儿童急性髓细胞性白血病WAVE1基因对耐药相关基因表达影响的研究  

Interaction of WAVE1 and genes involved in multiple drug resistance in children with acute myeloblastic leukemiaInteraction of WAVE1 and genes involved in multiple drug resistance in children with acute myeloblastic leukemia

作　　者：杨明华[1] 赵明一[1] 贺钰磊[1] 汪敏慧[1] 王卓[1] 谢岷[1] 吴秀山[2] 曹励之[1] 

机构地区：[1]中南大学湘雅医院儿科,长沙410008 [2]湖南师范大学生命科学学院

出　　处：《中华儿科杂志》2010年第3期175-179,共5页Chinese Journal of Pediatrics

基　　金：国家自然科学基金面上资助项目（30571982,30772353,30973234）;教育部博士点基金资助项目（20070533042）

摘　　要：目的探讨儿童急性髓细胞性白血病（AML）中WASP家族Verprolin同源蛋白1（WAVEl）基因参与P糖蛋白（P—gP）、多药耐药相关蛋白1（MRP1）、肺耐药相关蛋白（LRP）、乳腺癌耐药蛋白（BCRP）表达和调控的机制。方法将52例患儿依据是否在随访期内持续缓解（CCR）分为难治／复发组和缓解组，应用实时定量PCR（RQ-PCR）法和蛋白印迹（Western blot）分别检测52例AML患儿治疗前及疾病发展过程中骨髓单个核细胞（BMMCs）中WAVE1、P—gP、MRP1、LRP及BCRP蛋白和基因mRNA的表达水平。将PCDNA3．1-WAVEl真核表达质粒转染HL60细胞构建WAVEl高表达HL-60细胞，将WAVEl基因的特异性小片段干扰RNA（WAVEl siRNA）转染HL60／ADR构建WAVE1低表达HL60／ADR细胞；Western blot和RQ—PCR法检测基因转染前后白血病细胞系grAVE1、MRP1及BCRP蛋白及基因的表达水平。结果①复发／难治组病例的WAVE1及耐药相关基因水平明显高于缓解组患儿WAVE1基因表达水平（P〈0．05）；②同一患儿初治及复发时的WAVE1基因水平明显高于缓解期表达水平（P〈0．05）；③与HL60细胞相比较，HL60／ADR细胞WAVE1mRNA及蛋白表达水平增加3．53倍及95％；④转染WAVE1后HL60细胞系MRP1 mRNA和蛋白表达水平分别增加16．54倍、129％，BCRPmRNA和蛋白表达水平分别增加4．93倍及96％，而转染WAVE1 siRNA的HL60／ADR细胞MRP1 mRNA和蛋白的表达水平为干扰前的0．11倍及43％，BCRPmRNA和蛋白的表达水平为转染前的0．14倍及71％。结论WAVE1与儿童AML的病程及预后相关，参与了髓系白血病细胞多药耐药的形成并可影响MRP及BCRP1等多个重要的耐药相关基因的表达和作用。Objective Multidrug resistance （MDR） is one of the primary causes of suboptimal outcomes in chemotherapy of children with acute myeloblastic leukemia （AML）. The mechanisms of drug transport resistance may chiefly contribute to MDR. Expression and/or activity of P-glycoprotein （ P-gp）, multiple resistance-associated protein-1 （ MRP1 ） , lung-resistance related protein （LRP） and breast cancer resistance protein （BCRP） have been considered to be associated with unfavourable outcomes in pediatric AML patients. In previous studies, we found WASP-family verprolin-homologous protein-1 （WAVE1）was involved in the MDR mechanisms in K562/A02 leukemia cells. To investigate the expression of WAVE1, P- gp, MRP1, LRP/MVP and BCRP; and if WAVE1 is involved in MDR of human leukemia cell. Methods WAVE1, P-gp, MRP1, LRP, BCRP mRNA and protein expression in bone marrow mononuclear cells （BMMCs） were measured by real-time fluorescence quantitative PCR （RQ-PCR） and Western blot in a cohort of 52 children with acute myeloblastic leukemia. During follow-up, of the 52 patients, 21 were documented as being relapsing or refractory, and 31 were induced into complete continuous remission.Furthermore, HL60 cells and HL60/ADR ceils were transiently transfected with PCDNA3.1-WAVE1 reconstructed plasmid and specifically siRNA to WAVE1 respectively, and the expression of WAVE1, MRP1 and BCRP before and after transfection was assessed by real-time PCR and Western blot analysis. Results① The expression levels of WAVE1, P-gp, MRP, LRP and BCRP in refractory/relapsing group were much higher than that in complete continuous remission （CCR） group. ② WAVE1 mRNA and protein expression in BMMCs of children were at higher levels when they were newly diagnosed or relapsed, compared with complete continuous remission. ③ The WAVE1 expression at mRNA and protein level in HL60/ADR cells was increased by about 353% and 95% respectively as compared with that in HL60 cells. ④ Overexpression of WAVE1 in HI360 cell lin

关 键 词：白血病 髓样 抗药性 多药 RNA 小分子干扰 基因 WAVE1 

分 类 号：R733.71[医药卫生—肿瘤]