Bex1抑制结肠癌肝转移的功能、机制及转化研究

Liver metastasis accounts for much of the colon cancer mortality. What makes it challenging to address this issue is that the molecular mechanism of metastasis is not fully understood. Our previous study found that a small molecular protein Bex1 was significantly silenced in metastatic colon cancer tissues, compared to non-metastatic colon cancer tissues. Bex1 expression was decreased statistically in hepatic metastatic cells versus its primary tumor cells, Which were all originated from the orthotopic colon cancer liver metastasis animal model. Further investigation also confirmed that suppressed expression of Bex1 in colon cancer cells promotes liver metastasis in subcutaneous tumor model. Bex1 was supposed to be a suppressor of liver metastasis in colon cancer based upon these studies. To fully understand of the function and mechanism of Bex1 plays in liver metastasis of colon cancer, we aim to evaluate the ability of liver metastasis of Bex1-knockout colon cancer cell line in vitro and in vivo. Specifically, we will use several kinds of methods including Transwell assay, Wound healing assay, 3-D culture, etc for in vitro study, and animal model such as orthotopic colon cancer liver metastasis mouse model for in vivo study. Multiple molecular biology techniques including gene microarray, Real time PCR, Immunohistochemistry, etc will be utilized to screen the downstream target genes of Bex1 inhibited liver metastasis. We plan to explore the possibility of reversing liver metastasis through inhibiting the expression of the up-regulated target genes with shRNA or over-expressing the down-regulated target genes by in vitro and in vivo tests, respectively. The role and molecular mechanism of Bex1 inhibited liver metastasis of colon cancer will be confirmed eventually. The feasibility of using antibody or inhibitor against Bex1 targets for preventing or treating liver metastasis will also be explored. The elucidation of Bex1's function and mechanisms could lead new insights into the comprehensive therapy of colorectal liver metastases.

肝转移是导致结肠癌患者死亡的重要原因，但目前对肝转移的分子机制尚不明确。我们前期研究发现，与非转移性结肠癌组织相比，一个小分子蛋白Bex1在转移性组织中显著沉默。原代结肠癌肝转移细胞系中Bex1的表达也明显低于原代盲肠原位肿瘤细胞。动物模型也证实Bex1表达抑制后结肠癌细胞肝转移率明显增加。我们推测：Bex1表达可抑制结肠癌肝转移。本研究拟构建Bex1完全敲除结肠癌细胞系，应用细胞迁移侵袭实验及三维培养等体外实验平台，盲肠原位肿瘤肝转移等动物模型，评估Bex1被完全敲除后细胞肝转移能力；进一步运用基因芯片、定量PCR、免疫组化等技术筛选Bex1下游靶基因；用shRNA抑制上调的靶基因或过表达下调的靶基因后，在细胞和动物模型中验证可否逆转转移，最终明确Bex1在结肠癌肝转移中的功能和作用机制。我们也将探索Bex1靶基因抗体/抑制剂预防/治疗肝转移的可行性，为结肠癌肝转移的综合治疗探索新靶点。

本项目前期研究发现，与非转移性结肠癌组织相比，小分子蛋白BEX2在转移性组织中显著沉默。原代结肠癌肝转移细胞系中BEX2的表达也明显低于原代盲肠原位肿瘤细胞。动物模型也证实BEX2表达抑制后结肠癌细胞肝转移率明显增加。我们推测: BEX2表达可抑制结肠癌肝转移。本项目研究内容为构建BEX2完全敲除结肠癌细胞系，利用体内外实验，评估BEX2完全敲除细胞肝转移能力；进一步运用基因芯片、定量 PCR、免疫组化等技术筛选BEX2作用信号通路以及下游靶基因，为结肠癌肝转移的综合治疗探索新靶点。我们首先利用Crispr/cas9技术，成功构建BEX2稳定敲除的肠癌细胞系DLD1-BEX2-/-。通过划痕实验、Transwell迁移和侵袭实验，发现BEX2敲除后肠癌细胞的迁移和侵袭能力明显提高。我们再将BEX2稳定敲除的肠癌细胞系重新表达BEX2后，通过Transwell迁移实验发现肠癌细胞迁移能力明显提高的现象可被逆转。进一步利用动物肝转移模型，我们发现BEX2敲除后的肠癌细胞发生体内肝转移的比例明显增高，生存分析也提示接种BEX2敲除后肠癌细胞的裸鼠总生存较差。通过RNA测序技术，对比BEX2敲除前后基因表达谱差异，同时利用GO分析、KEGG信号通路分析等技术，提示Hedgehog信号通路等通路显著激活。进一步通过定量PCR以及蛋白免疫印迹等技术发现BEX2敲除后Hedgehog信号通路的SMO、GLI2等关键分子的活化。通过核浆分离技术，PCR等技术我们进一步发现BEX2敲除后可能促使ZIC2入核增加，从而促进Hedgehog通路的转录活化，导致肠癌细胞的迁移和侵袭能力增强。本课题深入揭示BEX2敲除后促进肠癌肝转移发生机理，有助于进一步深入理解结直肠癌肝转移的分子生物学机制。本课题目前接收及发表研究论著8篇，授权专利1项，出国交流学习2人，与国家顶级肿瘤学专家建立良好的合作伙伴关系；培养研究生5名。