候选抑癌基因BMP3在结直肠癌中的作用机制研究

Secretive BMP growth factors play an important role in tumorigenesis. The applicant of this grant found for the first time that BMP3 gene was frequently methylated in colorectal neoplasms, but rarely in normal epithelia, and the expression of BMP3 gene was silenced by aberrant methylation in its promoter region. The applicant further hypothesized that BMP3 might function as a tumor suppressor in colorectal cancer (CRC). This project is designed to prove this hypothesis and further explore its regulatory mechanisms in vitro and in vivio using cell models, mouse models，and tissue specimens. After BMP3 expression is restored in methylated CRC cell lines by transfecting with plasmid containing BMP3 cDNA or silenced in unmethylated CRC cell lines using siRNA, cell and mouse models will be used to investigate changes in cell/tumor growth and BMP-Smad pathway activity. Gene chip will be employed to screen for downstream target genes. The expression of the identified target genes will be artificially managed to observe their influence on the biological behavior of cancer cells and on the function of BMP3 gene. Then, the interactions among BMP3, target genes, and other relevant genes in BMP-Smad pathway will be verified in colorectal tissue specimens. The association of alterations in BMP-Smad pathway with clinical characteristics of CRC will be analyzed. The diagnostic value of methylated BMP3 as a biomarker will also be evaluated. The success of this project will provide scientific evidence to the early diagnosis and treatment of CRC.

BMP分泌型生长因子在肿瘤的形成中起着重要作用。申请人首次发现了BMP3基因在结直肠肿瘤中发生高频率的肿瘤特异性甲基化，且高甲基化沉寂了BMP3的表达，因而率先提出BMP3在结直肠癌中发挥潜在抑癌作用的假设。本课题将在细胞-动物-组织标本三个层次求证这一假设并深入探索其调控机制。通过转染高表达BMP3和siRNA沉寂BMP3表达，在结直肠癌细胞和小鼠模型中研究BMP3表达改变对癌细胞生长特性及BMP-Smad信号通路活性的作用；进一步采用基因芯片筛选出下游靶基因，观察改变靶基因表达对癌细胞生物学行为和BMP3功能的影响。然后在组织标本中验证BMP3、靶基因和相关BMP-Smad通路分子之间的相互作用，分析它们与结直肠癌临床病理特征的关系，评估甲基化BMP3作为诊断标志物的临床意义。本课题将为结直肠癌的早期诊断和治疗提供科学依据。

已知分泌型生长因子BMP与肿瘤的发生和发展相关。课题组在结直肠组织和结直肠癌细胞中，检测了BMP3基因的甲基化情况和蛋白表达水平，明确了在结直肠肿瘤中BMP3基因启动子区发生高频率甲基化，而且由于畸变甲基化导致了BMP3基因表达的沉默。为了深入研究BMP3在结直肠肿瘤中的生物学功能，建立了BMP3高甲基化高表达的HCT-116-BMP3稳定细胞模型和BMP3低甲基化低表达的km12-shBMP3稳定细胞模型，通过BMP3表达水平的改变研究其生物学特性的变化，发现BMP3的表达水平与结直肠癌细胞的增殖、迁移、侵袭，以及体内成瘤能力负相关，同时，BMP3的表达水平上调还会引起肿瘤细胞的凋亡，提示BMP3是结直肠癌的一个抑癌基因。本课题组又深入探索了BMP3的调控机制，有趣的是BMP3并非通过常见报道的BMP-Smad通路进行调控，而是通过BMP3-ActR2B-Smad2信号轴在结直肠肿瘤中发挥重要作用。为了进一步探讨BMP3在结直肠肿瘤中的作用方式，课题组又利用人类全基因组芯片技术，初步筛选出了10个BMP3调控的潜在下游靶基因，正在对它们进行验证工作。.本课题验证了BMP3基因在结直肠肿瘤中发生高频甲基化，明确了BMP3是结直肠肿瘤的抑癌基因，深入探索了其分子调控机制。BMP3作为结直肠肿瘤的潜在生物标志物，将为结直肠癌的早诊断、早治疗提供坚实的科学基础。