| Objective:To study the relationship between ARK5 and the multidrug resistance of gastric cancer cells at cellular level,and to determine whether silencing ARK5 gene can effectively reverse the drug resistance in gastric cancer multidrug-resistance cells SGC7901/DDP.Methods:1.Design RNA interference(RNAi)sequence and negative control(NC)sequence which could specifically target ARK5 gene,and successfully construct LV-ARK5-RNAi recombinant lentivirus and negative control lentivirus.2.The multidrug-resistant gastric cancer cell SGC7901/DDP was infected by negative control lentiviruses with fluorescence(GFP).Then the optimal infection conditions were screened out by observing the fluorescence inversion microscopy.3.Western Blot was used to detect the expression of ARK5 in parental gastric cancer cells SGC7901 and DDP-induced multidrug-resistant cells SGC7901/DDP,and to verify the gene silencing efficacy of lentiviral vector on ARK5 gene.4.The parental gastric cancer cells SGC7901,multidrug resistant cells SGC7901/DDP,lentivirus-infected cells SGC7901/DDP-shARK5 and negative virus infected cells SGC7901/DDP-NC were treated with cisplatin(DDP),5-fluorouracil(5-Fu),adriamycin(ADR)and docetaxel(DR)respectively.Observing the effect of silencing ARK5 gene on drug resistance of multi-drug resistant cells in gastric cancer by detecting these following changes:(1)CCK colorimetric assay was used to detect the survival rate of cells in each experimental group and the half maximal inhibitory concentration(IC50)of cells to the four chemotherapeutic drugs mentioned above;(2)colony formation assay was used to detect the colony forming ability of cells in each experimental group;(3)flow cytometry was used to detect the apoptotic rate of cells in each experimental group.5.The accumulation and retention of adriamycin in SGC7901 cells,SGC7901/DDP cells,SGC7901/DDP-shARK5 cells and SGC7901/DDP-NC cells were respectively detected by flow cytometry,which could also observe the effect of silencing ARK5 gene on the active drug efflux ability of multidrug resistant gastric cancer cells.Results:1.Pre-experimental results of viral infection: The optimum conditions for LV-ARK5-RNAi recombinant lentivirus infection as follows,firstly,the MOI(multiply of infection)value is 20,secondly,the infection system is the 1640low-sugar medium with 10% FBS and HiTransG A,also,the infection time is 72hours;2.Western Blot results showed that,the expression of ARK5 protein in multidrug resistant gastric cancer cell SGC7901/DDP was significantly higher than that in parental gastric cancer cell SGC7901.Moreover,LV-ARK5-RNAi lentiviral vector could significantly reduce the expression of ARK5 protein in multidrug resistant gastric cancer cell SGC7901/DDP,.Meanwhile,the expression level of ARK5 in multi-drug resistant gastric cancer cell SGC7901/DDP which was infected with negative lentivirus showed no significant change.3.After treating with the four chemotherapy drugs,DDP,5-Fu,ADR and DR,the cell viability,concentration of IC50,number of cell colony formation and apoptotic rate of multidrug-resistant gastric cancer cells SGC7901/DDP with high expression of ARK5 were higher than those of parental gastric cancer cells SGC7901 with low expression of ARK5.After silencing ARK5 gene,compared with multidrug-resistant gastric cancer cells SGC7901/DDP,the ARK5 lentiviral-infected cells SGC7901/DDP-shARK5 showed a significant decrease in cell viability,concentration of IC50,number of cell colony formation and apoptotic rate(p < 0.01).4.The drug pumping rate of multi-drug resistant gastric cancer cell SGC7901/DDP with high expression of ARK5 was significantly higher than that of its parental cell SGC7901.After silencing the ARK5 gene,the drug pumping rate of ARK5 lentivirus infection group SGC7901/DDP-shARK5 was significantly lower than that of multidrug resistant gastric cancer cell SGC7901/DDP.Conclusion:After the silencing of ARK5 gene,the sensitivity of multidrug resistant gastric cancer cell SGC7901/DDP to chemotherapeutic drugs increased significantly while the drug resistance decreased significantly.The results indicated that the ARK5 is closely related to the multidrug resistance of gastric cancer.Additionally,silencing the expression of ARK5 gene could effectively reverse the multidrug resistance of gastriccancer cells SGC7901/DDP,and the mechanism might be connected with that interfering with the expression of ARK5 gene can promote the apoptosis and inhibit the active drugs efflux ability of multidrug resistance cells. |
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