microRNAs expression profile in acute promyelocytic leukemia cell differentiation induced by all-trans retinoic acid and arsenic trioxide / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression profile of microRNAs in acute promyelocytic leukemia (APL) cells during differentiation.</p><p><b>METHODS</b>Differentiation of APL cell line NB4 cells was induced by all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3). Morphological and immunological assay was performed by Wright-Giemsa staining and flow-cytometric analysis of CD11b surface expression. During in vitro NB4 differentiation induced by ATRA and As2O3, microRNA expression profiles (miR-15b, miR-16, miR-34a, miR-107, miR-124a, miR-146, miR-155, miR-181a, miR-223, miR-342, let7c) were detected by real time RT-PCR, and the relative expression level of microRNAs were quantitatively analyzed by using 2(-ΔΔCt), and compared with that of control group. Meanwhile, the microRNA expression profiles were also detected in 15 newly diagnosed APL patients and 15 complete remission (CR) APL cases by real time RT-PCR, and the relative expression level of microRNA was quantitated by using 2(-ΔCt), and compared with that of control group (newly diagnosed APL as control group). These data were expressed as x(-) ± s, and differences between groups were examined using t test. P < 0.05 was considered statistically significant.</p><p><b>RESULTS</b>The expression levels of miR-15b, miR-16, miR-107, miR-223 and miR-342 in NB4 differentiation group were obviously up-regulated (3.40, 4.22, 5.41, 20.03 and 5.29 folds higher in ATRA treated NB4 cells than that of control group respectively, and 3.62, 2.49, 2.58, 4.27 and 1.94 folds higher in AS2O3 treated NB4 cells than that of control group respectively), except for miR-15b, the expression levels of miR-16, miR-107, miR-223 and miR-342 in ATRA treated group was significantly higher than that in As2O3 treated group. The relative expression levels of miR-15b, miR-16, miR-107, miR-181a, miR-223 and miR-342 were 0.4137, 0.6367, 0.1260, 0.0522, 0.6611, 0.0280 in APL CR group, and 0.0751, 0.2022, 0.0425, 0.3064, 0.1733, 0.0090 in newly diagnosed APL group, respectively. The expression level of miR-15b, miR-16, miR-107, miR-223 and miR-342 in APL CR group were significantly upregulated compared with that of newly diagnosed APL groups (P < 0.05), while the expression level of miR-181a was significantly downregulated (P < 0.05).</p><p><b>CONCLUSION</b>Specific expression of microRNA profiles is a key contributing factor in the differentiation of APL.</p>

Packaging of lentivirus carrying gene hβc and overexpression of gene hβc in NB4 cells / 中国实验血液学杂志

This study was aimed to overexpress gene hβc in NB4 cells via the method of lentivirus-mediated gene transfer, to observe the differentiation behaviour change of hβc over-expressing NB4 cells treated with IL-3 or GM-CSF, to explore the relationship between hβc gene and the differentiation behaviour of NB4 cells. The targeted hβc gene was amplified by PCR from the cloned vector carrying ORF of hβc. The PCR product containing PmeI and BstBI site introduced by primer was digested, and then cloned into lentivirus vector pRRLSIN.cPPT.PGK/IRES/GFP.WPRE to construct a lentiviral vector carrying hβc, named pLV-hβc. And the pLV-hβc plasmid was confirmed by restriction and sequencing. The recombinant lentivirus was produced by co-transfecting three plasmids into 293T packing cells. After transfection, the lentiviral supernatant was collected to transfect NB4 cells. GFP expression was examined by fluorescent microscope and the expression of hβc gene was detected by Western blot. Then, the NB4 cells over-expressing hβc were treated with IL-3 (10 ng/ml), GM-CSF (10 ng/ml), ATRA (1 µmol/L) respectively, and the CD11b expression, morphology and differentiation behaviour changes of every groups were observed by flow cytometry and microscopy, while NB4 cells transfected with blank lentivirus (NB4-blank cells) were used as controls. The results showed that the recombinant lentivirus vector carrying hβc gene could efficiently transfect NB4 cells and made NB4 cells to stably over-express hβc gene. The expression of CD11b was up-regulated in NB4-hβc cells treated with of IL-3 or GM-CSF, but it was not as obvious as the effect of ATRA, and no morphological change was observed in NB4 hβc cells treated with the IL-3 or GM-CSF. It is concluded that IL-3 or GM-CSF can induce NB4 cells over-expressing hβc to differentiate to neutrophils, but can not make them fully matured.

