Research on restoration of C/EBPα activity and induction of granulocytic differentiation in 32 D-P210 cells by hnRNP E2 decoy RNA
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/EBPα) 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 electropora-tion 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,CD11 b 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-1 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 CD11b 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 granulocytic differentiation of 32D-P210 cells. The mechanisms may be that the decoy RNA specifically blocks hnRNP E2's binding to the untranslated region of C/EBPα mRNA,leading to the regulation of C/EBPα mRNA translation and restoration of the expression of 42 ku-C/EBPα,and up-regulation of its downstream differentiation-oriented genes such as G-CSFR and MPO. G-CSF accelerates these effects and hence facilitates the granulocytic differentiation of 32D-P210 cells.