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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

    摘要
  • 摘要:
      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LipofectamineTM2000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(IC50); 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(OD450 nm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。

     

    Abstract:
      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 LipofectamineTM2000 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 (m6A) content of cells in each group; CCK-8 method was used to detect the half inhibitory concentration (IC50) 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 wild-type 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 (OD450 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 EZH2 m6A 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.

     

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