摘要
目的:探讨细胞周期检测点激酶1(checkpoint kinase 1,CHEK1)参与拮抗慢性粒细胞白血病(chronic myelogenous leukemia,CML)细胞凋亡的分子机制。方法:通过GEO(Gene Expression Omnibus)数据库内置的R语言程序进行CML细胞的基因表达数据分析,选取差异表达的靶基因。采用实时荧光定量PCR法及蛋白质印迹法检测8例CML患者外周血中CHEK1 mRNA和蛋白的表达水平,并分析其与CML病情进展的关系。用针对CHEK1的抑制剂SCH900776抑制CHEK1磷酸化后,采用FCM法检测K562和Ku812细胞周期及凋亡的变化,并用蛋白质印迹法检测下游相关分子的表达水平变化。结果:通过对GEO数据库中3项CML基因表达数据进行分析,得到共同的差异表达基因共21个,其中12个为表达上调,9个为表达下调;选取CHEK1作为研究对象。CML患者外周血中CHEK1 mRNA和蛋白表达水平均明显高于健康体检者(P值均<0.05),且急变期CHEK1 mRNA表达水平明显高于慢性期(P<0.05)。CHEK1及其磷酸化蛋白的表达水平随病情进展亦有增高的趋势,且在伊马替尼处理后未有明显变化。抑制CHEK1磷酸化可激活caspase-3,并显著诱导K562和Ku812细胞凋亡(P值均<0.01)。抑制CHEK1磷酸化后,S期细胞减少,G2/M期细胞增多,S期相关蛋白细胞周期蛋白E(cyclin E)表达水平下调,而DNA损伤标志物γ-H2XA 表达水平升高,同时p53家族成员中p53和p73的表达均明显上调(P值均<0.05)。结论:CHEK1去磷酸化失活可逆转细胞S期阻滞,破环DNA损伤修复,进而导致P73升高,诱导细胞凋亡。
Objective:To explore the molecular mechanism of checkpoint kinase 1(CHEK1)mediating apoptosis resistance of chronic myelogenous leukemia(CML)cells.Methods:CHEK1 was selected as the target by analyzing 3 gene expression data studies of CML cells in Gene Expression Omnibus(GEO)database using the inner R program.The expression of CHEK1 mRNA and protein in peripheral blood of 8 patients with CML was detected by real-time fluorescent quantitative PCR and Western blotting,and the relationship between CHEK1 expression and the progression of CML was analyzed.After phosphorylation of CHEK1 was inhibited by CHEK1 inhibitor SCH900776,the cell cycle and apoptosis of K562 and Ku812 cells were detected by FCM,and the expressions of downstream related molecules were detected by Western blotting.Results:Out of 21 genes that differentially expressed in all 3 studies of GEO database(9 down and 12 up),CHEK1 was selected as an upregulated gene for the further research.Both CHEK1 mRNA and protein levels were increased in CML patients as compared with the healthy controls.Besides,the CHEK1 protein and phosphorylated CHEK1 were further upregulated in CML patients at blast crisis stage(P<0.05),which were not affected by imatinib treatment.Inhibition of CHEK1 phosphorylation significantly triggered the apoptosis of K562 cells(P<0.01)and Ku812 cells(P<0.001)through Caspase-3 activation.Dephosphorylation of CHEK1 attenuated the expression of S-phase related protein cyclin E,resulting to the raise of G2/M phase and the failure of DNA damage repair which was reflected by up-regulatingγ-H2XA level.In addition,the expressions of two P53 family members P53 and P73 were obviously enhanced after CHEK1 dephosphorylation(P<0.05).Conclusion:Dephosphorylation of CHEK1 can reverse S phase arrest and destroy DNA damage repair,leading to increase of P73 level and induction of apoptosis.
作者
王方
王昕
王亚文
WANG Fang;WANG Xin;WANG Yawen(Department of Laboratory Medicine,First A liated Hospital of Xi’an Jiaotong University,Xi’an 710061,Shaanxi Province,China;Biobank,First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061,Shaanxi Province,China)
出处
《肿瘤》
CAS
CSCD
北大核心
2020年第11期758-766,共9页
Tumor
基金
国家自然科学基金资助项目(编号:81600134)
