烷基化修复蛋白B同源物5调控果蝇Zeste基因增强子人类同源物2对K562/阿霉素细胞耐药的影响

Effect of alkylation repair protein B homolog 5 on drug resistance of K562/adriamycin cells by regulating enhancer of Zeste homolog 2

目的探讨烷基化修复蛋白B同源物5(ALKBH5)对K562/阿霉素(ADM)细胞耐药的影响及其作用机制。方法以人白血病细胞系K562和ADM抗性细胞K562/ADM细胞为研究对象,利用Lipofectamine;2000将K562/ADM细胞分为对照组、si-NC组、si-ALKBH5-1组、si-ALKBH5-2组、空载体组(转染pcDNA3.1)、ALKBH5-WT组(转染pcDNA3.1-ALKBH5-WT)、ALKBH5-MUT组(转染pcDNA3.1-ALKBH5-MUT)、si-ALKBH5-2+空载体组、si-ALKBH5-2+pcDNA3.1-EZH2组。采用比色法检测细胞中N6-甲基腺苷(m6A)含量;采用CCK-8法检测细胞的半数抑制浓度(IC_(50));采用荧光光度计法检测ADM外排;采用Western blot检测细胞中ALKBH5、果蝇Zeste基因增强子人类同源物2(EZH2)、P糖蛋白(P-gp)、多药耐药基因1(MDR1)的蛋白表达;采用RNA免疫共沉淀(RIP)实验验证ALKBH5与EZH2 mRNA的相互作用;采用甲基化RNA免疫共沉淀(MeRIP)检测EZH2 m6A水平。结果与K562细胞比较,K562/ADM细胞对ADM的耐药性及细胞中ALKBH5蛋白表达水平升高,m6A含量降低,差异有统计学意义(P<0.01);沉默ALKBH5可增加K562/ADM细胞中m6A含量,过表达野生型ALKBH5可减少m6A含量,而过表达突变型ALKBH5(H204A)对m6A含量无明显影响。与对照组、si-NC组比较,si-ALKBH5-1组、si-ALKBH5-2组细胞在450 nm处的光密度值(OD_(450nm)值)、P-gp、MDR1蛋白表达水平降低,细胞内荧光强度升高,差异有统计学意义(P<0.05)。在K562/ADM细胞中,ALKBH5蛋白能与EZH2 mRNA相互作用;沉默ALKBH5可上调K562/ADM细胞中EZH2 m6A水平,下调EZH2蛋白表达水平,过表达野生型ALKBH5则呈相反趋势;EZH2过表达逆转了沉默ALKBH5对K562/ADM细胞活力及耐药性的影响。结论沉默ALKBH5通过促进EZH2的甲基化来下调EZH2的表达,进而降低K562/ADM的耐药性。

Objective To investigate the effect of alkylation repair protein B homolog 5(ALKBH5) on drug resistance of K562/adriamycin(K562/ADM) cells and its mechanism. Methods The human leukemia cell line K562 and ADM resistant K562/ADM cells were selected as the research objects, and Lipofectamine;2000 was used to assign K562/ADM cells into control group, si-NC group, si-ALKBH5-1 group, si-ALKBH5-2 group, empty vector group(transfected with pcDNA3.1), ALKBH5-WT group(transfected with pcDNA3.1-ALKBH5-WT), ALKBH5-MUT group(trans fected with pcDNA3.1-ALKBH5-MUT), si-ALKBH5-2+empty vector group and si-ALKBH5-2+pcDNA3.1-EZH2 group. Colorimetric method was used to detect the N6 methyladenosine(m6 A) content of cells in each group;CCK-8 method was used to detect the half inhibitory concentration(IC_(50)) of cells;fluorescence photometer method was used to detect ADM efflux;Western blot was used to detect the protein expressions of ALKBH5,enhancer of Zeste homolog 2( EZH2),P glycoprotein( P-gp)and multidrug resistance gene 1( MDR1) of cells;RNA immunoprecipitation( RIP) experiment was used to verify the interaction between ALKBH5 and EZH2 mRNA;methylated RNA immunoprecipitation( MeRIP) was used to detect the level of EZH2 m6A. Results Compared with K562 cells,the resistance of K562/ADM cells to ADM and the protein expression level of ALKBH5 in the cells were significantly increased,and the m6A content was significantly decreased( P < 0. 01);silencing ALKBH5 was able to increase m6A content in K562/ADM cells,and overexpression of wildtype ALKBH5 was able to reduce m6A content,but overexpression of mutant ALKBH5( H204A)had no significant effect on m6A content. Compared with the control group and the si-NC group,the absorbance value at 450 nm( OD_(450 nm)) and protein expression levels of P-gp and MDR1 in the cells of the si-ALKBH5-1 group and si-ALKBH5-2 group were significantly reduced,while the intracellular fluorescence intensity was significantly increased( P < 0. 05). ALKBH5 protein was able to interact with EZH2 mRNA in K562/ADM cells;silencing ALKBH5 was able to up-regulate the EZH2m6A level and down-regulate the protein expression level of EZH2 in K562/ADM cells,while overexpression of wild-type ALKBH5 showed the opposite trend;EZH2 overexpression reversed the effect of silencing ALKBH5 on the viability and drug resistance of K562/ADM cells. Conclusion Silencing ALKBH5 can down-regulate the expression of EZH2 by promoting the methylation of EZH2,thereby reducing the resistance of K562/ADM.