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

作　　者：武坤 马晓波[1,2,3] 程沈菊 杨金荣[4,5] 贺振新 郭翀[1,2,3] WU Kun;MA Xiao-bo;CHEN Shen-ju;YANG Jin-rong;HE Zhen-xin;GUO chong(Yunnan Key Laboratory of Laboratory Medicine,Kunming 650032,China;Yunnan Provincial Innovation Team of Clinical Laboratory and Diagnosis,Kunming 650032,China;Department of Laboratory Medicine,The First Affiliated Hospital of Kunming Medical University,Kunming 650032,China;Department of Hematology,The First Affiliated Hospital of Kunming Medical University,Kunming 650032,China;Yunnan Hematological Disease Research Center,Kunming 650032,China)

机构地区：[1]云南省检验医学重点实验室,昆明650032 [2]云南省医学检验临床医学研究中心,昆明650032 [3]昆明医科大学第一附属医院医学检验科,昆明650032 [4]昆明医科大学第一附属医院血液科,昆明650032 [5]云南省血液病研究中心,昆明650032

出　　处：《现代免疫学》2024年第4期295-301,共7页Current Immunology

基　　金：云南省科技厅科技计划项目(202201AY070001-058)。

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-γ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。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-γwere 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-γlevel 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 

分 类 号：R735.3[医药卫生—肿瘤]