摘要
目的:通过MeRIP联合逆转录实时定量PCR(RT-qPCR)技术,证明WTAP介导的RNA m^(6)A修饰对伴t(8;21)急性髓系白血病(AML)细胞中KDM4B基因的直接调控作用。方法:采用靶向WTAP或KDM4B基因的短发夹RNA(small hairpin RNA,shRNA)慢病毒载体沉默t(8;21)AML细胞系Kasumi-1和SKNO-1中WTAP或KDM4B基因表达,以转染随机打乱序列(scramble)的shRNA的细胞为对照。用超纯RNA提取试剂盒(DNaseⅠ)提取细胞RNA,Magna MeRIP^(TM)m^(6)A Kit试剂盒富集甲基化修饰片段,并通过RT-qPCR检测m^(6)A甲基化修饰的RNA区域;采用蛋白免疫印迹实验(Western blot)和逆转录实时定量PCR(RT-qPCR)技术检测细胞中WTAP、KDM4B蛋白和mRNA的表达水平。采用克隆形成实验检测细胞体外克隆形成能力。结果:沉默Kasumi-1细胞中WTAP的表达后,m^(6)A甲基化修饰在KDM4B mRNA 3’UTR的富集程度显著下降(P<0.01),沉默Kasumi-1和SKNO-1细胞中WTAP的表达可显著抑制KDM4B蛋白(P<0.001)和mRNA表达水平(Kasumi-1:P<0.001;SKNO-1:P<0.01)、细胞体外克隆形成能力下降(Kasumi-1:P<0.001;SKNO-1:P<0.01)。结论:t(8;21)AML细胞系中,WTAP通过调控KDM4B基因mRNA 3’UTR的m^(6)A修饰调控KDM4B的表达,沉默KDM4B表达可以抑制t(8;21)AML细胞增殖。
Objective:To confirm the direct regulatory effect of WTAP-mediated RNA m^(6)A 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 MeRIP^(TM) m^(6)A Kit was used to enrich methylated modified fragments,and detect the m^(6)A 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 m^(6)A 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 m^(6)A modification of the 3′UTR of KDM4B mRNA,and silencing the expression of KDM4B could inhibit the cellular proliferation in vitro.
作者
李雨晴
邵杨柳
李梦月
王莉莉
高晓宁
LI Yu-Qing;SHAO Yang-Liu;LI Meng-Yue;WANG Li-Li;GAO Xiao-Ning(Department of Hematology,The Fifth Medical Center,Chinese PLA General Hospital,Beijing 100071,China;Medical School of Chinese PLA,Beijing 100853,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2024年第2期382-388,共7页
Journal of Experimental Hematology
基金
国家自然科学基金(82070149,81870109)
北京市自然科学基金(7202191)。
