GSDME在DADS诱导白血病K562细胞焦亡中的作用研究  

Role of GSDME in DADS-induced pyroptosis in leukemic K562 cells

作　　者：周灵艳 胡轩源 刘银花 殷小成 ZHOU Lingyan;HU Xuanyuan;LIU Yinhua;YIN Xiaocheng(Department of Pediatrics,the First Affiliated Hospital of Hengyang Medical College,University of South China,Hunan Hengyang 421600,China.)

机构地区：[1]南华大学衡阳医学院附属第一医院儿科,湖南衡阳421600

出　　处：《现代肿瘤医学》2024年第15期2730-2737,共8页Journal of Modern Oncology

基　　金：国家自然科学基金面上项目(编号:31271482);湖南省卫生健康委员会科技计划重点项目(编号:20201977)。

摘　　要：目的:研究DADS(Diallyl disulfide, DADS)诱导K562细胞焦亡(pyroptosis)及其分子机制。方法:Western Blot检测Kasumi、K562、THP-1、HL-60、NB4细胞的Gasdermin家族蛋白E(Gasdermin E, GSDME)、Gasdermin家族蛋白D(Gasdermin D,GSDMD);甲基特异性PCR(Methylation-specific PCR,MSP)法检测K562及NB4细胞GSDME基因启动子检测区甲基化情况;CCK-8检测DADS对K562细胞增殖活力的影响;Western Blot、LDH释放实验检测DADS处理K562细胞后GSDME、GSDME-N、IL-1β、Cleaved IL-1β蛋白表达水平及乳酸脱氢酶(Lactic dehydrogenase, LDH)释放量变化;慢病毒转染技术构建GSDME敲低的稳转K562细胞株;Western Blot检测下调GSDME对DADS处理K562细胞GSDME-N端蛋白表达水平的影响。结果:Kasumi、K562、THP-1、HL-60、NB4中GSDME、GSDMD的蛋白表达水平存在差异;NB4细胞的GSDME基因启动子检测区甲基化扩增出阳性条带,而非甲基化扩增为阴性;K562细胞GSDME基因启动子检测区甲基化未能扩增出阳性条带,而非甲基化扩增为阳性;DADS处理后K562细胞的LDH释放量、GSDME-N、Cleaved-IL-1β蛋白均显著升高(P<0.01);与NC组相比,敲低组的K562细胞中mRNA及蛋白表达水平均明显降低(P<0.01);DADS处理后,NC组的GSDME-N蛋白水平明显上升(P<0.001),QD组GSDME-N蛋白的水平无显著差异(P>0.05);与NC-DADS组相比,经过DADS处理的QD组GSDME-N蛋白水平明显下降(P<0.001)。结论:DADS诱导K562细胞发生细胞焦亡,其分子机制可能与GSDME介导的焦亡通路有关。Objective:To investigate the molecular mechanisms of DADS-induced pyroptosis in K562 cells.Methods:We used Western Blot to detect the overall expression levels of GSDME and GSDMD proteins in Kasumi,K562,THP-1,HL-60,and NB4 cells.Methylation-specific PCR(MSP)was employed to assess the methylation status of the GSDME gene promoter region in K562 and NB4 cells.CCK-8 assays were conducted to evaluate the proliferative viability of K562 and NB4 cells after treatment with varying concentrations of DADS.Western Blot and LDH release experiments were utilized to measure changes in protein expression levels of GSDME,GSDME-N,IL-1β,Cleaved-IL-1β,as well as LDH release,in K562 cells following DADS treatment.Lentiviral transfection was utilized to establish stable cell lines with knockdown of GSDME in K562 cells.Western Blot was then performed to detect changes in the expression level of the GSDME-N terminal protein in the stable cell lines treated with DADS.Results:Significant differences in the expression levels of GSDME and GSDMD were observed among Kasumi,K562,THP-1,HL-60 and NB4 cells.MSP analysis positive bands for GSDME gene promoter methylation were detected in NB4 cells,while unmethylated amplification was negative.In K562 cells,unmethylated amplification was positive,but positive bands for GSDME gene promoter methylation was not detected.After DADS treatment the LDH release GSDME-N ,Cleaved-1L-1β protein of K562 cells were significantly increased ( P <0.01).qRT-PCR and Western Blot knockdown of GSDME significantly reduced its mRNA and protein expression in K562 cells compared to NC groups ( P <0.01).DADS treatment significantly increased GSDME-N protein levels in NC groups ( P <0.001),and there was no significant difference in GSDME-N protein levels in the QD group after DADS treatment ( P >0.05).The QD group showed a significant decrease in GSDME-N protein levels after DADS treatment compared to the NC-DADS group ( P <0.001). Conclusion: DADS induces pyroptosis in K562 cells,and its molecular mechanism is related

关 键 词：DADS K562 细胞焦亡 GSDME 

分 类 号：R733.7[医药卫生—肿瘤]