Effects Of ARK5 Gene Silence Combined With 5-fluorouracil On Biological Behaviors Of Gastric Cancer Cells

Objective: The tumor microenviroment is always in a ischemia, hypoxia and energy shortage condition due to the excessive proliferation of tumor cells.Meanwhile studies revealed that ARK5 was involved in the tolerance of tumor cells to local metabolic changes, and promoted tumor cells survival, invasion and metastasis. In the study, we will establish the lentiviral vectors of ARK5-RNAi to explore the effects of ARK5 gene silence combined with 5-fluorouracil on biological behaviors of gastric cancer cells.Methods: We designed three RNA interference(RNAi) sequences targeting the ARK5 gene and a negative control(NC) sequence and constructed the lentiviral vectors; The negative control lentivirus with GFP was used to infect the gastric cancer cells in order to get the optimum conditions of infection, and then we chosen the most effective ARK5-RNAi vector by Western Blot and transfected to SGC7901 cells; MTT colorimetric assay was used to test the cell growth inhibition rates in order to determine the concentration of 5-fluorouracil combined with ARK5 gene silence. The cultured SGC7901 gastric cancer cells were randomly divided into 6 groups: Control group, NC group, ARK5-RNAi group, Control+5-FU group, NC+5-FU group and ARK5-RNAi+5-FU group, we measured cell proliferation by MTT colorimetric assay, cell migration by scratch assay, cell invasion by transwell, cell apoptosis and cycle by flow cytometry, each group was repeated six times.Results: The result of gene sequencing showed that three ARK5-RNAi recombinant lentiviral vectors had been constructed successfully; When the SGC7901 cells were infected with viral particles in the presence of ENI.S. and Polybrene at a multiplicity of infection(MOI) of 50, the infection efficiency was optimal; Compared with negative control group, the expression of ARK5 in the ARK5-RNAi-3 group decreased mostly(p < 0.01); MTT assay showed that cell growth inhibition rates of SGC7901 increased as concentration of 5-FU increasing, and ARK5-RNAi+5-FU group inhibited the cell proliferation more significantly than ARK5-RNAi group and control+5-FU group, in addition, ARK5 gene silence combined with 10 to 160 μg/ml 5-FU could produce synergistic effects, however, this synergy could be most obvious when the concentration of 5-FU was 40 μg/ml; Cell scratch assay and transwell assay further stated that the capabilities of cell migration and invasion in combined group were lower than other groups(p<0.01), detection of flow cytometry also comfirmed that the rate of apoptosis and cell cycle arrest in combined group changed more obviously(p<0.01).Conclusion: After ARK5 gene silencing, the biological behavior of gastric cancer cell line SGC7901 was inhibited significantly. That demonstrated that ARK5 played an important role in promoting gastric cancer occurrence and development. Furthermore, treated by 5-fluorouracil together with ARK5 silening, the biological behavior of tumor cells was more inhibited, indicating that ARK-RNAi and 5-fluorouracil used in combination had a synergistic inhibitory effect on the biological behavior of gastric cancer cells.