Gene expression profiling of HL-60 cells following knockdown of nucleostemin using DNA microarrays

Abstract

Nucleostemin (NS) plays an important role in tumorigenesis and progression. Most studies consider that NS plays its role through combining with p53 and inhibiting it, however our previous studies revealed that NS could also function without the existence of p53. To date, few studies have focused on the p53-independent pathway of NS, and its molecular mechanism remains unknown. The aim of the present study was to investigate the p53-independent pathway of NS in the human acute myeloid leukemia cell line HL-60 which was p53-null by using the DNA microarray technique. Lentivirus-mediated RNA interference technique was used to knock down NS expression in HL-60 cells, and then DNA microarray and bioinformatics were used to analyze the gene expression profiling changes. The microarray data showed that after knocking down NS in HL-60 cells, 2,628 differentially expressed genes were identified through ≥ 2 or ≤ 0.5-fold-change, in which 818 genes were upregulated and 1,810 genes were downregulated. Real-time quantitative polymerase chain reaction (qPCR) validated the reliability of DNA microarray data. Pathway analysis showed extensive signal pathways in HL-60 cells were influenced by inhibiting NS expression. In particular, the inhibition of PI3K-AKT pathway, JAK-STAT pathway, RAS-RAF-MEK-ERK1/2 pathway and activation of JNK pathway, p38 MAPK pathway may associate with the apoptosis of HL-60 cells after knocking down NS. The findings of this study provide insight to further explore the specific molecular mechanism of NS function in p53-null leukemia and they also lay the foundations for exploring new therapeutic targets for p53-null leukemia and even p53-null tumors.

Gene expression profiling of NB4 cells following knockdown of nucleostemin using DNA microarrays

“…In addition, several of the upregulated genes were enriched in the MAPK signaling pathway ( Fig. 4 ), which was a similar finding to previous observations in HL-60 cells ( 20 ). MAPK pathways mainly consist of the JNK, the RAS/RAF/MEK/ERK2 and the p38MAPK pathways, while activation of JNK and p38MAPK pathways is known to induce cell apoptosis ( 31 ).…”

“…However, the underlying mechanisms of the function of NS in p53-inactivated tumor cells have largely remained elusive. A previous study by our group reported that inhibition of the JAK/STAT, the PI3K/AKT and the RAS/RAF/MEK/ERK1/2 pathways, as well as the activation of the p38MAPK and JNK pathways may participate in the induction of apoptosis following NS knockdown in p53-null HL-60 cells ( 20 ).…”

“…Thus, it is urgent to explore effective treatment targets for tumors which are p53-null or p53-mutant. A previous study by our group has performed gene expression profiling of the p53-null HL-60 leukemia cell line following NS knockdown ( 20 ). In order to further explore the p53-independent NS pathway, the present study subjected the p53-mutant NB4 leukemia cell line to DNA microarray analysis and gene expression profiling following NS knockdown.…”

Proteomic Investigation of the Role of Nucleostemin in Nucleophosmin-Mutated OCI-AML 3 Cell Line

“…In the acute promyelocytic leukaemia cell line NB4, NS silencing was shown to induce differentiation and potentiate all-trans-retinoic acid-mediated cell death [ 16 ]. NS expression was also detected in the p53-null acute myeloid leukaemia (AML) HL-60 cell line; also, in this case NS silencing was associated with apoptosis, possibly through activation of the JNK pathway [ 17 ]. Importantly, transcript levels of NS were analysed in the bone marrow of newly diagnosed AML patients; they were found to be higher as compared to normal bone marrow in all FAB subtypes and to correlate with blast percentages [ 18 ].…”