results The results showed that the expression of VEGF in stable transfectant A549 cell line was obviously inhibited over 90% by pSilencer-VEGF-2. Two months later the inhibition rate was still above 80%. Although growth of the pSilencer-VEGF-A549 cells was hard inhibitedt, the angiogenesis of endothelial cells was decreased in vitro. Compared with the control, the tumor growth was significantly slow(P<0.05) and the survival was obviously enlonged in the mice injected pSilencer-VEGF-2-A549(P<0.05)in vivo.In the mechanism study, we found that the P53, Bcl-2 and Bax expression did not change in the stable transfected A549 cell lines. And we did not observe the apoptosis of stable transfected A549 cells. Compared with the control group, the expression of Bax was not changed and the Bcl-2 expression was decreased, however the P53 expression increased with apoptosis in the stable transfected A549 cell lines under the HUVEC.Conclusion The shRNA vector pSilencer-VEGF which could silence the VEGF expression in lung cancer effectively was successfully constructed. Proliferation of the lung cancer cells and development of the lung cancer were inhibited with RNAi silencing VEGF expression both in vivo and vitro. The possible mechanism might be due to the block of VEGF/VEGFR resulting in apoptosis of the cancer cells.