同源异型盒基因Prrx1通过调控EMT/MET表型转化促进乳腺癌转移的研究

Breast cancer is the most common malignancy with the highest incidence among women in the world. Metastasis is the major reason for breast cancer-related deaths. Targeted therapy for metastatic disease is clinically unavailable, because the molecular mechanism underlying metastasis remains unclear. In recent years, phenotypic changes of cells in the special microenvironment are found to be important in tumor metastasis: epithelial cells acquire fibroblast-like properties and exhibit reduced cell-cell adhesion and increased motility via epithelial-mesenchymal transition (EMT), which facilitates the escape of tumor cells from primary tumors. Subsequently, the disseminated mesenchymal tumor cells undergo the reverse transition, mesenchymal-epithelial transition (MET), at the site of metastases for colonization. Homeobox gene Prrx1 has an important role as a mediator of cell phenotype in embryonic limb bud formation and skeletal development. Based on phenotypic changes of tumor cells and embryogenesis similarity, our previous research has already demonstrated that homeobox protein Prrx1 over-expressed in breast cancer and decreased in metastasis focus，which significantly associated with α-SMA and E-cadherin expression. In this study, we sought to investigate the role of Prrx1 on regulation of cell phenotype in breast cancer and its mechanism, explore the effect of Prrx1 on the migration and invasion of breast cancer. Furthermore, the molecule mechanism of Prrx1 on regulation of TGF-β/Smad signal pathway will be investigated. The results of the present study will be valuable for future investigations of effective prevention and treatment of breast cancer, and provide cues and information for studying the mechanisms of other malignant tumor metastasis.

乳腺癌是女性最常见的恶性肿瘤之一，其远处转移是导致患者死亡的主要原因。近年来研究发现肿瘤细胞在不同的微环境中发生EMT/MET表型转化是癌细胞转移播散的重要机制。同源异型盒基因Prrx1作为细胞表型的调控者在胚胎发育中发挥重要作用，基于肿瘤细胞表型转化与胚胎发育的相似性，我们前期的研究发现Prrx1在乳腺癌组织中高表达，且与淋巴结转移、TNM分期密切相关；而转移淋巴结中Prrx1表达下降，统计分析发现其在癌组织及转移淋巴结中差异表达与细胞表型标志物α-SMA、E-钙粘素呈线性相关，初步证实Prrx1与肿瘤细胞表型转化的相关性。本课题在此基础上欲阐明Prrx1在不同微环境中对乳腺癌细胞表型的调控作用及对肿瘤细胞转移播散的影响，探讨其对TGF-β/Smad通路调节作用，分析调控Prrx1的分子机制。研究结果将为防治乳腺癌转移提供新的靶点和理论依据，亦为研究其它恶性肿瘤的转移机制提供线索和参考。

乳腺癌是女性最常见的恶性肿瘤，远处转移是导致患者死亡的主要原因。本课题重点研究Prrx1在不同微环境中对乳腺癌细胞表型的调控作用及对肿瘤细胞转移播散的影响，分析调控Prrx1关键分子和作用机制。发表论文23篇，SCI收录10篇，培养研究生5人。研究结果不仅拓展Prrx1的生物学功能，加深对乳腺癌分子病理学的认识，为今后更有效的防治乳腺癌转移提供新的分子靶点和理论依据，亦可为研究其它恶性肿瘤的转移机制提供借鉴和参考。.1. 寻找到乳腺癌EMT/MET证据：筛查43例乳腺癌及淋巴结标本，发现肿瘤原发病灶中边缘区域较中心区域癌细胞间质细胞标志物弹性蛋白表达上调，上皮细胞标志物细胞角蛋白、E-钙粘素表达下降；而转移淋巴结中相反，寻找到EMT/MET直接证据。.2. 阐明乳腺癌中Prrx1临床意义及与EMT关系分析：免疫组化发现Prrx1在乳腺癌中阳性表达高于癌旁非癌组织。Prrx1 表达与淋巴结转移、临床分期有显著性相关。Prrx1与E-钙粘素表达呈负性相关，与α-SMA表达呈正相关。.3. 证实Prrx1过表达诱导EMT促进乳腺癌侵袭迁移：Prrx1过表达组与病毒对照组、空白对照组波形蛋白、E-钙粘蛋白差异有统计学意义，过表达组波形蛋白mRNA水平是空白组的2.27倍，E-钙粘素是空白对照组的0.71倍；Prrx1过表达组迁移细胞数为明显高于对照组。.4. 靶向阻断Prrx1b诱导MET促进乳腺癌粘附能力：靶向阻断Prrx1b后癌细胞由“纺锤体样”向“铺路石样”形态改变，上皮表型蛋白表达升高和间质表型蛋白表达下降，抑制乳腺癌细胞增殖能力，降低细胞迁移、侵袭能力，但细胞粘附能力增强。荧光素酶实验说明Prrx1b可能通过调节Wnt/β-catenin信号通路发挥作用。.5. 揭示miR-655是Prrx1上游关键调控因子：生物软件预测miR-655与Prrx13'UTR区具有结合位点。将野生型和miR-655mimics共转染，显著降低荧光素酶活性，而Prrx13'UTR于突变型组荧光素酶活性无明显变化。共转染anti-miR-655和野生型 Prrx13'UTR荧光素酶活性升高，而突变型组荧光素酶活性无明显变化；蛋白检测发现沉默miR-655可以上调Prrx1的表达。