小分子肽A8在重建p53抑癌功能中的作用和机制

iASPP is a specific protein inhibitor which against the normal tumor suppressor function of P53. Currently researches have found that, iASPP stops the tumor suppressor function of P53 by mutually combine. Hence, the de-assembling of iASPP and P53, and the releasing of dissociative P53 subsequently, might be the effective way that rescues the tumor suppressor function of P53 back to the normal. The competitive binding ability of small molecule peptide-A8 top53 is used and studied in this project, and use technologies such as report-gene, CHIP, RNAi, immunofluorescence, flow cytometry, and transplanted tumor model etc. to explore the interaction between A8 and iASPP both in vivo and in vitro. Moreover, through those influences that observed in cell growth, proliferation and apoptosis, as well as in the pre-apoptosis gene expression and promoter activity, we are trying to explain the mechanism in rehabilitation and reconstruction of the tumor suppressor function of P53 by the over-expression of small molecule peptide-A8.

iASPP是特异性抑制p53正常抑癌功能的蛋白，前期研究发现，iASPP通过与p53的结合，从而抑制了p53的正常抑癌功能。因此，解除iASPP与p53的结合，释放出游离的p53,可能是p53的正常抑癌功能得到恢复的有效方法。本项目利用小分子肽A8能与p53竞争iASPP结合位点的特性，采用报告基因、CHIP、RNAi、免疫荧光、流式细胞、移植瘤模型等技术，从体内体外两方面研究小分子肽A8与iASPP的相互作用，观察小分子肽过表达对细胞生长、增殖和凋亡的影响以及对前凋亡基因表达和启动子活性的影响，阐明小分子肽A8在恢复和重建p53抑癌功能中的作用和机制。由于50%的人类肿瘤中都存在着野生型p53，因此本项目的完成有望为使用小分子肽在含野生型p53的肿瘤中恢复和重建p53抑癌功能提供新的思路和治疗靶点。

iASPP对p53蛋白有抑制功能。iASPP促进肿瘤发生的主要机制是通过抑制p53转导功能。针对iASPP和p53的相互作用，可能存在恢复p53活性的潜在治疗方法。因此，我们构建了一种iASPP的衍生肽，命名为A8,它来源于iASPP的C端。这样，我们转染了含有野生型p53的两种细胞中，U2OS和A549，并检测凋亡细胞。运用免疫共沉淀，双荧光素酶，染色体免疫共沉淀进一步验证了A8影响凋亡的机制。RT-PCR和免疫印迹实验也证明了凋亡相关基因的mRNA和蛋白的表达。本研究表明了，A8会增加野生型p53细胞系的凋亡。通过双荧光素酶报告基因实验证明A8可以增强启动子 Bax 和 PUMA的活性。并且，A8对裸鼠的移植瘤模型中的肿瘤也有抑制生长的作用。综上所述，这些结果为表达野生型p53的肿瘤细胞恢复p53的抑制功能提供了一种新的方向，从而为肿瘤的治疗提供了一种新的治疗方法。