An Study On Inhibitory Effect Of VEGF-C And Raf-1 Gene Corporate Silence To The Proliferation Of Human Lung Cancer Cell Line A549

Objective To explore the effects and possible mechanisms of RNA interference targeting VEGF-C and RAF-1 mRNA on the proliferation, invasion and cell cycle of human lung cancer cell line A549.Methods According to the basic principle of RNA interference, plasmid which expressed short hairpin RNA (shRNA) corresponding to VEGF-C and RAF-1 were respectively constructed by DNA recombining technology. After restriction endonuclease digestion and sequence were identified, the plasmid was transfected into A549 cell. Then the expression of mRNA and protein was detected by RT-PCR and Western blot. According to the result, the effective sequence was selected and the stable cell line which expressed shRNA was screened out. Plasmid vector with both VEGF-C and RAF-1 were constructed, and the stable cell line transfected with that were built. MTT was used to test the cell growth, Boyden methods was used to dectect the the invasion ability of A549 cell, flowcytometry was used to observe the expression level of protein, cell cycle and apoptosis, and Western blot was using to test the the level of associated pathway such as Caspase3, PDK1, AKT, RAF and ERK phosphorylation.Results The expressions of mRNA and protein of A549-VEGF-Ce and A549-RAF-le cell line which was transfected with shRNA targeting VEGF-C or RAF-1 gene were significantly inhibited. The inhibited effect of A549-RAF-1-VEGF-Ce cell line which contained two kinds of shRNA was same as A549-VEGF-Ce or A549-RAF-le cell line. According to MTT results, the cell proliferation ability of A549-Raf-1-VEGF-Ce, A549-Raf-le and A549-VEGF-Ce cell lines were significantly lower than the control group.The average number of A549-RAF-le cell line was a little lower than that of A549-VEGF-Ce, but there was no significant difference between them. Nevertheless, the proliferation ability of A549-Raf-1-VEGF-Ce cell was dramaticly lower than that of A549-VEGF-Ce. In the invasion test in vitro, the invasion cell number of A549-Raf-le, A549-VEGF-Ce and A549-Raf-1-VEGF-Ce was lower than control cell group, but there were no significant difference among them. In cell cycle tested by flowcytometry, the proportion of cell number in G1 phase of A549-VEGF-Ce was higher than other cell groups and a apoptosis peak was emerged in A549-Raf-le group.The A549-Raf-1-VEGF-Ce cell line with lower proportion of G1 phase cell than A549-VEGF-Ce had obviously more apoptosis cell. Further examination about the apoptosis level of all cell lines showed that the apoptosis ratio of A549-Raf-1-VEGF-Ce (24%) was higher than that of A549-Raf-le (17%) and the ration of other groups which kept in line with cell cycle test had no significant changes. Western blot results showed that actived large subunit of Caspase3 was caught both in 549-Raf-1-VEGF-Ce and A549-Raf-le group, and the phosphorylation level of AKT, PDK1 and RAF in all cell groups had no significant difference exception of the obvious decrease of phosphorylation level of ERK in A549-Raf-1-VEGF-Ce cell line.Conclusion shRNA targeting VEGF-C and RAF-1 gene could inhibit the proliferation and invasion abilities of A549 cell and regulate the cell cycle and apoptosis of tumor cell. And RNA interference targeting on VEGF-C and RAF-1 gene simultaneously had a cooperative effect mainly due to the increased level of Caspase3 and phosphorylation form of ERK.