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

CHEN Li; JI Huijuan; REN Libin; ZHANG Hongfeng; SUO Xiaohui; LIU Hongfeng; XIA Limin

doi:10.7619/jcmp.20213953

Journal of Clinical Medicine in Practice > 2022 > 26(7): 87-92. > DOI: 10.7619/jcmp.20213953

CHEN Li, JI Huijuan, REN Libin, ZHANG Hongfeng, SUO Xiaohui, LIU Hongfeng, XIA Limin. Effect of alkylation repair protein B homolog 5 on drug resistance of K562/adriamycin cells by regulating enhancer of Zeste homolog 2[J]. Journal of Clinical Medicine in Practice, 2022, 26(7): 87-92. DOI: 10.7619/jcmp.20213953

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

1.
the First Department of Hematology, Handan City Central Hospital in Hebei Province, Handan, Hebei, 056001
2.
Department of General Surgery, Handan City Central Hospital in Hebei Province, Handan, Hebei, 056001
3.
Section of Scientific Research, Handan City Central Hospital in Hebei Province, Handan, Hebei, 056001
4.
Department of Breast Surgery, Handan City Central Hospital in Hebei Province, Handan, Hebei, 056001

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Received Date: October 07, 2021
Available Online: April 14, 2022
Published Date: April 14, 2022

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Abstract

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 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.
- N6 methyladenosine,
- alkylation repair protein B homolog 5,
- enhancer of Zeste homolog 2,
- adriamycin,
- P glycoprotein,
- multidrug resistance gene 1

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References

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