摘要
目的:构建人凋亡抑制蛋白2(hIAP-2)基因的小分子干扰RNA(siRNA)的表达质粒,并检测其对乳腺癌细胞MCF-7的作用。方法:利用含U6启动子的mU6pro载体构建hIAP-2 siRNA表达质粒,RT-PCR和Western印迹法检验其对MCF-7细胞的RNA干扰(RNAi)效果。用MTT法分析其对细胞增殖,流式细胞仪检测其对细胞周期,以及Hoechst染色法观察其对细胞形态的影响。同时,用胱天蛋白酶-3(caspase-3)Ac-DEVD-pNA底物法检测caspase-3活性的变化,Western印迹法检测一些相关蛋白质的表达变化。结果:hIAP-2 siRNA表达质粒高效而特异地剔降MCF-7细胞中hIAP-2的表达,抑制肿瘤细胞增殖(P<0.01),阻断MCF-7细胞在G1期。随着hIAP-2基因被沉默,MCF-7细胞变得多核化和巨核化,caspase-3酶原表达升高并被激活(P<0.01)。此外,IκBα和p21waf1蛋白表达升高,NF-κB(p65)则降低,Cyt C维持不变。结论:RNA干扰hIAP-2对MCF-7细胞增殖和细胞周期调控有重要影响,细胞裂亡可能是导致其细胞死亡的主要原因,DNA受损后其细胞周期阻滞在G1期可能与上调p21waf1有关。
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 IκBα（p37） and p21wall increased,of NF-αB（p65） decreased and Cyt C remained unchanged. Conclusion： hlAP-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; G1 arrest after DNA damage might be associated with the p21wall increase.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2006年第2期150-155,共6页
Academic Journal of Second Military Medical University
基金
广东省教育厅高校重点学科项目(9306)
广东省中医药局科研立项课题(1050051)~~
关键词
人凋亡抑制蛋白2
SIRNA
乳腺肿瘤
细胞裂亡
human inhibitor of apoptosis protein 2, siRNA
breast neoplasmsmitotic cell death
