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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.
OBJECTIVE: To block the aberrant translation of the CCAAT enhancer binding protein alpha (C / EBPα) gene by targeting the decoy RNA of hnRNP E2 and to induce the differentiation of murine leukemia-like 32D-P210 cells into myeloid differentiation Methods: The wild-type and mutant hnRNP E2 decoy RNA plasmids were transfected into 32D-P210 cells by electroporation, and the stable strains were selected by G418.C / EBPa, Granulocyte colony-stimulating factor receptor (G-CSFR) and myeloperoxidase (MPO) mRNA levels were detected by Western Blot. Wright’s staining was used to observe the expression of granulocyte colony-stimulating factor G-CSF), and the expression of Gr-1 and CD11b were detected by flow cytometry.Results: TG and TGA cell lines stably expressing wild-type or mutant hnRNP E2 decoy RNA were screened. Compared with untransfected 32D-P210 cells, the level of C / EBPa mRNA in TG cells did not change, but the levels of 42ku-C / EBPa protein, G-CSFR mRNA and MPO mRNA increased by 43.8 ± 4.9% 69.1 ± 3.2)%, and (37.8 ± 4.2)%, respectively (P <0.05). After G-CSF stimulation, middle and late promyelocytes were even appeared in TG cells Meanwhile, the expression of Gr-1 differentiated antigen increased to 40.3%, while the expression of Gr-1 in non-transfected 32D-P210 cells was 5.5% (P <0.05); however, the expression of CD11b in all three groups was (P> 0.05) .There was no significant difference between the above parameters in the 32D-P210 cells (P> 0.05) .Conclusion: hnRNP E2 decoy RNA can induce 32D-P210 cells to myeloid differentiation, the mechanism may be specifically with decoy RNA block hnRNAE2 and C / EBPα mRNA untranslated region binding, regulating C / EBPα mRNA translation, restore 42ku-C / EBPα expression, upregulation of its downstream G- CSFR and MPO and other differentiation genes related to the expression.G-CSF can accelerate this role in order to promote the 32D-P210 cell differentiation process.