In Vivo Study Of Inhibitor Of Apoptosis Protein-like Protein-2 Promoting Breast Cancer Cell Growth

Inhibitor of apoptosis protein-like protein-2(ILP-2)is a member of the inhibitors of apoptosis proteins(IAPs).Located at human chromosomes(19q13.3-13.4),its gene is highly conserved,whose encoded product is able to inhibit cell apoptosis.Our previous study has found that ILP-2 is highly expressed in breast cancer serum and can promote the growth of breast cancer cells,suggesting that ILP-2 is of great importance to the research of occurrence and development of breast cancer.This study intends to further explore the effect of ILP-2 on breast tumor growth and verify its relevant molecular mechanism through experiments in vivo,providing experimental basis for the identification of ILP-2 as a molecular target for targeted treatment of breast cancer and the development of corresponding anticancer drugs.In this study,a lentiviral vector which knocked down ILP-2 constructed using lentiviral vector mediated RNAi technology,was transferred into human breast cancer MCF-7 cells and the stably strains of knockdown of ILP-2 expression were screened by puromycin.Western Blot was used to detect the expression of ILP-2 in stably transfected cells,and it was found that the expression of ILP-2 in MCF-7-ILP-2-sh5 strain was significantly decreased,displaying that a stably transfected MCF-7 strain of silenced ILP-2 was successfully established.CCK-8 method was further applied to test growth activity of the three groups of the stable strains.The result was that the growth rate of stably transfected cells with targeted knocked down ILP-2 was slowed apparently,declaring that ILP-2 promotes the growth of breast cancer cells.Strains of MCF-7-ILP-2-sh5 and MCF-7(MCF-7-control)were respectively transplanted into the subcutaneous tissues of nude mice,what was shown was that volume of the transplanted tumor in the interference group was evidently smaller than that in the control group,declaring that knockdown the expression of ILP-2 can effectively inhibit the growth of human breast cancer xenograft tumor in nude mice.The mechanism of ILP-2 promoting breast tumor growth was further investigated by Western Blot,Quantitative Real-time PCR(RT Q-PCR)and immunohistochemistry.The result revealed that down-regulation of ILP-2 did not affect expression of Chk2 while it up-regulated p-Chk2 and Caspase-3,indicating that ability of ILP-2 promoting the growth of breast tumor is associated with the signaling pathway regulated by Chk2.