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Published on: October 21, 2012
[Effects of silent nucleostemin gene expression on apoptosis of HL-60 cells in vitro]
Bao-Hong Yue1, Li-Na Yu, Yuan-Yuan Wang
1Department of Clinical Laboratory Examination, Zhengzhou University, Zhengzhou 450052, Henan Province, China. ybh2002@163.com
Abstract:
This study was aimed to explore whether the apoptosis of leukemia cells can be induced by targeting silencing nucleostemin gene in vitro. HL-60 cells were taken as the model, and were directly transfected with nucleostemin short hairpin RNA (NS-shRNA). Sequences unrelated with NS gene were taken as control. The blocking effect of NS-shRNA was detected by RT-PCR, the morphology changes in living cells were observed under inverted microscope, and the changes of cell shape and nucleus were detected by Wright-Giemsa staining. The amount of apoptotic cells were assayed by flow cytometer (FCM) and TUNEL technique, and the positive rate of apoptosis was determined meanwhile. The results showed that two NS-shRNA were synthesized in vitro, and the more effective one was selected to be transfected into HL-60 cells. The blocking rate of NS-mRNA reached to 74.94%. After transfection for 48 hours, Wright-Giemsa staining showed nuclear fragmentations and "apoptosis body" in cells. The apoptosis rate in transfected group detected by flow cytometry and TUNEL method were (25.32 +/- 3.06)% and (27.3 +/- 3.21)% respectively, but were only (3.12 +/- 0.38)% and (3.30 +/- 1.52)% in control group, the difference between the transfected group and the control group was significant (p < 0.01). It is concluded that the apoptosis of HL-60 leukemia cells can be induced by the silencing NS gene expression in vitro, which provides a theoretical basis for using NS gene as a candidate target gene in therapy of malignant tumor.
Insights
Silencing the nucleostemin (NS) gene in leukemia cells using NS short hairpin RNA (NS-shRNA) in vitro effectively induces apoptosis. This finding supports NS gene as a potential therapeutic target for leukemia and other malignant tumors.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Context:
- Leukemia is a significant health concern, necessitating novel therapeutic strategies.
- Targeting specific genes involved in cell survival pathways is a promising approach in cancer therapy.
- Nucleostemin (NS) plays a crucial role in cell proliferation and has been implicated in various cancers.
Purpose:
- To investigate the efficacy of targeting and silencing the nucleostemin (NS) gene in inducing apoptosis in leukemia cells in vitro.
- To evaluate the potential of NS gene as a therapeutic target for malignant tumors.
Summary:
- The study utilized HL-60 leukemia cells and transfected them with nucleostemin short hairpin RNA (NS-shRNA) to silence NS gene expression.
- RT-PCR confirmed effective NS gene silencing (74.94% blocking rate).
- Morphological analysis revealed characteristic apoptotic features, including nuclear fragmentation and apoptotic bodies.
- Flow cytometry and TUNEL assays demonstrated a significant increase in apoptosis rates in NS-shRNA transfected cells (25.32% and 27.3% respectively) compared to control cells (3.12% and 3.30%).
Impact:
- The findings provide strong evidence that silencing NS gene expression can induce apoptosis in leukemia cells.
- This research establishes a theoretical foundation for exploring the NS gene as a novel therapeutic target for leukemia and potentially other malignant tumors.
- The study contributes to the development of targeted gene therapies for cancer treatment.
