Construction of hIAP-2 siRNA plasmid and its effects on breast cancer cell line MCF-7
- Author:
Li-Ping LI
1
Author Information
1. Institute of Biochemistry and Molecular Biology
- Publication Type:Journal Article
- From:
Academic Journal of Second Military Medical University
2006;27(2):150-155
- CountryChina
- Language:Chinese
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Abstract:
Objective: To construct an expression plasmid of siRNA against gene hIAP-2 and to assess its effect on breast cancer cell line MCF-7. Methods: A hIAP-2 siRNA plasmid was constructed with mU6pro vector containing U6 promotor and its RNAi effect on MCF-7 cells was detected by RT-PCR and Western blot. The effects of the plasmid on MCF-7 proliferation were analyzed by MTT assay, on the cell cycle by flow cytometry, and on the cell morphology by Hoechst staining. The activity of caspase-3 was determined with its substrate Ac-DEVD-pNA and the expression of some related proteins was detected by Western blot. Results: The hIAP-2 siRNA plasmid knocked down hIAP-2 expression in MCF-7 cells specifically and obviously, inhibited tumor cell proliferation and arrested the MCF-7 cells at G1 phase. After hIAP-2 gene was silenced, MCF-7 cells became multinuclear and had macronucleus; procaspase-3 was activated and its expression was increased. The protein levels of IκBa (p37) and p21waf1 increased, of NF-κB (p65) decreased and Cyt C remained unchanged. Conclusion: hIAP-2 in MCF-7 cel ls plays a crucial role in cell proliferation and cell cycle progression. Mitotic cell death might be the main way of the cell death; G1 arrest after DNA damage might be associated with the p21waf1 increase.
