摘要
目的观察三氧化二砷(ATO)对急性髓系白血病细胞KG1a细胞表面NKG2D配体UL16结合蛋白1(ULBP1)表达的影响,探讨其调节ULBP1表达的分子机制。方法常规体外培养KG1a细胞,采用细胞增殖/毒性检测试剂(CCK8)检测不同浓度ATO对KG1a细胞的增殖抑制率;应用实时荧光定量反转录(RT)-PCR方法检测KG1a细胞ULBP1 mRNA表达的影响;流式细胞术检测ATO对KG1a细胞表面ULBP1蛋白表达的影响。加入毛细血管扩张性共济失调突变和RAD3相关激酶(ATM/ATR)抑制剂咖啡因,观察其是否影响ATO对KG1a细胞ULBP1 mRNA及蛋白的表达。采用Western blot法观察ATO对KG1a细胞中CHK1、CHK2蛋白及其磷酸化表达的影响。结果不同浓度(1、2、3、4、5μmol/L)ATO均可抑制KG1a细胞的增殖,呈浓度依赖性,其对KG1a细胞的半数抑制浓度(IC50值)为2.7μmol/L。荧光定量RT-PCR法检测结果示ATO可使KG1a细胞ULBP1 mRNA的相对表达水平升高,ATO在浓度分别为1、2、3、4、5μmol/L时,与未加药组比较,ULBP1 mRNA相对表达水平分别升高至(1.86±0.30)倍、(3.02±0.71)倍、(3.16±0.75)倍、(4.80±0.70)倍、(3.70±0.89)倍,差异均有统计学意义(均P<0.05);与未加药组相比,当ATO浓度分别为1、2、3、4、5μmol/L时,ULBP1蛋白相对表达水平均显著升高,差异均有统计学意义(均P<0.05)。咖啡因预先处理KG1a细胞2 h再联合ATO共同孵育KG1a细胞24 h,ULBP1 mRNA和蛋白表达水平均显著降低。在咖啡因浓度为8 mmol/L时,ULBP1 mRNA相对表达水平由不用咖啡因处理组的(9.55±0.38)倍降为(6.36±0.93)倍,差异有统计学意义(P<0.05);流式细胞术检测结果发现,当咖啡因浓度分别为2、4、8 mmol/L时,ULBP1蛋白相对表达水平由不用咖啡因处理组的(3.50±0.08)倍分别降为(2.17±0.07)倍、(2.02±0.06)倍、(1.75±0.06)倍,差异均有统计学意义(均P<0.05)。CHK1和CHK2蛋白相对表达水平均随ATO浓度的升高而降低,而CHK1和CHK2磷酸化蛋白的相对表达水平均随ATO浓度的升高而升高。结论ATO诱导上调KG1a细胞ULBP1 mRNA及蛋白的表达,ATM/ATR-CHK1/CHK2通路可能参与这一过程。
Objective To observe the effect of arsenic trioxide(ATO)on the expression of NKG2D ligand UL16 binding protein 1(ULBP1)in acute myeloid leukemia KG1a cells,and explore the molecular mechanism for its regulation of ULBP1 expression.Methods KG1a cells were cultured in vitro.Then,the inhibition of KG1a cell proli-feration by different concentrations of ATO was detected by cell counting kit-8(CCK8)assay,and the expression of ULBP1 mRNA and surface protein in KG1a cells were examined by real-time RT-PCR and flow cytometry,respectively.After that,the blocking effects of ataxia telangiectasia mutated and RAD3-related kinase(ATM/ATR)inhibitor caffeine on ATO-upregulated expression of ULBP1 mRNA and surface protein expressions were investigated,and the effects of ATO on the expression of CHK1 and CHK2 proteins and their phosphorylation in KG1a cells were observed by Western blot method.Results Different concentrations(1,2,3,4,5μmol/L)of ATO could inhibit the proliferation of KG1a cells,which was concentration dependent,and the half inhibitory(IC50)concentration to KG1a cells was 2.7μmol/L.The expression of ULBP1 mRNA on KG1a cells were increased when incubated with ATO at concentration 1,2,3,4,5μmol/L,compared without ATO group,ULBP1 mRNA expression level relatively increased respectively to(1.86±0.30)times,(3.02±0.71)times,(3.16±0.75)times,(4.80±0.70)times and(3.70±0.89)times,and the differences were statistically significant(all P<0.05).Furthermore,ATO(1,2,3,4 and 5μmol/L)upregulated ULBP1 protein expression on KG1a cells compared with that in the group without caffeine,and the differences were statistically significant(all P<0.05).After caffeine pretreat KG1a cell 2 h and ATO incubate KG1a cell 24 h,ULBP1 mRNA and protein expression levels were significantly reduced.When caffeine concentration was 8 mmol/L,ULBP1 mRNA expression level relatively reduces from(9.55±0.38)times to(6.36±0.93)times compared with that in the group without caffeine,and the difference was statistically significant(P<0.05).When caffeine concentration was 2,4 and 8 mmol/L respectively,the expression of ULBP1 protein was reduced from that in the group without caffein treatment(3.50±0.08)times to(2.17±0.07)times,(2.02±0.06)times and(1.75±0.06)times,respectively,and the differences were statistically significant(all P<0.05).The expression of CHK1 and CHK2 proteins decreased with the increase of ATO concentration,while p-CHK1 and p-CHK2 are increased as ATO.Conclusions ATO upregulate the expression of ULBP1 mRNA and protein in KG1a cells,and the ATM/ATR-CHK1/CHK2 pathway may be involved in it.
作者
姬曼曼
董家行
崔姗姗
司晓慧
李雅慧
牛新清
Ji Manman;Dong Jiaxing;Cui Shanshan;Si Xiaohui;Li Yahui;Niu Xinqing(The Third Clinical College of Xinxiang Medical University,Xinxiang 453003,Henan Province,China;Henan Key Laboratory of Immune and Targeted Drugs,School of Laboratory Medicine,Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
出处
《中华实用儿科临床杂志》
CSCD
北大核心
2020年第3期231-235,共5页
Chinese Journal of Applied Clinical Pediatrics
基金
国家自然科学基金(81800139)
河南省自然科学基金(182102311054)。
