Study On Effect Of Nm23-H1 Gene On Differentiation Of Human Chronic Myeloblastic Leukemia Cell Line K562 By Using RNA Interference Technology

Objective: To investigate the effects and mechanism of nm23-H1 gene on the differentiation of human chronic myeloblastic leukemia cell line K562 by the technology of RNA interference, providing evidences for nm23-H1 targeted gene therapy of leukemia.Methods: Eukaryotic expression vector pSilencer^TM4. 1-CMV-siNM526 which can express siRNA targeting nm23-H1 was transfected into human chronic myeloblastic leukemia cell line K562 with lipofectamine. Cell pools that can stably silence nm23-H1 gene was screened out by G418. The recombinant of siRNA expression vector and cell genome was identified ty PCR method. Expression of nm23-H1 was identified by Real-time PCR and immunohistochemistry. Growth curves were drawn by MTT method to examine the effects on proliferation. Cell cycle was analyzed by flow cytometry. After the transfected cell strains induced by PMA, the morphologic changes were observed by Giemsa-wright's dyeing, the differentiation extent was tested by NBT reduction assay, and the expression of differentiation antigen CD41 on cell surface was detected by flow cytometry. The relationship between nm23-H1 and c-myc was analysed by Real-time PCR.Results: The cell polls can stably silence nm23-H1 gene was successfully established and named K562-siNM23. The inhibition rate was over 70 persents. Silencing of nm23-H1 gene obviously inhibited the proliferation of K562, and increased the percentage of phase G₀/G₁ cells but didn't induce apotosis. The K562-siNM23 cells were induced to differentiate into monocaryon and megacaryocyte according to their morphologic changes, it's sensitivity to the didderetiation inducer PMA was enhance. Also the expression of differentiation antigen CD41 was increased. On the other hand, the silencing of nm23-H1 down regulated the expression of c-myc gene.Conclusions: nm23-H1 gene was relative to cell differentiation. Silencing nm23-H1 gene can inhibit the growth and proliferation of K562, enhance the sensitivity to differentiation inducer PMA. And it also can promot PMA-induced K562 differentiate to megakaryocyte. There was a positive correlation between the mRNA lever of nm23-H1 and c-myc. Silencing nm23-H1 promoted cell differentiation was probably by interfering with cell cycle and down regulated the expression of c-myc.