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[Inhibition of osteosarcoma cell proliferation by a short hairpin RNA targeting proliferation cell nuclear antigen]
Qi-liang Zhang1, Shu-hua Yang, Hong-yun Liu
1Department of Orthopedics, Xiehe Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. zhangqiliang2004@yahoo.com.cn
Objective:
To construct a plasmid vector expressing the short hairpin RNA (shRNA) targeting proliferation cell nuclear antigen (PCNA), and to investigate its effect in vitro on the expression of PCNA, proliferation and apoptosis of human osteosarcoma cells.
Methods:
A plasmid vector expressing the short hairpin RNA targeting at PCNA was constructed and transfected into human osteosarcoma cell line MG-63 by dosper liposomal method. PCNA mRNA and protein expressions were examined using RT-PCR and immunohistochemical staining, respectively. Inhibition of the cell proliferation was studied by MTT method and colony forming assay. DNA synthesis was analyzed by (3)H-TdR incorporation and the cell cycle was determined by flow cytometry. The apoptotic cells were stained with acridine orange.
Results:
Expression of PCNA mRNA after the transfection was markedly inhibited by 80.51% with a PI value of 25.68% of that of the control group. PCNA shRNA inhibited MG-63 cell growth detected by using MTT method, with an inhibition rate of 61.78% at 48 h. DNA synthesis rate also decreased in the (3)H-TdR incorporation test. Flow cytometry analysis showed an increase of the percentage of G(0)/G(1) phase cells, along with a decrease of cell population in the S phase. The apoptosis rate of cells transfected with the plasmid vector was 16.54%.
Conclusions:
PCNA shRNA significantly suppresses the expression of PCNA at both mRNA and protein levels, corresponding to an inhibition of the proliferation of MG-63 cell and an increase of the cellular apoptosis.
Insights
Short hairpin RNA targeting proliferation cell nuclear antigen (PCNA) significantly inhibits PCNA expression in human osteosarcoma cells. This suppression reduces cell proliferation and induces apoptosis, offering a potential therapeutic strategy.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Silencing
Context:
- Osteosarcoma is a primary bone cancer with limited treatment options.
- Proliferation cell nuclear antigen (PCNA) is a key regulator of DNA replication and cell proliferation, often overexpressed in cancers.
- Targeting PCNA offers a potential strategy for inhibiting osteosarcoma growth.
Purpose:
- To construct a plasmid vector for short hairpin RNA (shRNA) against PCNA.
- To evaluate the in vitro effects of PCNA-targeting shRNA on human osteosarcoma cells (MG-63).
- To assess the impact on PCNA expression, cell proliferation, and apoptosis.
Summary:
- A plasmid vector expressing PCNA-targeting shRNA was successfully constructed and transfected into MG-63 cells.
- PCNA mRNA and protein levels were significantly reduced post-transfection.
- Cell proliferation, DNA synthesis, and cell cycle progression (S phase) were inhibited, while apoptosis increased.
Impact:
- Demonstrates the efficacy of PCNA shRNA in suppressing osteosarcoma cell proliferation and inducing apoptosis.
- Highlights PCNA as a viable therapeutic target for osteosarcoma.
- Provides a foundation for further in vivo studies and potential clinical applications.
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