癌基因iASPP-SV参与乳腺癌的形成

摘要

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 asso-ciated 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 ampliifcation 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 iASPP-SV 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 lfuorescent quantitative polymerase chain reactive (RTFQ-PCR) was used to de-tect 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 identiifed 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 identiifed that similarly to iASPP (828), iASPP-SV promoted tumor cell proliferation and acted as an onco-gene. RTFQ-PCR result showed that the median values of iASPP-SV and iASPP (828) in breast cancers with wild-type p53 were more signiifcantly 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 ifndings indicate that iASPP-SV may be a potential target for breast cancer thera-py.%背景与目的：p53凋亡刺激蛋白抑制剂(inhibitor of apoptosis-stimulating protein of p53，iASPP)属于ASPP家族成员，其与p53结合，抑制p53靶基因的转录活性，抑制细胞凋亡，与肿瘤形成相关。先前该研究所在课题组发现了iASPP的一个新亚型iASPP剪切变异体(iASPP splice variant，iASPP-SV)，其是包含407个氨基酸残基的核蛋白，能与p53结合、抑制p53的转录活性，但是其与乳腺癌细胞增殖的作用还不清楚。因此，该研究旨在探讨iASPP-SV在乳腺癌发生、发展中的作用。方法：用5’-基因末端快速扩增(rapid ampliifcation of cDNA ends，RACE)方法检测乳腺癌细胞系MCF-7的iASPP-SV mRNA的5’末端序列。将pFLAG-iASPP-SV和pFLAG-iASPP(828)分别转染HEK 293细胞，采用蛋白[质]印迹法(Western blot)检测HEK293细胞及8种人类肿瘤细胞iASPP-SV的表达情况。建立稳定表达FLAG-iASPP-SV和FLAG-iASPP(828)的NIH 3T3细胞系，细胞增殖分析、克隆形成实验和软琼脂集落形成实验检测iASPP-SV、iASPP(828)是否促进细胞增殖、是否是癌基因。用实时荧光定量聚合酶链反应(real-time lfuorescent quantitative polymerase chain reactive，RTFQ-PCR)的方法检测原发性乳腺癌iASPP-SV和iASPP(828) mRNA表达水平，确定人乳腺癌中iASPP-SV是否上调。用荧光素酶报告基因实验检测iASPP-SV、iASPP(828)、p53与NF-κB/p65的关系。结果：5’-RACE方法显示，MCF-7细胞中的iASPP-SV由RAI序列(DQ986418.1)编码。Western blot实验显示，多种人类肿瘤细胞系表达内源性iASPP-SV。细胞增殖分析、克隆形成实验和软琼脂集落形成实验证实iASPP-SV与iASPP(828)能促进肿瘤细胞增殖，是癌基因。RTFQ-PCR实验显示，p53野生型乳腺癌组织iASPP-SV表达水平的中位值比p53突变型显著升高。荧光素酶报告基因实验证实iASPP-SV、iASPP(828)能抑制NF-κB/p65转录，因此iASPP可能是双功能蛋白。结论：iASPP-SV可能作为乳腺癌治疗的有效靶点。