诱骗RNA靶向阻断RNA结合蛋白E2诱导K562细胞向粒系分化

Targeted blockage of RNA binding protein E2 by decoy RNA induces the granulocytic differentiation of K562 cells

目的应用诱骗RNA（decoy RNA）靶向阻断RNA结合蛋白E2（hnRNP E2）调节CCAAT增强子结合蛋白（C／EBPα）基因异常翻译的作用，诱导白血病细胞株K562细胞向粒系分化，并初步探讨其分子机制。方法将已构建的hnRNP E2 decoy RNA表达质粒经阳离子脂质体的介导转染K562细胞，用G418筛选出稳定表达细胞株，用RT—PCR和Western blot方法检测该细胞株C／EBPα和粒细胞集落刺激因子受体（G—CSFR）的表达，通过瑞特-姬姆萨染色观察细胞形态，免疫细胞化学法分析细胞粒系分化抗原CD13、CD15的表达。结果筛选到稳定表达细胞株pG细胞，与未转染的K562细胞相比，其C／EBPα mRNA水平无改变，但相对分子质量42×10^3的C／EBPα蛋白（42kD—C／EBPα）水平升高了（49．7±5．5）％（P〈0．05）；G—CSFR mRNA水平升高了（42．1±3．6）％（P〈0．05），其蛋白水平也升高了（37．4±6．2）％（P〈0．05）；细胞形态学方面观察到pG细胞出现中、晚幼粒细胞甚至成熟粒细胞的特征，且免疫细胞化学法检测显示粒系分化抗原CD13、CD15表达增高[阳性细胞百分比分别为（18、7±2．5）％和（15．2±2．6）％]。结论hnRNP E2 decoy RNA能够诱导K562细胞向粒系分化，G—CSF对其分化过程有促进作用，其作用机制可能与decoy RNA靶向阻断hnRNP E2，调节C／EBPα mRNA翻译，恢复42kD—C／EBPα表达，上调42kD-C／EBPα下游分化基因G-CSFR的表达有关。

Objective To use a decoy RNA targeted blockage of the RNA binding protein E2 （hnRNP E2） resulting in the CCAAT/enhancer-binding protein α （C/EBPα） gene＇s abnormal translation and investigate its effect on the granulocytic differentiation of K562 cells and the probable molecular mechanism. Methods The hnRNP E2 decoy RNA expression plasmid was constructed and transfected into K562 cells with cationic liposome, and stable expression cells were obtained by G418 selection. The changes of C/EBPα and granulocyte colony-stimulating factor receptor（G-CSFR） gene expression were detected by RT- PCR and Western blot. The morphologic changes were observed after Wright-Giemsa staining. The expression of granulocytic differentiation antigens CD13 and CD15 was studied by immunocytochemistry. Results The stably expressed pG cells were obtained. Its C/EBPα mRNA level remained unchanged, while 42kD-C/EBPα protein expression was increased by （ 49.7 ± 5.5 ） % （ P 〈 0.05 ） ; and G-CSFR mRNA was increased by （42.1 ± 3.6） % （ P 〈 0.05 ）, and its protein was increased by （ 37.4 ± 6.2 ） % （ P 〈 0.05 ） compared to that in the K562 control cells. The characteristics of polymorphonuclear neutrophils appeared in pG cells and CD13 and CD15 positive cell ratios were （ 18.7 ± 2.5） % and （26.3 ± 2.9） % respectively. Conclusions HnRNP E2 decoy RNA can induce granulocytic differentiation of K562 cells, and G-CSF promotes this effect. The mechanisms may be that decoy RNA specifically blocks hnRNP E2, hence regulates the translation of C/ EBPα mRNA, restores the expression of 42kD-C/EBPα, and then up-regulates the expression of G-CSFR gene.