Irx5抑制乳腺癌转移的分子机制及其转录调控网络的研究

IRX5, Iroquois homeobox 5, is a member of the Iroquois family of homeobox transcription factors that play multiple roles in patterning and regionalization of embryonic tissues during development and are highly conserved from Drosophila to mammals. Several IRX family members are involved in carcinogenesis. Both Irx1 and Irx4 were identified as metasatasis suppressor gene in gastric cancers or prostate cancers, respectively， and IRX3 was down-regulated in LNCaP cells. However, the role of IRX5 in breast cancers has not yet been elucidated so far. .Our preliminary studies showed that Irx5 gene was down-regulated in highly metastatic human and mouse cancer cell lines, and had high expression in poorly metastatic cell lines. Based on the data from ONCOMINE, The negative correlation of Irx5 gene expression levels and metastasis potential was confirmed in human breast cancer tissures. Moreover, we found that ectopic over-expression of Irx5 in MDA-MB-231(a high metastatic cell line with lower expression of Irx5) could suppress the cell invasion and cell migration in vitro, and no significant difference in cell proliferation was observed between the model cell line and the vector control. So, these findings indicate that Irx5 may be a promising metasatasis suppressor gene in breast cancer. In this study, We will also establish a model cell line with Irx5 knock-out based on the non-metastatic mouse 67NR cell line (higher expression of Irx5)using a shRNA technique, its role in these two kinds of model cell lines will be further investigated in cell proliferation, colony formation, cell migration, and invasion in vitro and in long-distance metastasis using a xenograft experimental mouse model to confirm our hypothesis that Irx5 is a novel metastasis suppressor gene, and then the effects on epithelial-mesenchymal transitions，cell signalling and transcriptional regulation will be investigated so that we can elucidate the molecular mechanisms of Irx5 to suppress the breast cancer metastasis. In the meanwhile, the transcriptional regulatory networks of Irx5 gene will be constructed using chromatin immunoprecipitation (ChIP), gene expression profiling, and computational methods..Successful completion of this project will further ourstanding of breast cancer initiation, progression and metastasis and may open the way to new lines of research aimed at developing approaches to predict, diagnose or prevent the metastatic breast cancers.

Irx5基因是伊洛魁基因家族中的重要成员，该家族中的Irx和Irx4已被证实分别是胃癌和前列腺癌转移的抑制基因。申请人已有的工作表明Irx5基因在一系列高转移人、鼠乳腺癌细胞系中低表达，而在低转移或不转移的乳腺癌细胞系中高表达，且Irx5的mRNA水平与患者癌细胞的转移潜力负相关；过表达Irx5明显降低MDA-MB-231细胞系的浸润和转移能力,但对其细胞增殖无影响，预示Irx5可能是乳腺癌转移的抑制基因。本课题将进一步充实体外和体内实验，利用自建的Knock-in和Knock-out两套细胞系，从细胞增殖、浸润和转移能力等方面展开研究，确认Irx5基因对乳腺癌转移的抑制作用，并通过对细胞信号传导、上皮细胞-间质细胞转换、转录-翻译调控等方面的研究，阐明其发挥作用的分子机制，并绘制出以Irx5基因为核心的转录调控网络，从而为转移性乳腺癌的提前预测、分子诊断、个体化治疗等奠定理论基础。

IRX5 (Iroquois homeobox gene family)基因是伊洛魁同源盒基因家族中的一员，该家族有6个成员。有报道表明IRX家族的一些成员与人多种肿瘤的发生有关，其中IRX1和IRX4已被证实分别是胃癌和前列腺癌转移的抑制基因。前期的研究表明IRX5基因可能是一个乳腺癌转移的抑制基因。为了进一步研究IRX5基因在乳腺癌细胞发生、发展及转移过程中的作用，我们构建了IRX5基因过表达MDA-MB-231、SUM159乳腺癌细胞株和IRX5基因沉默的MCF7乳腺癌细胞株。细胞增殖、迁移和侵袭等体外实验的结果显示IRX5基因可明显抑制乳腺癌细胞的转移，确认IRX5基因是乳腺癌转移的抑制基因。在此基础上，对过表达IRX5的MDA-MB-231细胞及其对照的蛋白质组学进行了研究，筛选到差异蛋白（大于1.5倍或小于0.67倍）146个，其中多数差异基因与肿瘤的发生、发展和转移直接相关；生物信息学结果显示8个上调蛋白 (HKDC1, AKAP12, ANXA1, ALDOA, HSPA1B, HSPH1, HSP90AA1, and HSP90AB1)和3个下调蛋白(STAT1, ALDH3A1 and PTK7)可能是IRX5转录调控网络中的重要成员，但最终结果的证实和功能确认还需进一步研究。