Effect of nucleophosmin（NPM） 1 knockdown on the apoptosis of OCI/AML3 leukemic cell with mutant NPM1 and its mechanisms

Abstract:

Objective：To investigate the effect of nucleophosmin 1（NPM1） on the apoptosis in leukemic cells and the relevant molec－ular mechanisms. Methods：OCI/AML3 cell lines（NPM1 mutant type） and HL60 cell lines（NPM1 wild type） were served as experi－mental group and negative control group. Mock group，pGIPZ group，shNPM group were established in the meantime. Expressions of the NPM1 mutant A（NPM1-mA） gene and protein were detected by qRT-PCR，Western blot and immune cytochemistry. Apoptosis analy－sis was determined by flow cytometry and Wright-Giemsa staining. The gene and protein expression of apoptosis related molecular（Bax/Bcl-2） was detected by qRT-PCR and Western blot and the activity of p-ERK was also assessed by Western blot. The percent－age of apoptotic cells was detected after treatment with MAPK/ERK pathway inhibitor（PD98059） by flow cytometry. Results：The re－sults showed significant down-regulation of NPM1-mA mRNA levels（F=20.078，P=0.000；PMock vs. shNPM=0.001，PpGIPZ vs. shNPM=0.003，PMock vs. pGIPZ=0.333） and protein levels after NPM1 silencing，accompanying by reduce of cytoplasm NPM mutant protein of OCI/AML3 cells. In OCI/AML3 cells，down-regulation of NPM1-mA resulted in increase of cell apoptotic rate（F=25.236，P=0.000；PMock vs. shNPM=0.001，PpGIPZ vs. shNPM=0.001，PMock vs. pGIPZ=0.729） and morphological changes including apoptosis bodies under light microscopy. NPM1 gene si－lencing led to increase of pro-apoptotic protein Bax expression（F=7.649，P=0.000；PMock vs. shNPM=0.015，PpGIPZ vs. shNPM=0.015，PMock vs. pGIPZ=0.984） and decrease of anti-apoptotic protein Bcl-2 expression at mRNA and protein levels（F=5.190，P=0.000，PMock vs. shNPM=0.034，PpGIPZ vs. shNPM=0.029，PMock vs. pGIPZ=0.909）. NPM1 gene silencing can also down-regulate the expression of p-ERK. Furthermore，PD98059 promoted OCI/AML3 leukemic cell apoptosis（F=25.643，P=0.007）. Conclusion：NPM1-mA plays an important role in leukemic cell anti-apoptosis，which involving MAPK signal pathway and Bax/Bcl-2 expression.