Knock-Down of Nucleostemin Gene Expression by RNAi Induced Differentiation and/or Apoptosis in Leukemic Stem Cell Models

Objective: Leukemia is an important disease with stem cell origin which arises in CD34+ cells. Identification and targeting of signaling pathways involved in proliferation, differentiation and apoptosis of leukemic stem cells are new frontiers in treatment of CML patients. In this case, nucleostemin...

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Bibliographic Details
Published in:Cell journal (Yakhteh) 2011-01, Vol.13, p.45-45
Main Authors: Gogani, S N, Moosavi, MA, Bonab, M N
Format: Article
Language:English
Subjects:
Apoptosis
bromides
CD34 antigen
Cell culture
Cell proliferation
Colorimetry
Cytoplasm
Differentiation
Double-stranded RNA
Gene silencing
GTP-binding protein
Latex
Leukemia
Microscopy
Molecular modelling
Nucleoli
nucleostemin
Phagocytes
Polymerase chain reaction
RNA-mediated interference
Signal transduction
siRNA
Stem cells
Transfection
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Summary:Objective: Leukemia is an important disease with stem cell origin which arises in CD34+ cells. Identification and targeting of signaling pathways involved in proliferation, differentiation and apoptosis of leukemic stem cells are new frontiers in treatment of CML patients. In this case, nucleostemin (NS), a novel nucleolar GTP-binding protein, plays a critical role in controlling cell-cycle progression, self-renewal and proliferation of both stem cells and cancerous cells. Expression of NS gene in leukemic stem cells has been demonstrated, but there is no comprehensive research in NS mechanisms of action yet. The aim of the present study was to introduce NS-siRNA into human leukemic stem cell models (K562 and NB4) and investigated effects of NS gene silencing in growth, differentiation and apoptosis of leukemic stem cells. Materials and Methods: NS specific double-stranded small interfering RNA (NS-siRNA) was transfected into leukemic stem cell models (K.562 and NB4). Changes in NS gene expression pattern evaluated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). [3-(4, 5'-dimethyltiazol-2-yl)-2, 5-diphenyltetrazolium bromide] colorimetric assay (MTT) and trypan blue exclusion test were used for cell proliferation and viability evaluation. Flow cytometery was used for evaluation of cell-cycle states of treated cells. Differentiation was studied using Wright-Giemsa staining and latex particle phagocytic activity. Apoptosis was studied using fluorescent microscopy. Results: The results showed that NS gene was highly expressed in K562 and NB4 cells. After 72 hours transfection of 200 nM siRNA, growth was inhibited up to 30-40% in both cell lines. In addition, viability was significantly reduced up to 30-40% after 72 hours in K562 cells. But, no substantial reduction on viability of NB4 cells was observed. Mechanistic study showed NS-siRNA treatment of K562 cells resulted in G1 cell-cycle arrest followed with apoptosis after longer transfection times. Whereas, differentiation characteristics such as decrease in nuclear cytoplasm ratio and ingestion of latex particles were observed in NB4 cells. Overally these results suggested that apoptosis in K562 and differentiation in NB4 were ultimate effects of NS inhibition. Conclusion: Based on apoptosis and differentiating effects of NS-siRNA in human leukemic stem cell models, NS may have potential application in molecular therapies based on induction of differentiation and apoptosis in
ISSN:2228-5806