Effect Of Silencing Atg5 On Chemosensitization Of Adriamycin In K562/ADM Cells

Objective:To investigate the effect of Atg5 on autophagy and chemotherapy sensitivity of K562/ADM cells by down-regulating the expression of Atg5.Methods: 1.K562/ADM cells,NC-K562/ADM cells and Atg5siRNA-K562/ ADM cells were cultured in vitro.2.The specific Atg5 siRNA and scramble siRNA/negative control siRNA without any gene homology targeting Atg5 was designed and synthesized by small interfering RNA(siRNA)technology,and then transfect them into K562/ADM cells mediated by LipofectamineTM2000,continue to culture for 48 hours,the expression level of Atg5 protein in each cell grop(K562/ADM,NC-K562/ADM cells,Atg5siRNA-K562/ADM)was examined by Western Blotting,to verify the effect of siRNA silencing of Atg5 gene expression.3.On the basis of down-regulation of Atg5 protein expression,detecting the cell proliferation of K562/ADM cells,NC-K562/ADM cells and Atg5siRNA-K562/ADM cells after adriamycin treatment with different concentrations for 48 hours under the same conditions by Cell Counting Kit 8(CCK-8).4.After downregulating the expression of Atg5 protein,using Western Blotting to detect the expression level of autophagy marker protein LC3-II in K562/ADM cells,NC-K562/ADM cells and Atg5siRNA-K562/ADM cells,and then compare the changes of autophagy level between the cells of each group.Results: 1.Compared with K562/ADM cells and NC-K562/ADM cells,the difference of Atg5 protein expression in Atg5siRNA-K562/ADM cells was significant(p<0.05),the expression level of Atg5 protein in Atg5siRNA-K562/ADM cells was obviously decreased,which demonstrated that Atg5 siRNA can effectively silence Atg5 gene expression in K562 / ADM cell.2.Based on Atg5 siRNA interference Atg5 expression,under the same conditions with different concentrations of adriamycin treated cells,compared with K562/ADM cells and NC-K562/ADM cells,the cell viability of Atg5siRNA-K562/ADM cells was significantly decreased,that is to say,the drug sensitivity of Atg5siRNA-K562/ADM cells to adriamycin increased significantly.3.After down regulating the expression of Atg5 protein,Western Blotting detected that compared with K562/ADM cells and NC-K562/ADM cells,the expression level of LC3-II in Atg5siRNA-K562/ADM cells decreased significantly,namely cell autophagy is obviously inhibited.Conclusion: Atg5 siRNA can effectively down-regulate the expression of Atg5 gene in K562/ADM cells and enhance the sensitivity of K562/ADM cells to adriamycin chemotherapy by inhibiting autophagy.