Construction of hIAP-2 siRNA plasmid and its effects on breast cancer cell line MCF-7
- VernacularTitle:hIAP-2 siRNA表达质粒的构建及其对乳腺癌细胞MCF-7的作用
- Author:
Liping LI
- Publication Type:Journal Article
- Keywords:
human inhibitor of apoptosis protein 2;
siRNA;
breast neoplasms;
mitotic cell death
- From:
Academic Journal of Second Military Medical University
1982;0(02):-
- CountryChina
- Language:Chinese
-
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 G_(1) 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 I?B?(p37) and p21~(waf1) increased,of NF-?B(p65) decreased and Cyt C remained unchanged.Conclusion: hIAP-2 in MCF-7 cells plays a crucial role in cell proliferation and cell cycle progression.Mitotic cell death might be the main way of the cell death;G_(1) arrest after DNA damage might be associated with the p21~(waf1) increase.
