miR-192恢复特定位点突变型p53功能的研究

The p53 tumor suppressor gene is the most frequently mutated gene in cancer. It is such a potent tumor suppressor that it or its pathways must be inactivated by mutation for cancer to proceed. Most p53 mutations are missense point mutations located in the DNA-binding core domain with abundant amount. One can therefore envisage abundant mutant p53 as a "loaded gun" present in the tumor cells. However, the gun's trigger is locked by a mutation. Several research groups have reported that anti-tumor effects of mutant p53 may be restored by various strategies, such as specific antibody binding, short peptides and small molecules treating. However, the molecules identified so far have some limits which compromise their clinical application, including problems related to delivery, potency and toxicity. Interestingly, our previous studies indicated that miR-192 exhibited potent anti-tumor effect in tumor cells with Y220C mutant p53 (p53/Y220C). Besides, the anti-tumor effect of miR-192 in these cells was worked in a p53/Y220C-dependent manner. Moreover, compared with the anti-tumor effect of miR-192 in tumor cells with wild-type p53, its effect was more potent in tumor cells with p53/Y220C. The Y220C mutation is one of the more prevalent oncogeneic mutations in p53 and presents an almost ideal paradigm for searching for and studying small molecules that stabilize p53 in a mutation-specific way. To the best our knowledge, there is no report about reactivation of mutant p53 by miRNAs. Compared with previous reported molecules, miR-192 has some advantages to be used as a novel molecule to rescue mutant p53. Firstly, the anti-tumor effect of miR-192 is potent in tumor cells with mutant p53/Y220C. Secondly, miR-192 is a natural molecule existing in normal cells, so its administration will be safer compared with other chemo-drugs which have more or less toxicity to normal cells, especially at high concentration. Thirdly, some studies have indicated that miRNA is relatively stable in terms of other RNAs which are usually degraded rapidly in plasma or body fluids, giving miRNA a advantage to be delivered systematically. Given to the results of our pilot research and the advantages of miR-192 as a novel mutant p53 activator, we intent to explore the internal link between miR-192 and reactivation of mutant p53, and to elucidate the underlying mechanism within the rescuing process. This project will provide evidences to support that miR-192 would be served as an efficient, potent, and highly selective anti-tumor pre-drug.

超过一半的人类肿瘤中存在TP53基因的突变。p53突变体在肿瘤细胞中往往富集，大多为单碱基突变。如此强大的抑癌基因，仅仅因为几百个氨基酸中的一个发生改变而失去抑癌活性，这的确是个让人遗憾又值得回味的病理学现象。我们前期研究发现：miR-192在含有特定位点突变的p53(Y220C)的细胞中可以发挥强效的抑瘤作用，且该效应是p53(Y220C)依赖的。我们推测：miR-192不仅如已有的报道那样，是野生型p53依赖的抑癌基因，它还可能通过恢复特定位点突变型p53的功能，发挥抑瘤作用。更有意义的是，由于突变型p53在肿瘤细胞中富集，miR-192的抑瘤作用较之在野生型p53的细胞中更为强大。miRNA恢复突变型p53功能的研究目前未见报道。本研究旨在细胞和整体水平验证miR-192对突变性p53功能的恢复效应，探讨其中机制，为miR-192成为强效且高选择性的抗肿瘤生物干预分子靶提供理论依据。

TP53基因的表达产物p53蛋白几乎参与了所有肿瘤生物学特性的调控，是许多关键信号通路的分子枢纽，其功能之复杂和强大使之成为当之无愧的明星抑癌基因，也是迄今所发现的人类肿瘤中突变率最高的抑癌基因。寻找有效的、易于给药且毒性小的物质来“扣动抑癌基因p53的扳机”是一项有着良好应用前景和实用价值的工作。.本课题探讨了miR-192的强效抑瘤效应与p53之间的关系。项目最主要的研究成果是:阐明了miR-192可通过直接靶向性抑制Rictor来增进和恢复p53的功能。与以往发现的促进和恢复p53功能的小分子化合物比较，miR-192有易于给药且毒性小的特点。.本课题中我们通过精确靶点验证发现miR-192可以直接靶向Rictor，在mRNA和蛋白水平抑制Rictor的表达。恢复miR-192的表达及抑制Rictor的表达（siRictor）均可以显著抑制肿瘤的特性生物学行为（细胞的增殖、迁移减弱，肿瘤的血管形成减少）。进一步的研究发现，miR-192促进p53的功能有赖于其对Rictor的抑制作用。通过免疫荧光共定位、免疫共沉淀及pull down等技术，我们反复印证了本课题最重要的发现即：Rictor和p53分子之间存在直接的绑定作用，这种绑定可以显著抑制p53的功能。以上的研究分别在细胞和整体水平进行，获得了比较一致的结果。对于临床标本的分析我们进一步阐明：miR-192在人肝癌组织标本中明显低表达，且与Rictor的表达呈负相关；在肝癌中Rictor的过表达和p53的失活相关，且与不良预后相关。更有趣的是我们发现miR-192不仅可以促进野生型p53的功能，还可以恢复Y220位点突变的p53的功能，我们研究结果提示Rictor可以和突变型p53结合，从而解绑定的突变型p53，这一过程可能恢复了p53原有的野生构像而发挥了抑瘤作用。目前这一部分研究仍在进行中。.作为青年项目，我们的研究是卓有成效的，有较好的原创性，相关的研究还有进一步拓展和深入的潜能。我们寄希望在现有成果的研究基础上，进一步探讨miR-192作为小分子物质增进和恢复p53的功能的可能性，我们亟待解决的问题包括：如何在整体水平更高效的递送miR-192到靶向的肿瘤细胞；分析miR-192对不同状态的p53的肝癌抑瘤效应，为精准医学治疗提供更多、更可行的实验依据。