OBJECTIVETo construct a stable nm23-H1-knock-down cell model with K562 cell line and study its differentiation toward megakaryocyte.

METHODSEukaryotic expression vector pSilencer 4.1-CMV-sinm23 expressing siRNA targeting nm23-H1 was transfected into K562 cells with lipofectamine2000. Cells with stably nm23-H1 silence were screened out by G418. Real-time quantitative PCR, immunocytochemistry, western blot were used to confirm the nm23-H1-knock-down K562 model. Cell differentiation capacity was detected by NBT reduction assay. Surface antigen Gp IIb-IIIa (CD41) of knock-down cells treated with phorbol 12-myristate 13-acetate was analyzed by flow cytometry. Western blot was used to detect the ERK1/2 signal pathway after the stimulation of phorbol 12-myristate 13-acetate.

RESULTSEndogenous nm23-H1 was silenced by pSilencer 4.1-CMV-sinm23 and the silence efficiency was up to 75% and 70% in mRNA and protein levels respectively compared with the mock cells. Under phorbol 12-myristate 13-acetate treatment, the knock-down cells displayed a significantly increased differentiation ability toward megakaryocyte compared with control. The NBT reduction values were (0.31 +/- 0.07) and (0.23 +/- 0.05) respectively. Further results revealed that nm23-H1 gene regulating the megakaryocytic differentiation was due in part to the increased ERK1/2 phosphorylation.

CONCLUSIONSA stable nm23-H1-knock-down K562 cell model is successfully constructed. nm23-H1 involves in regulating the megakaryocytic differentiation of K562 cell line.