Study On The Reversal Of Drug Resistance Of Leukemic Cell Line K562/ADM By Physcion

Objective: The aim of this study is to evaluate the reversal effect of physcion on multidrug resistance of chronic myelogenous leukemia(CML)drug-resistant cell line K562/ADM and its mechanism Methods :1.Cell culture :CML cell line of K562 cells and resistant K562/ADM cells were cultivated by vitro culture of tumor cells.2.CCK8(cell counting kit-8,CCK8):test(1)the drug resistance multiple between K562 cells and K562/ADM cells;(2)The cell viability of K562/ADM cells interfered by different concentrations of physcion;(3)The cell viability of K562 cells interfered by ADM after miR-146 a is knockdown by miR-146 a inhibitor and CXCR4 is knockdown by corresponding siRNA in K562 cells;(4)the effect of physcion combined with ADM on the activity of K562/ADM cells.3.Colony formation assay:test the influence of K562/ADM cells’ viability by using the union of physcion and ADM.4.FMC(flow cytometry)and the effect of physcion combined with ADM on the activity of K562/ADM cells.Hoechst 33258 was performed to detect apoptotic cells: test(1)after the K562 cells was interfered by miR-146 ainhibitor.Test the apoptosis of the K562 cells、the K562+NC cells、the K562+mi R-146 a inhibitor cells;(2)after the K562/ADM cells was interfered by miR-146 a mimic test the apoptosis of the K562/ADM cells、the K562/ADM+NC cells、the K562/ADM +miR-146 a inhibitor cells;(3)the miR-146 a and miR-146 a inhibitor’s influence of the apoptosis of the K562 cells;(4)the influence of K562/ADM cells apoptosis by using the union of physcion and ADM;(5)the influence of physcion about the apoptosis of cells induced by ADM after decreasing miR-146 a expression in the K562/ADM cells.5.quantitative real-time PCR(qRT-PCR):test(1)the expression of miR-146 a in K562 cells and K562/ADM cells;(2)the expression of miR-146 a in K562 cells was downregulated by transfection of mi R-146 a inhibitor in the expression of miR-146 a in K562 cells.;(3)the expression of mi R-146 a in the K562/ADM cells after transfection miR-146 a mimic to over expression miR-146a;(4)after downregulating the expression of mi R-146 a in K562 cells,the expression of P-glycoprotein(P-glycoprotein,P-gP),multidrug resistance protein-1(multidrug resistant related protein,and resistant)in K562 cells of each group was observed;(5)the expression of P-gP,MRP-1 and CXCR4 in the K562/ADM cells of each group after increasing the expression of mi R-146 a in K562/ADM cells;(6)the expression of mi R-146 a in K562/ADM cells under the intervention of physcion;(7)the expression of CXCR4 in all cells after transfection of CXCR4 target to siRNA,miR-146 a inhibitor and transfected CXCR4 targeting siRNA+mi R-146 a inhibitor in K562 cells;(8)the influence inCXCR4 expression in different concentrations of physcion.6.Western blot: detect(1)down regulation of miR-146 a expression in K562 cells and the protein expression of CXCR4 in K562 cells in each group;(2)up-regulation the expression of miR-146 a in K562/ADM cells and the protein expression of CXCR4 in K562/ADM cells in each group;(3)the expression of CXCR4 protein in the cells of K562/ADM cells after different concentrations of physcion;(4)expression of CXCR4 protein in all cells after transfection of CXCR4 target to siRNA,miR-146 a inhibitor and transfected CXCR4 targeting siRNA+miR-146 a inhibitor in K562 cells.7.Transwell: evaluation of the effect of physcion on CXCR12 binding to CXCR4 in K562/ADM cells.8.Surface CXCR4 expression:test(1)down regulation of miR-146 a expression in K562 cells and the expression of CXCR4 on the surface of K562 cells in each group;(2)the expression of CXCR4 on the surface of K562 / ADM cells in each group after increasing the expression of miR-146 a in K562/ADM cells.Results1.miR-146 a is downregulated in K562/ADM cells.After 48 hours of ADM intervention,the IC50 of K562 cells and K562/ADM cells were 0.15 and 3.5 M respectively,indicating that the drug resistance of K562/ADM was 23 times of that of K562 cells.The expression of miR-146 a in K562/ADM was significantly lower than that in K562 cells.2.Downregulation of mi R-146 a can enhance the K562 cells resistant to ADMThe expression level of mi R-146 a was significantly decreased after transfection of miR-146 a inhibitor,and the survival rate of K562 cells after downregulation of mi R-146 a was significantly higher than that of normal K562 cells and negative control group(IC50 = 2.8 M,0.15 M,and 0.18 M respectively).Down regulation of miR-146 a can significantly enhance the resistance of K562 cells to ADM induced apoptosis.3.Upregulation of mi-R146 a can reinforce the sensitivity of K562/ADM cells to ADMThe expression of miR-146 a in K562/ADM cells transfected with miR-146 a mimic increased significantly.Compared with K562/ADM cells and K562/ADM cells transfected with blank plasmid,the sensitivity of K562/ADM cells to ADM increased significantly(IC50 was 0.42 M,3.5 u M and 3.2 U),respectively.It shows that up regulation of mi RNA-146 a can make K562/ADM cells more sensitive to ADM induced apoptosis.4.miR-146 a participates ADM resistance in K562 cells mainly through CXCR4Down regulation of miR-146 a in K562 cells or up regulation of miR-146 a in K562/ADM did not cause significant changes in mRNA of P-gp and MRP-1,and mi R-146 a expression was negatively correlated with CXCR4 mRNA expression,and CXCR4 was the direct target of CXCR4.Targeting CXCR4 siRNA can inhibit the expression of CXCR4 in cells,miR-146 a inhibitors can induce up regulation of CXCR4 expression and obviously enhance the drug resistance of K562 cells.5.Physcion strengthens the sensitivity of K562/ ADM to ADMThe effect of physcion on the activity of K562/ADM cells was dose-dependent,and the reversal multiple of drug resistance was 2.03 times and5.3 times at the concentration of 2 and 5 M respectively.Compared with the cells treated with ADM alone,the colony number and colony size of the cells decreased significantly when the physcion and ADM were combined with K562/ADM.Compared with the single ADM intervention,the combined intervention concentration significantly increased the apoptosis of K562/ADM cells after the action of 2 and 5 M emodin methyl ether on the cells..6.Physcion reverses drug-resistance through upregulating mi R-146 a and interfering CXCL12/ CXCR4 signal pathAfter interfered by physcion,the level of miR-146 a in K562/ADM cells increased in a dose-dependent manner.The effect of emodin methyl ether on the sensitivity of K562/ADM cells to the sensitivity of ADM was almost completely disappeared after the miR-146 a inhibitor.The prognosis of emodin methyl ether(emodin)results in a dose-dependent reduction in the expression of chemokine receptor CXCR4 in K562/ADM cells.Significantly impaired migration after emodin methyl ether.Conclusion1.Down regulation of miR-146 and up-regulated CXCR4 may be associated with resistance to K562/ADM cells.2.Pyscion can reverse the drug resistance of K562/ADM cells to ADM by inhibiting the expression of CXCL12/CXCR4 by inducing the expression of miR-146 a.