关键词: p53凋亡刺激蛋白抑制剂, p53凋亡刺激蛋白抑制剂剪切变异体, p53, NF-&kappa, B, 乳腺癌

Abstract: Background and purpose: Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is one of the ASPP family. It binds to p53 to inhibit the transcriptional activity of p53-target genes and cell apoptosis, which is associated with tumor formation. Previously, we found a new subtype of iASPP, iASPP splice variant (iASPP-SV), which is a nuclear protein containing 407 amino acid residues and can bind to p53, inhibiting p53 transcriptional activity. However, the relationship of iASPP-SV and breast cancer is still obscure. Therefore, the purpose of this research was to study the role of iASPP-SV on breast cancer tumorigenesis and progression. Methods: 5’-rapid amplification of cDNA ends (RACE) was used to identify the 5’-end of iASPP-SV mRNA in MCF-7 cells. HEK 293 cells were transfected with pFLAG-iASPP-SV and pFLAG-iASPP (828). Then Western blot was used to identify whether endogenous iASPPSV was expressed in HEK 293 cells and 8 types of human tumor cell lines. This study established the stable clones of NIH 3T3 expressing FLAG-iASPP-SV and FLAG-iASPP (828). Cell proliferation assay, colony formation and soft agar colony formation assay were used to identify whether iASPP-SV and iASPP (828) can promote cell proliferation and iASPP-SV is an oncogene. Real-time fluorescent quantitative polymerase chain reactive (RTFQ-PCR) was used to detect the levels of iASPP-SV and iASPP (828) mRNA in primary breast cancers. Luciferase assays were used to identify the relationships between iASPP-SV, iASPP (828), p53 and NF-κB p65. Results: The study identified that iASPP-SV was encoded by previously reported NF-κB p65 subunit (RelA)-associated inhibitor (RAI), and endogenously expressed in many human cancer cell lines. Analysis of cell proliferation, colony formation assay and soft agar assay for colony formation identified that similarly to iASPP (828), iASPP-SV promoted tumor cell proliferation and acted as an oncogene. RTFQ-PCR result showed that the median values of iASPP-SV and iASPP (828) in breast cancers with wild-type p53 were more significantly over-expressed than those of mutant p53. Luciferase assays showed that iASPP-SV and iASPP (828) could suppress NF-κB p65 transcriptional activity. Thus iASPP family may participate in the regulation of p53 and NF-κB activity, which imply that iASPP perhaps shows pro- or anti-survival activities when it interacts with different proteins. Conclusion: These findings indicate that iASPP-SV may be a potential target for breast cancer therapy.

Key words: Inhibitor of apoptosis-stimulating protein of p53, Inhibitor of apoptosis-stimulating protein of p53 splice variant, p53, NF-κB, Breast cancer