【摘要】 目的:在慢性粒细胞白血病(chronic myeloid leukemia,CML)由慢性期向急性期的过渡阶段伴随有多聚胞嘧啶结合蛋白E2[poly(rC)-binding protein E2,hnRNP E2]的异常表达。本研究初步探讨hnRNP E2诱骗RNA(decoy RNA)对32D-BCR/ABL细胞增殖的影响及其可能的分子机制。方法:用电转染方法将hnRNP E2诱骗RNA野生序列和突变序列的表达载体(pGD和pGM)转入32D-BCR/ABL细胞,用G418筛选出稳定表达诱骗RNA的细胞。锥虫蓝染色法和克隆形成实验检测细胞的增殖能力。FCM分析细胞周期,RT-PCR和Western印迹法检测下游CCAAT增强子结合蛋白α(CCAAT/enhancer-bindingproteinα,C/EBPα)和c-Myc的表达。结果:筛选出稳定表达野生型和突变型诱骗RNA的32DP210-pGD细胞和32DP210-pGM细胞。野生型32DP210-pGD细胞与未转染32D-BCR/ABL细胞相比,其细胞增殖抑制率为(69.48±5.21)%,克隆形成能力明显减弱,细胞周期由G0/G1期向S期进展受阻;C/EBPαmRNA水平无改变,但在蛋白水平上42 ku-C/EBPα的表达增加(43.83±4.91)%;c-Myc mRNA水平下降(35.67±6.64)%,蛋白水平降低(30.91±3.84)%。突变型32DP210-pGM细胞与未转染32D-BCR/ABL的细胞相比,其上述各指标无明显差异。结论:hnRNP E2诱骗RNA能够抑制32D-BCR/ABL细胞的增殖,其机制可能是诱骗RNA阻断hnRNP E2和C/EBPαmRNA的结合,引起42 ku-C/EBPα表达增加,进而引起其下游靶基因c-Myc下调。更多还原

【Abstract】 Objective:Expression of poly(rC)-binding protein E2(hnRNP E2) was abnormal from chronic phase to blast crisis of chronic myeloid leukemia(CML).The present study was to investigate the effect of hnRNP E2 decoy RNA on the proliferation of 32D-BCR/ABL cells and the possible molecular mechanisms.Methods:Plasmids expressing wild sequence(pGD) and mutation sequence(pGM) of hnRNP E2 decoy RNA were transfected into 32D-BCR/ABL cells with electroporation method.Stably transfected cells were selected with G418.The cell reproductive activity was detected by cell growth curve using trypan blue staining and colony formation test;the cell cycle was analyzed by flow cytometry;the mRNA and protein expressions of downstream CCAAT/enhancer-binding protein α(C/EBPα) and c-Myc were determined by RT-PCR and Western blot.Results:The stably expressed 32DP210-pGD cells and 32DP210-pGM cells were selected.The proliferation of 32DP210-pGD cells was inhibited compared with untreated 32D-BCR/ABL cells.The inhibitory rate was(69.48±5.21)%.The clony formation ability of 32DP210-pGD cells was weakened.The 32DP210-pGD cells were arrested at G0/G1 phase.The expression of C/EBPα mRNA of 32DP210-pGD cells remained unchanged,but the 42 ku-C/EBPα protein expression was elevated by(43.83±4.91)%;mRNA and protein expression levels of c-Myc were decreased by(35.67±6.64)% and(30.91±3.84)%,respectively.However,there was no significant difference between 32DP210-pGM cells and untreated 32D-BCR/ABL cells in the above mentioned parameters.Conclusion:hnRNP E2 decoy RNA inhibited the proliferation of 32D-BCR/ABL cell.The action mechanism of hnRNP E2 decoy RNA may be related with its effect of blocking the binding of hnRNP E2 with C/EBPα mRNA and subsequent elevation of 42 ku-C/EBPα and down-regulation of c-Myc gene expression.更多还原