Synergistic Effect Of Silencing ARK5 Gene And 5-flourouracil On Tumor Biological Behaviors Of AGS Gastric Cancer Cells

Objective:In this study,in order to detect the expression of ARK5 protein,we will construct the lentiviral vectors of ARK5-RNAi which was used to infect the human gastric cancer AGS cell lines.The basic-treatment drug of gastric cancer was selected and the effect of the 5-fluorouracilwas detected on human gastric cancer AGS cell lines.We used the experimental design of gene interference and common gastric cancer treatment drugs to explore the synergistic effect of silencing ARK5 gene and using 5-fluorouracil on tumor biological behaviors such as proliferation,migration,invasion and the cell cycle,cell apoptosis in human gastric cancer AGS cells.Methods:1.RNA interference(RNAi)sequence targeting the ARK5 gene was designed and a negative control(NC)sequence-lentiviral vectors silencing ARK5 were successfully constructed;2.The human gastric cancer AGS cells were infected with the negative control lentivirus of GFP in order to detect the best condition of infection in preliminary experiments;3.Western Blot analysis was used to detect the silencing effect of ARK5-RNAi;4.Detecting the cell proliferation inhibition and determining the most obvious inhibition of 5-FUconcentration which the human gastric cancer cells AGS were infected by ARK5-RNAi in combination with 5-FU drugs via MTT colorimetric assay;5.Based on the above experimental results,the experiment was randomly divided into six groups: Control group,NC group,ARK5-RNAi group,Control+5-FU group,NC+5-FU group and ARK5-RNAi+5-FU group.(1)To observe the ability of migration on human gastric cancer AGS cell via the scratch assay;(2)To examine the effect of invasion on human gastric cancer AGS cell by the Transwell;(3)To detect the cell cycle by flow cytometry;(4)In order to observe the cell apoptosis of human gastriccancer AGS cell via flow cytometry,each group was repeated six times.Results:1.Preliminary experimental results: with fluorescent negative lentiviral to infect the human gastric cancer cells AGS,the result of preliminary experiments showed that the best infective efficiency was in the presence of diet culture medium containing 10%FBS+Polybrene at a multiplicity of infection(MOI)of 100;2.The western blot showed that the expression of ARK5 in the ARK5-RNAi group decreased mostly,compared with noninfectious group(p<0.01),but negative group was compared to control group that was no significant difference(P >0.01);3.MTT assay results showed that cell growth inhibition rates of the human gastric cancer cells AGS increased when 5-FU concentration was increasing,and the inhibited effect of cell proliferation of ARK5-RNAi+5-FU group significantly enhanced compared to ARK5-RNAi group and control+5-FU group.Moreover,the synergistic effect was the most obvious when the 5-FU concentration was 80ug/ml,but the same effect was not be reached when the drug concentration>80ug/ml;4.Cell scratch assay revealed that the capabilities of cell migration after ARK5 gene silencing,ARK5-RNAi group and 5-FU drug were lower than Control groups,furthermore,NC group in compared with Control group was very similar;when ARK5 gene silencing in combination with 5-FU drugs,ARK5-RNAi+5-FU group compared with Control+5-FU group or 5-FU drug significantly reduced migration ability on tumor cells,the above results had significant statistical significance(P <0.01);5.Transwell invasion experiment showed that the capabilities of cell invasion after ARK5 gene silencing,ARK5-RNAi group and 5-FU drug were lower than Control groups and NC group,besides,NC group in compared with Control group was very similar;at the same time,when ARK5 gene silencing in combination with5-FU drugs,ARK5-RNAi+5-FU group compared with Control+5-FU group or 5-FU drug significantly reduced invasion ability on tumor cells,the above results had significant statistical significance(P < 0.01);6.Flow cytometry test results revealedthatARK5 gene silencing promoted the apoptosis and retarded the cell cycle of human gastric cancer AGS cell lines,theresults have significant statistical significance(P<0.01);apoptosis rate was higher and cell cycle arrest was stronger after ARK5 gene silencing in combination with 5-FU drug compared with ARK5-RNAi or 5-FU drug all alone on human gastric cancer AGS cell lines,the difference was statistically meaning(P<0.01).Conclusion: The ARK5-RNAi combined with 5-FU drug could significantly inhibit the malignant tumor biological behaviors of human AGS gastric cancer cell compared with using ARK5 gene silencing or 5-FU drug,the effects of inhibition were characterized by obviously inhibiting gastric cancer cell proliferation,migration and invasion,blocking the cell cycle,distinctly promoting apoptosis of gastric cancer cells.The above results indicated that ARK5-RNAi in combination with 5-FU had a synergistic inhibitory effect on malignant tumor biological behaviors of human AGS gastric cancer cells.