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

  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^TM2000 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 (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 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.