BMP-4诱导肿瘤细胞上皮－间质转化促进肝细胞癌侵袭转移及其分子机制的研究

Epithelial mesenchymal transition (EMT) plays a critical role in the invasion and metastasis of human hepatocellular carcinoma (HCC). Bone morphogenetic protein-4 (BMP-4) is an important growth factor regulating cellular EMT, differentiation, and migration. Our previous experiments found that BMP-4 expression levels in cirrhotic liver tissues were insignificantly increased via the secretion of BMP-4 by activated hepatic stellate cells. The biomarkers of HCC epithelial and mesenchymal phenotypes were also regulated by BMP-4. However, the effects and the molecular mechanisms of BMP-4 on HCC EMT, metastasis, and the patients' prognosis remain to be elucidated. Employing multi-techniques including real-time RT-PCR, Western blotting, immunofluorescence staining, flow cytometry, confocal laser scanning microscope, in vivo imaging in small animal, TaqMan? Low Density Arrays, and Bio-Plex Protein Suspension Array in the experiments on HCC cells, nude mice model and clinical HCC specimens, the present study aims to test our scientific hypothesis that BMP-4 promotes tumor invasion and metastasis through its EMT induction, so as to be associated with a poor prognosis of HCC patients. Meanwhile, the interacted signaling pathways and the targeting downstream EMT-associated genes of BMP-4 in HCC EMT regulation will be also investigated. The findings will help us to further understand the molecular mechanisms of HCC metastasis, and the correlation between liver cirrhosis and HCC prognosis. Meanwhile, the results achieved from this project will provide us with more theoretical basis and experimental evidences to improve the patients' prognosis through HCC metastasis prevention by the regulation of BMP-4 expression and signaling in liver.

上皮－间质转化（EMT）能显著提高肝细胞癌（HCC）转移能力，骨形态发生蛋白4（BMP-4）对细胞EMT、分化与迁移具有重要调控作用。我们前期发现肝星状细胞分泌BMP-4导致肝硬化组织中BMP-4表达显著增强，BMP-4可调控HCC细胞的上皮与间质表型标志物的表达，但它对HCC的EMT、侵袭转移、患者预后的作用及其分子机理尚不明确。本项目拟运用定量RT-PCR、Western杂交、免疫荧光、流式细胞术、激光共聚焦显微镜、低密度基因芯片、蛋白悬液芯片、小动物活体成像等技术，在细胞学、裸鼠模型、临床样本三个层次，验证BMP-4通过诱导EMT，促进HCC侵袭转移，导致患者预后不良，以及BMP-4的HCC细胞信号传导与EMT相关下游靶基因(群)及其相互作用的分子机理。从而帮助我们进一步了解HCC转移机制及肝硬化对HCC患者预后的影响，为阻断HCC转移与改善肝硬化HCC患者预后提供理论基础和依据。

骨形态发生蛋白4（BMP-4）对细胞EMT、分化与迁移具有重要调控作用。我们前期发现肝星状细胞分泌BMP-4导致肝硬化组织中BMP-4表达显著增强，BMP-4可调控肝细胞癌（HCC）细胞的上皮与间质表型标志物的表达，但它对HCC的EMT、侵袭转移、患者预后的作用及其分子机理尚不明确。. 我们采用体外和体内细胞增殖、侵袭转移、细胞周期与凋亡、回顾与前瞻性临床大样本分析等手段，通过对肝癌细胞、肝癌转移裸鼠模型、500余例肝癌组织标本的检测。除应用常规医学实验技术，还综合并灵活运用定量RT-PCR、 Western 杂交、免疫荧光、流式细胞术、激光共聚焦显微镜、PCR Array、小动物活体成像等技术，在细胞学、裸鼠模型、临床样本三个层次，证实了BMP-4通过诱导 EMT，促进HCC侵袭转移，导致患者预后不良。首次证明了BMP4可直接上调ID2 的表达，从而介导细胞周期调节蛋白CDKN1B的表达，BMP4可通过ID2/CDKN1B信号通路促进细胞周期进程从而导致肝细胞癌的增殖；BMP4可通过MEK / ERK / ELK1信号通路诱导HCC 的EMT和OXA化疗耐药，对于接受基于OXA化疗的HCC患者，BMP4可能成为有价值的治疗靶点。. 本项目研究成果进一步明确了BMP-4通过诱导 EMT，促进HCC侵袭转移，导致患者预后不良的分子机理。从而帮助我们进一步了解 HCC 转移机制，为通过调控肝脏中BMP-4的表达和信号通路预防肝癌转移提供了提供理论基础和依据。