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ONCOLOGY / BASIC RESEARCH
KDM1A represses apoptosis in osteosarcoma cells via demethylating Bcl-2/c-Myc
1
Department of Orthopedics Surgery, Xixi Hospital of Hangzhou, Hangzhou, Zhejiang Province, China
2
Department of Orthopedics Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
3
Orthopedics Research Institute of Zhejiang University, Hangzhou, Zhejiang Province, China
4
Key Laboratory of Motor System Disease Research and Precision Therapy of Zhejiang Province, Hangzhou, Zhejiang Province, China
Submission date: 2023-03-17
Final revision date: 2023-07-17
Acceptance date: 2023-07-22
Online publication date: 2023-08-07
Corresponding author
Wei Gao
Department of Orthopedics Surgery, Xixi Hospital of Hangzhou, No. 2 Hengbu Street, Liuxia town, Xihu District, Hangzhou 310012, Zhejiang Province, China
Department of Orthopedics Surgery, Xixi Hospital of Hangzhou, No. 2 Hengbu Street, Liuxia town, Xihu District, Hangzhou 310012, Zhejiang Province, China
KEYWORDS
osteosarcomalysine demethylase 1Aapoptosisreactive oxygen speciesendoplasmic reticulum stressdemethylationBcl-2/c-Myc
TOPICS
ABSTRACT
Introduction:
Considering the poorly understood role of lysine demethylase 1A (KDM1A) in osteosarcoma (OS), we conducted this study to elucidate the underlying mechanism.
Material and methods:
Following the appropriate transfection, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and flow cytometry assays were used to determine the viability and apoptosis of MG-63 OS cells, in which the generation of reactive oxygen species (ROS) and the binding between KDM1A and Bcl-2/ cellular Myc (c-Myc) were separately confirmed via the DCF-DA (2,7-dichlorodihydrofluorescein diacetate) method and chromatin immunoprecipitation-PCR. Reverse-transcription quantitative PCR and western blot were finally introduced to quantify the levels of KDM1A/Bcl-2/c-Myc and endoplasmic reticulum (ER) stress-related factors.
Results:
Overexpressed KDM1A enhanced the viability (48 h) yet repressed the apoptosis and ROS generation, with downregulation of ER stress-related factors (C/EBP homologous protein [CHOP]; proline-rich extensin-like receptor kinase (PERK) and activating transcription factor 4 [ATF4]) but elevation of Bcl-2/c-Myc, while its depletion exerted contrary effects. More importantly, KDM1A could act as the demethylase of Bcl-2/c-Myc, as reflected by the results showing that the depletion of KDM1A decreased the enrichment of Bcl-2/c-Myc promoter using the antibody against KDM1A yet increased the enrichment by the antibody targeting H3K9me2. Bcl-2/c-Myc silencing, conversely, promoted ROS generation and apoptosis, elevated the levels of ER stress-related factors and abolished the effects of KDM1A on OS cells.
Conclusions:
KDM1A exerts a repressive effect on the apoptosis of MG-63 OS cells by inhibiting ROS generation and ER stress via demethylation of Bcl-2 and c-Myc.
Considering the poorly understood role of lysine demethylase 1A (KDM1A) in osteosarcoma (OS), we conducted this study to elucidate the underlying mechanism.
Material and methods:
Following the appropriate transfection, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and flow cytometry assays were used to determine the viability and apoptosis of MG-63 OS cells, in which the generation of reactive oxygen species (ROS) and the binding between KDM1A and Bcl-2/ cellular Myc (c-Myc) were separately confirmed via the DCF-DA (2,7-dichlorodihydrofluorescein diacetate) method and chromatin immunoprecipitation-PCR. Reverse-transcription quantitative PCR and western blot were finally introduced to quantify the levels of KDM1A/Bcl-2/c-Myc and endoplasmic reticulum (ER) stress-related factors.
Results:
Overexpressed KDM1A enhanced the viability (48 h) yet repressed the apoptosis and ROS generation, with downregulation of ER stress-related factors (C/EBP homologous protein [CHOP]; proline-rich extensin-like receptor kinase (PERK) and activating transcription factor 4 [ATF4]) but elevation of Bcl-2/c-Myc, while its depletion exerted contrary effects. More importantly, KDM1A could act as the demethylase of Bcl-2/c-Myc, as reflected by the results showing that the depletion of KDM1A decreased the enrichment of Bcl-2/c-Myc promoter using the antibody against KDM1A yet increased the enrichment by the antibody targeting H3K9me2. Bcl-2/c-Myc silencing, conversely, promoted ROS generation and apoptosis, elevated the levels of ER stress-related factors and abolished the effects of KDM1A on OS cells.
Conclusions:
KDM1A exerts a repressive effect on the apoptosis of MG-63 OS cells by inhibiting ROS generation and ER stress via demethylation of Bcl-2 and c-Myc.
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