The Role And Mechanism Of UVRAG On Drug Resistance In K562/ADM Cells

Objective: To investigate the role of UVRAGsi RNA on autophagy and drug resistance by down-regulating the expression of UVRAG gene in K562/ADM cells and to reveal preliminarily the possible mechanism of UVRAG participating in K562/ADM cells multidrug resistance(MDR) formation.Methods:1.K562 cells and K562/ADM cells were cultured in vitro. UVRAG protein levels were measured by Western Blotting in K562 cells and K562/ADM cells and the highly UVRAG-expressed cells was selected out as appropriate transfected model of the cell lines.2.Specific interference UVRAGsi RNA and Scrambled si RNA without any gene homology were designed and synthesized, LipofectamineTM 2000-mediated si RNA transfected K562/ADM cells,then si-UVRAG-5562/ADM cells and NC-K562/ADM cells were successfully established. The UVRAG expression levels in transfected K562/ADM ce1 ls and untransfected cells were assessed by Western Blotting.3.The influences of chemotherapeutic adriamycin on cell viability and half inhibitory concentration50(IC50) of K562 cells,K562/ADM cells, NC-K562/ADM cells and si-UVRAG-K562/ADM cells were detected by CCK-8 kit.4. The formation of autophagic vacuoles of K562 cells, K562/ADM cells and si-UVRAG-K562/ADM cells were observed by MDC staining and fluorescence microscope. Further Through Western Blotting to measure the autophagy-related protein(Beclin-1,P62)and P-gp protein expression levels of K562 cells, K562/ADM cells and the transfected K562/ADM ce1 ls.Results:1. The expression level of UVRAG protein in K562/ADM cells was higher than K562 cells with significant difference(P<0.05). The UVRAG protein expression level of K562/ADM cells was obviously decreased after transfection with UVRAGsi RNA.2.In comparison with NC-K562/ADM cells group, K562/ADM cells group and K562 cells group, the resistance of adriamycin in si-UVRAGK562/ADM group was declined and IC50 obviously decreased.3.MDC staining and fluorescence microscope observation showed that the autophagic vacuoles in endochylema of K562/ADM cells were more than K562 cells. Compared with the K562/ADM cells, autophagic vacuoles in si-UVRAG-K562/ADM cells were signifycantly reduced after UVRAGsi RNA transfection. Further findings showed that the expression levels of Beclin-1 and P-gp and the degradation of P62 were higher than K562 cells, the expression levels of Beclin-1 and P-gp were singnificantly decreased and the degradation of autophagic substrate P62 was also reduced after inhibition expression of UVRAG gene(P all<0.05).Conclusion:1. The expression level of UVRAG protein in K562/ADM cells was higher than K562 cells, suggesting that the highly expressed UVRAG protein may be closely related to the formation of MDR in K562/ADM cells.2. Transfection with UVRAGsi RNA downregulate the UVRAG protein expression in K562/ADM cells,which can reduce drug resistance to chemotherapeutic adriamycin and decrease cell viability after chemotherapy. It may be involved in the inhibition of autophagy and downregulation expression of P-gp.