p300调控Skp2诱导EMT促进非小细胞肺癌远处转移的作用及机制

Distant metastasis is the main cause of death in patients with NSCLC, but the molecular mechnisms are far from clearly elucidated. P300, a histone acetyltransferase, is involved in regulating many transcription factors and tumorgenesis of human malignancies. Our previous studies demonstrated that p300 expression associated with distant metastasis in human NSCLC, in vitro assays also demonstrated that over expression of p300 promoted cell migration and invasion in NSCLC cell lines, and furtherly showed that p300 could induce EMT in NSCLC. Our recent study found that p300 regulated the stability of Skp2 in NSCLC cell lines, while other studies in literature reported Skp2 was involved in degration of E-cadherin, whose down-regulation is the molecular phenotype of EMT. Taken together, we suppose that p300 may promote distant mestatastasis of NSCLC by regulating Skp2 to inducing EMT. In this project, we will use the newly advanced in vitro cell culturing system and molecular biological techniques, combined with orthotopic implantation of lung cancer in mice, to demonstrate the function of p300 in inducing EMT and promoting distant metastasis, to identify the moleculars and signaling pathways that directly interacting with p300, and elucidate the mechanisms of p300 in promoting distant metastasis of NSCLC, with the purpose to find potential molecular targets for the treatment of NSCLC.

远处转移是NSCLC致死的主要原因，但其分子机制仍待深入研究。P300是一种组蛋白乙酰基转移酶，通过调控转录因子参与恶性肿瘤的发生发展。我们的前期研究发现p300表达与NSCLC远处转移相关，体外也证实p300高表达促进NSCLC细胞的迁移和侵袭能力，并发现p300能诱导NSCLC细胞发生EMT。进而我们还发现p300可调控NSCLC细胞Skp2的表达，且已有文献显示Skp2可降解E-cadherin。而E-cadherin下调是EMT的分子表型。由此我们提出：p300可能通过调控Skp2诱导EMT促进NSCLC远处转移。本项目将应用最新的体外培养系统和分子生物学手段，结合小鼠原位移植瘤模型，获得p300高表达诱导EMT促进NSCLC远处转移的可靠证据，寻找并证实此过程中p300直接作用的分子和信号通路，阐明p300促进NSCLC远处转移的机制，为研发NSCLC靶向治疗药物提供新的思路。

组蛋白乙酰基转移酶（HAT）p300是一种转录共活化因子，参与协调和整合多个细胞信号与转录复合物形成，使细胞增殖、分化、凋亡等相关基因活性在各种生理及病理刺激下保持适当水平。近年的研究发现p300能促进恶性肿瘤的发生和发展，其高表达是多种恶性肿瘤预后不良的重要因素。本研究将探讨p300在非小细胞肺癌（NSCLC）侵袭性发展过程中的生物学功能及分子作用机制。用携带p300 shRNA及对照序列慢病毒感染H1975、H1993细胞，构建p300敲降表达细胞株H1975/shP300、H1993/shP300。将携带p300基因序列的质粒P300-pcDNA3.1-EGFP和空载体质粒瞬时转染入H460细胞，构建了外源性过表达p300的细胞株H460/p300。用细胞增殖实验和克隆形成实验检测H1975/shP300、H1993/shP300、H460/p300和对照细胞的增殖能力。采用划痕实验和侵袭小室实验检测几对细胞的迁移和侵袭能力。在功能研究的基础上，检测上皮-间质转化相关蛋白的表达在p300敲降表达细胞和过表达细胞的改变，探索上皮-间质转化是否是p300促进非小细胞肺癌侵袭性发展的机制。细胞增殖实验的结果显示，H1975/shP300、H1993/shP300的细胞增殖能力较H1975/ctrl、H1993/ctrl明显下降，在软琼脂中形成的克隆数目明显减少；而H460/p300细胞的增殖能力较H460/ctrl明显提高。在细胞迁移、侵袭实验中，H1975/shP300、H1993/shP300在划痕损伤修复中的愈合速度减慢，在Transwell小室穿透Matrigel膜的能力也较H1975/ctrl、H1993/ctrl减弱；而H460/p300细胞显示较H460/ctrl升高的迁移和侵袭能力。在功能研究的基础上进一步发现，p300敲降表达的细胞H1975/shP300、H1993/shP300中上皮表型的标志物E-cadherin表达升高，间质表型的相关标志物Vimentin, Fibronectin, snail表达下降；而在p300过表达细胞H460/p300却呈上皮标志物表达减弱，间质标志物表达增强。研究证实p300表达促进了非小细胞肺癌增殖、迁移和侵袭能力的升高。上皮-间质转化是p300促进肺癌侵袭性发展的机制。