泛素结合酶E2T对急性淋巴细胞白血病细胞糖皮质激素耐药的影响  

Influence of UBE2T on glucocorticoid resistance in acute lymphoblastic leukemia cells

作　　者：宋传龙 岳迎宾 马志华[1] 宋曼 阿布来提·阿不都哈尔[1] SONG Chuan-long;YUE Ying-bin;MA Zhi-hua;SONG Man;Abulaiti ABUDUHAER(Department of Pediatric Acute and Critical Care Medicine,the First Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang Uygur Autonomous Region 830054,China;Department of Pediatrics,the First Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang Uygur Autonomous Region 830054,China)

机构地区：[1]新疆医科大学第一附属医院小儿急危重症医学科,新疆维吾尔自治区乌鲁木齐830054 [2]新疆医科大学第一附属医院儿内科,新疆维吾尔自治区乌鲁木齐830054

出　　处：《中华实用诊断与治疗杂志》2022年第6期589-595,共7页Journal of Chinese Practical Diagnosis and Therapy

基　　金：新疆维吾尔自治区自然科学基金(2019D01C249)。

摘　　要：目的探讨泛素结合酶E2T(ubiquitin-conjugating enzyme E2T,UBE2T)在急性淋巴细胞白血病细胞糖皮质激素耐药中的作用及其对泛素化介导的P53蛋白降解的影响。方法人急性淋巴细胞白血病细胞系糖皮质激素耐药株CEM-C1和糖皮质激素敏感株CEM-C7细胞,采用实时荧光定量PCR法检测2种细胞UBE2T、p53 mRNA相对表达量,采用Western blot法检测2种细胞UBE2T、P53蛋白相对表达量。对数生长期CEM-C1细胞分为空白组(不进行转染)、sh-NC组(转染sh-NC)、sh-UBE2T组(转染sh-UBE2T)、sh-UBE2T+sh-p53组(转染sh-UBE2T和sh-p53),转染48 h后采用实时荧光定量PCR法检测4组UBE2T、p53 mRNA相对表达量,采用Western blot法检测4组细胞UBE2T、P53蛋白相对表达量,采用CCK-8法检测4组细胞增殖率及地塞米松对CEM-C1细胞的半数抑制浓度,采用流式细胞术检测4组细胞凋亡率,采用Transwell实验检测4组迁移细胞数和侵袭细胞数;采用放线菌酮蛋白合成抑制实验检测sh-UBE2T组与sh-NC组细胞P53蛋白半衰期,采用免疫共沉淀实验检测sh-UBE2T组与sh-NC组细胞P53蛋白泛素化水平。结果CEM-C1细胞UBE2T mRNA和蛋白相对表达量(2.38±0.14、0.85±0.07)均高于CEM-C7细胞(1.00±0.03、0.43±0.02)(t=15.351,P=0.017;t=13.789,P=0.022),p53 mRNA和P53蛋白相对表达量(0.47±0.01、0.23±0.04)均低于CEM-C7细胞(1.00±0.02、0.61±0.06)(t=13.280,P=0.035;t=14.092,P=0.019)。sh-UBE2T+sh-p53组(0.33±0.04、0.28±0.01)、sh-UBE2T组(0.35±0.09、0.27±0.01)细胞UBE2T mRNA和蛋白相对表达量低于空白组(1.01±0.01、1.02±0.01)、sh-NC组(1.00±0.02、1.07±0.02)(P<0.05),sh-UBE2T+sh-p53组与sh-UBE2T组比较差异均无统计学意义(P>0.05);sh-UBE2T+sh-p53组细胞p53 mRNA(0.12±0.01)和P53蛋白(0.19±0.02)相对表达量均低于sh-UBE2T组(0.98±0.04、1.28±0.04)、空白组(1.00±0.01、0.25±0.01)、sh-NC组(1.00±0.03、0.26±0.02)(P<0.05);sh-UBE2T组细胞p53 mRNA相对表达量与空白组、sh-NC组比较差异均无统计学意�Objective To investigate the role of ubiquitin-conjugating enzyme E2 T(UBE2 T)in glucocorticoid resistance in acute lymphoblastic leukemia(ALL)cells and its influence on ubiquitination mediated degradation of P53 protein.Methods The relative expressions of UBE2 T and p53 mRNAs in CEM-C1 and CEM-C7 cells were detected by real-time fluorescence quantitative PCR,and the relative expressions of UBE2 T and P53 proteins were detected by Western blot.The CEM-C1 cells in logarithmic growth phase were divided into blank group(without transfection),sh-NC group(transfected with sh-NC),sh-UBE2Tgroup(transfected with sh-UBE2T)and sh-UBE2T+sh-p53group(transfected with sh-UBE2Tand sh-p53).After 48-h transfection,the relative expressions of UBE2Tand p53mRNAs were detected by real-time fluorescence quantitative PCR,and the relative expressions of UBE2Tand P53proteins were detected by Western blot.CCK-8assay was used to detect the proliferation rate and the half-maximal inhibitory concentration of dexamethasone to CEM-C1cells.Flow cytometry was used to detect the apoptosis rate.Transwell assay was used to detect the numbers of migrating cells and invading cells.The half-life of P53protein in sh-UBE2Tand sh-NC groups was detected by cycloheximide protein synthesis inhibition assay.Immunoprecipitation assay was used to detect the P53ubiquitination level in sh-UBE2Tand sh-NC groups.Results The relative expressions of UBE2T mRNA and protein were higher in CEM-C1cells(2.38±0.14,0.85±0.07)than those in CEM-C7cells(1.00±0.03,0.43±0.02)(t=15.351,P=0.017;t=13.789,P=0.022).The relative expressions of p53mRNA and P53protein were lower in CEM-C1cells(0.47±0.01,0.23±0.04)than those in CEM-C7cells(1.00±0.02,0.61±0.06)(t=13.280,P=0.035;t=14.092,P=0.019).The relative expressions of UBE2T mRNA and protein were lower in sh-UBE2T+sh-p53group(0.33±0.04,0.28±0.01)and sh-UBE2Tgroup(0.35±0.09,0.27±0.01)than those in blank group(1.01±0.01,1.02±0.01)and sh-NC group(1.00±0.02,1.07±0.02)(P<0.05),and showed no significant differences between

关 键 词：急性淋巴细胞白血病 泛素结合酶E2T P53蛋白 泛素化 糖皮质激素 耐药 

分 类 号：R733.7[医药卫生—肿瘤]