The Regulatory Effect of RNA m6A Methylation Modification on KDM4B Gene Expression in t(8;21)AML Cells by MeRIP-qPCR
10.19746/j.cnki.issn1009-2137.2024.02.009
- VernacularTitle:MeRIP-qPCR技术检测RNA m6A甲基化修饰对t(8;21)AML细胞中KDM4B基因表达的调控作用
- Author:
Yu-Qing LI
1
;
Yang-Liu SHAO
;
Meng-Yue LI
;
Li-Li WANG
;
Xiao-Ning GAO
Author Information
1. 中国人民解放军总医院第五医学中心血液病医学部,北京 100071;解放军医学院,北京 100853
- Keywords:
MeRIP-qPCR;
acute myeloid leukemia;
t(8;
21)chromosomal translocation;
KDM4B;
RNA m6A methylation modification
- From:
Journal of Experimental Hematology
2024;32(2):382-388
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To confirm the direct regulatory effect of WTAP-mediated RNA m6A modification on the KDM4B gene in t(8;21)acute myeloid leukemia(AML)cells through MeRIP combined with reverse transcription real-time quantitative PCR(RT-qPCR)technology.Methods:The lentivirus-mediated shRNA target WTAP or KDM4B gene was used to transfect the t(8;21)AML cell lines:Kasumi-1 and SKNO-1,and cells transfected with randomly shuffled shRNA as the control.Using the Ultrapure RNA Extraction Kit(DNase Ⅰ)to extract RNA.The Magna MeRIPTM m6A Kit was used to enrich methylated modified fragments,and detect the m6A methylated RNA regions by RT-qPCR,and the protein and mRNA expression levels of WTAP and KDM4B in cells were detected by Western blot and reverse transcription real-time quantitative PCR(RT-qPCR).Colony formation assays were used to detect the colony ability of cells in vitro.Results:Silencing the expression of WTAP in Kasumi-1 cells,the enrichment of m6A methylation modification was significantly decreased in the 3'UTR of KDM4B mRNA(P＜0.01),and the protein(P＜0.001)and mRNA(Kasumi-1:P＜0.001;SKNO-1:P＜0.01)expression levels of KDM4B were also significantly inhibited in Kasumi-1 and SKNO-1 cells upon WTAP knockdown(all P＜0.01),accompanied by a significant decrease in the colony-forming ability of both cell lines(both P＜0.01).Conclusion:In t(8;21)AML cell lines,WTAP could regulate the expression of KDM4B by regulating the m6A modification of the 3'UTR of KDM4B mRNA,and silencing the expression of KDM4B could inhibit the cellular proliferation in vitro.
