HnRNPE2 decoy RNA恢复C/EBPα活性诱导32D-P210的细胞粒系分化  

Research on restoration of C/EBPα activity and induction of granulocytic differentiation in 32D-P210 cells by hnRNP E2 decoy RNA

作　　者：魏容[1] 曾建明[1] 袁颖[2] 王雅珍[1] 黄世峰[1] 罗红伟[1] 肖青[3] 陈新敏[1] 冯文莉[1] 

机构地区：[1]重庆医科大学临床血液学教研室,临床检验诊断学教育部重点实验室,重庆400016 [2]重庆医科大学临床医学系,重庆400016 [3]重庆医科大学附属第一医院血液科,重庆400016

出　　处：《第四军医大学学报》2009年第10期877-880,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30600748)

摘　　要：目的:采用核不均一核糖核蛋白E2(hnRNP E2)decoy RNA靶向阻断CCAAT增强子结合蛋白alpha(C/EBPα)基因的异常翻译,诱导小鼠白血病样32D-P210细胞株向粒系分化,并进一步探讨其分子机制.方法:电穿孔法分别将野生型和突变型hnRNP E2 decoy RNA表达质粒转染入32D-P210细胞,经G418筛选出稳定株后,RT-PCR检测C/EBPa,粒细胞集落刺激因子受体(G-CSFR)与髓过氧化物酶(MPO)基因的mRNA水平;WesternBlot检测C/EBPa,G-CSFR的蛋白表达水平;瑞氏染色观察粒细胞集落刺激因子(G-CSF)刺激前后细胞形态;流式细胞仪检测分化抗原Gr-1,CD11b的表达.结果:筛选到分别稳定表达野生型或突变型hnRNP E2 decoy RNA的TG细胞株和TGA细胞株.与未转染组32D-P210细胞相比,TG细胞株C/EBPa mRNA水平无改变,但42ku-C/EBPa蛋白、G-CSFR mRNA和MPO mRNA水平分别升高了(43.8±4.9)%,(69.1±3.2)%和(37.8±4.2)%(P<0.05);G-CSF刺激后,TG细胞出现中、晚幼粒细胞甚至成熟粒细胞形态学特征;同时Gr-1分化抗原的表达率上升至40.3%,未转染组32D-P210细胞的Gr-1表达率5.5%(P<0.05);然而CD11b在3组细胞都是高表达,没有明显的变化(P>0.05);以上各参数在TGA细胞株与未转染组32D-P210细胞株间均无显著性差异(P>0.05).结论:hnRNP E2 decoy RNA能够诱导32D-P210细胞向粒系分化,其机制可能与decoy RNA特异性阻断hnRNAE2与C/EBPα mRNA非翻译区结合,调节C/EBPα mRNA翻译,恢复42ku-C/EBPα表达,上调其下游G-CSFR和MPO等分化基因的表达有关.G-CSF可加速这一作用从而促进32D-P210细胞的分化过程.AIM： To induce granulocytic differentiation of murine leukemia-like 32D-P210 cells through heterogeneous nuclear ribonucleoprotei E2 （hnRNP E2） decoy RNA aiming at the targeted blockage of the CCAAT/enhancer-binding protein alpha （C/EBPa） gene abnormal translation, and to further investigate the potential molecular mechanisms. METHODS： Both wild and mutant hnRNP E2 decoy RNA expression plasmids were respectively transfected into 32D-P210 cells by electroporation and the cells stably expressing the target genes were screened out by G418 selection. RT-PCR was employed to detect the changes in mRNA levels of C/EBPα, granulocyte colony-stimulating factor receptor（G-CSFR） and myeloperoxidase （MPO）. The protein expression changes of C/EBPα and G-CSFR were evaluated by Western Blot and the morphological changes were observed by light microscopy before and after Wright＇s staining. The expression levels of granulocyte differentiation antigens, CDllb and Gr-1 were detected by flow cytometry （FCM）. RESULTS：Cells stably expressing the hnRNP E2 decoy RNA, either wild or mutant, were screened out respectively and named respectively as TG cells and TGA cells. When compared with those of the untrans- fected 32D-P210 cells, C/EBPa mRNA levels in the TG cells remained unchanged, whereas the 42 ku-C/EBPa protein, G-CSFR mRNA and MPO mRNA expression levels respectively increased by （43.8±4.9）%,（69.1 ±3.2）% and （37.8 ±4.2）% （P〈 0.05）. Myelocytes, metagranulocytes and mature granulocytes appeared in TG cells stimulated by G-CSF. The expression level of granulocyte differentiation antigen Gr-I increased to 40. 3% and Gr-1 expression level in un-transfected 32D-P210 cells was 5.5% （P 〈 0. 05 ）. However, no difference was observed in CDllb expression level between the three groups and no significant difference was seen in all the above parameters for TGA cells when compared with those for untransfected 32D-P210 cells （P 〉 0.05）. CONLUSION： hnRNP E2 decoy RNA induces granulo

关 键 词：32D-P210 DECOY RNA 细胞分化 核不均一核糖核蛋白E2 

分 类 号：R285.5[医药卫生—中药学] R852.22[医药卫生—中医学]