DNMT1基因沉默增强NK-92对Jurkat细胞杀伤作用及其机制
Silencing DNMT1 by siRNA enhances the killing effect of NK-92 on Jurkat cells and the underlying mechanism
武坤 1马晓波 1程沈菊 1杨金荣 2贺振新 2郭翀1
作者信息
- 1. 云南省检验医学重点实验室,昆明 650032;云南省医学检验临床医学研究中心,昆明 650032;昆明医科大学第一附属医院 医学检验科,昆明 650032
- 2. 昆明医科大学第一附属医院血液科,昆明 650032;云南省血液病研究中心,昆明 650032
- 折叠
摘要
为了探讨沉默DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)增强NK-92对Jurkat细胞杀伤作用及机制,检测2020年1月-2021年12月35例急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)患者骨髓标本的DNMT1水平.si-DNMT1被转染至NK-92细胞,并与Jurkat细胞共培养,乳酸脱氢酶(lactate dehydrogenase,LDH)法检测对Jurkat细胞的杀伤作用,流式细胞术检测Jurkat细胞凋亡及NKG2D膜表达.ELISA检测细胞因子IFN-γ水平,Western blotting测定NK-92细胞Syk、ZAP70、p-Syk、p-ZAP70、穿孔素、颗粒酶B表达.结果显示,ALL患者DNMT1水平明显高于对照组(P＜0.05);DNMT1 对 ALL 具有诊断价值(AUC:0.692,95％CI:0.533-0.851,P=0.033).高 DNMT1 组患者 PFS 明显低于低DNMT1组患者(P＜0.05).转染si-DNMT1后,NK-92细胞对Jurkat细胞杀伤率、Jurkat细胞凋亡率明显高于si-NC组(P＜0.05),分泌NKG2D,及表达穿孔素、颗粒酶B能力明显升高(P＜0.05).si-DNMT1组NK-92细胞生成IFN-y水平明显高于si-NC组(P＜0.05),其p-Syk、p-ZAP70明显高于si-NC组(P＜0.05).综上,DNMT1在ALL中高表达,沉默DNMT1可增强NK-92细胞对Jurkat细胞杀伤作用,其机制可能与增强NK-92细胞记忆功能、激活Syk/ZAP70通路相关.
Abstract
This study aimed to investigate the effect and mechanism of silencing DNA methyltransferase 1(DNMT1)on the killing effect of NK-92 on Jurkat cells.The DNMT1 expression in the bone marrow of 35 patients with acute lymphocyte leukemia(ALL)from January 2020 to December 2021 were measured.The si-RNA-DNMT1 was transfected into NK-92 cells and co-cultured with Jurkat cells.The killing effect on Jurkat cells was detected by lactate dehydrogenase(LDH)method,and the apoptosis and membrane NKG2D of Jurkat cells were detected by flow cytometry.The levels of IFN-γ were estimated by ELISA.The expressions of Syk,ZAP70,p-Syk,p-ZAP70,perforin and granzyme B in NK-92 cells were measured by Western blotting.The results showed that the level of DNMT1 was increased in patients with ALL compared to the control group(P＜0.05),and it was of diagnostic value for ALL(AUC:0.692,95％CI:0.533-0.851,P=0.033).The progression free survival of patients with high DNMT1 was significantly lower than those with low DNMT1(P＜0.05).NK-92 transfected with si-RNA-DNMT1 showed a higher killing rate and cell apoptosis compared to those of the si-NC group(P＜0.05),along with the enhanced ability to secrete NKG2D and express perforin and granzyme B(P＜0.05).The IFN-γ level of NK-92 transfected with si-RNA-DNMT1 was significantly higher than that in the si-NC group(P＜0.05).The p-Syk and p-ZAP70 protein levels of si-RNA-DNMT1 transfected NK-92 were also significantly higher than those in the si-NC group(P＜0.05).In conclusion,DNMT1 expression increases in ALL and silencing DNMT1 promotes the killing ability of NK-92 cells on Jurkat cells.The mechanism may be related to enhanced function of NK-92 cells and activation of Syk/ZAP70 pathway.
关键词
DNA甲基转移酶1/急性淋巴细胞白血病/NK-92细胞/Jurkat细胞Key words
DNA methyltransferase 1/acute lymphocyte leukemia/NK-92 cells/Jurkat cells引用本文复制引用
基金项目
云南省科技厅科技计划项目(202201AY070001-058)
出版年
2024
NETL
NSTL
万方数据