Expression pattern of myeloid differentiation-related transcription factor mRNA in differentiation of NB4 and HL-60 cells induced by all-trans retinoic acid / 中国实验血液学杂志

Hematopoiesis is coordinated by a complex regulatory network of transcription factors that involves proliferation, differentiation and maturation of a very small population of pluripotent hematopoietic stem cells with self-renewing and differentiating into various specialized and distinct blood cell types. Malfunction of transcription factors may lead to diseases such as acute myeloid leukemia (AML). The purpose of this study was to investigate the expression pattern of transcription factor mRNA in acute myeloid leukemia (AML) cells during in vitro differentiation. The 2 human leukemic cell lines HL-60 and NB4 had been used as model cell lines. Differentiation of HL-60 and NB4 cells was induced by all-trans retinoic acid (ATRA) for 4 days. Morphological changes were observed by May-Grunwald Giemsa stainings, the CD11b expression level was detected by flow cytometry. Transcription factor mRNA profiles (PU.1, C/EBPα, ε, γ, GATA-1, GATA-2) were determined by real time RT-PCR during in vitro HL-60 and NB4 differentiation; The expression level of transcription factor mRNA was relatively quantitatively analyzed by using 2(-ΔΔCT) and compared with control group. The results showed that the expression levels of PU.1 and C/EBP ε mRNA in NB4 differentiation group were 5.75 and 6.16, respectively, which were significantly higher than those in untreated group; while the expression level of C/EBPα, γ, GATA-1, GATA-2 mRNA in NB4 differentiation group were 62%, 31%, 63% and 8.7% respectively, which were significantly lower than those in untreated group; In HL-60 differentiation group, the expression levels of PU.1, C/EBPα, ε were 1.97, 1.95 and 2.35 respectively, which were significantly higher than those in untreated group; while the expression levels of C/EBPγ, GATA-1, GATA-2 in HL-60 differentiation group were 20%, 21% and 18% respectively, which were significantly lower than those in untreated group. It is concluded that dysregulation of transcription factors is a key contributing factor in the pathogenesis of acute myeloid leukemia.

Expression change of IL-3 receptor system in all-trans retinoic acid induced differentiation of NB4 cells / 中国实验血液学杂志

Interleukin-3 receptor (IL-3R) is a heterodimeric membrane receptor. The α subunit is essential for ligand binding and confers ligand specificity to the receptor. The common beta chain (βc) subunit, which is shared by the granulocyte macrophage-colony stimulating factor (GM-CSF), IL-3 and IL-5 receptors, is required for high-affinity ligand binding and signal transduction, mediating growth and survival of hematopoietic progenitor cells and the production and activation of mature hematopoietic cells. In order to investigate the role of IL-3 receptor system (IL-3Rα, GM-CSFRα and hβc) in myeloid differentiation, the expression level of IL-3 receptor system gene in all-trans retinoic acid (ATRA)-induced NB4 cell differentiation was detected by quantitative real time RT-PCR. At the same time, DNA sequence change was analyzed by cDNA sequencing. The results showed that the expression level of IL-3Rα mRNA was obviously down-regulated in NB4 cells treated with ATRA for 24 hours, but during differentiation of ATRA induced NB4 cells, the expression level of IL-3Rα mRNA was gradually restored, while the expression levels of GM-CSFRα mRNA and hβc mRNA were gradually up-regulated. The sequence of IL-3Rα and GM-CSFRα gene did not change before and after NB4 cells differentiation, but the sequence of hβc gene changed when NB4 cells were treated with ATRA, the expression of hβc mRNA sequence before NB4 cell differentiation taken truncated mutation as dominant, as regards expression of hβc mRNA sequence after NB4 cell differentiation, the truncated mutation of hβc mRNA had restored to wild type. It is concluded that the IL-3 receptor abnormality exists in NB4 cells, over expression of IL-3Rα and truncated mutation of hβc may be involved in proliferation and differentiation block in NB4 cells.