大肠癌肝转移相关抑制基因筛选及其作用机制研究

Liver metastasis is one of the challenges in the treatment of colorectal cancer (CRC). It is urgent to clarify the mechanism of CRC liver metastasis. In our previous study, the genome-wide shRNA library and the second-generation sequencing were used to screen for metastasis suppressor genes in the nude mouse model of liver metastasis; global transcriptome analysis was used to find genes down-regulated in CRC liver metastasis; and then 6 novel CRC liver metastasis-suppressor gene candidates were found out by binding the above analyses together. Preliminary experimental results in vitro and in nude mice showed that knockdown of these genes promoted CRC cell migration and metastasis, and among these genes, FAM177A1 and HOXD4 genes had the strongest function. Further study revealed that the interaction of FAM177A1 and MYH9 , the transcriptional inhibition of HOXD4 on FOXQ1 gene may play an important role in their metastasis suppression. This project is intended to retrospectively study the mechanism and clinical significance of that the two metastasis suppressor genes down-regulate in CRC tissues in large clinical samples, so as to provide experimental evidence for their use as predictive markers of the metastasis and prognosis of CRC; and to investigate the effects of them on CRC metastasis and clarify the molecular mechanisms in molecular and cellular levels, clinical cases, liver metastasis model of nude mice and CRC model of tissue-specific gene knockout mice, so as to provide a theoretical basis for the targeted therapy of metastatic CRC.

肝转移是大肠癌(CRC)治疗的难题之一，阐明CRC肝转移机制是迫切需要解决的问题。我们前期在裸鼠肝转移模型中采用全基因组shRNA文库结合二代测序筛选转移抑制基因，通过全基因组表达谱分析寻找在CRC肝转移中表达下调的基因，两者相交，得到6种新的候选CRC肝转移抑制基因；体内外初步的功能实验结果表明，敲降这些基因表达均促进CRC细胞迁移/转移，其中FAM177A1和HOXD4功能最强；进一步研究发现，FAM177A1与MYH9相互作用、HOXD4抑制FOXQ1基因转录可能是其发挥作用的重要机制。本项目拟在临床上大样本回顾性研究这两个候选转移抑制基因在CRC中表达下调的机制及其临床意义，为将其作为CRC转移预测和预后判断的标志物提供实验依据；在分子、细胞、临床病例、裸鼠肝转移模型和组织特异性基因敲除小鼠CRC模型中揭示其对CRC转移的影响及其分子机制，为转移性CRC靶向治疗提供理论依据。

研究背景：大肠癌是人类最常见的恶性肿瘤之一，其远处转移（特别是肝转移）是患者治疗不佳及死亡的主要因素。因此，阐明其转移的分子机制，将为CRC转移的早期诊断（预测）、治疗及其靶向药物的研发提供重要参考。.主要研究内容：本项目利用全基因组RNAi文库加高通量测序结合表达谱差异筛选大肠癌肝转移抑制基因，在临床样本中研究候选基因FAM177A1和HOXD4表达下调的机制及其临床意义，为将其作为CRC转移预测和预后判断的标志物提供实验依据；在分子、细胞、动物模型及临床病例中揭示FAM177A1、HOXD4对CRC转移的影响及可能的作用机制。.研究结果：本研究筛选出一组可能的CRC肝转移抑制基因，其中FAM177A1和HOXD4功能最强；FAM177A1抑制CRC细胞迁移和侵袭及EMT，其可能是通过MYH9发挥作用，敲降MYH9可逆转FAM177A1对SW620细胞迁移的抑制作用。而HOXD4抑制CRC细胞体外迁移、侵袭及裸鼠体内肝转移；其发挥作用的机制可能是HOXD4结合于FOXQ1启动子TSS上游-153到-92bp区域（核心结合序列可能为SAMAGAAA），在转录水平抑制FOXQ1表达，抑制大肠癌细胞EMT和肿瘤干性，进而抑制大肠癌转移。.研究结论：FAM177A1和HOXD4在CRC组织中低表达，分别通过MYH9和FoxQ1反过来促进CRC的转移；肿瘤组织中HOXD4表达下调与大肠癌浸润和远处转移（特别是肝转移）相关。.科学意义或应用前景： 该研究从一种新的角度阐明了大肠癌转移的分子机制，并为预测CRC转移的早期诊断、治疗及其靶向药物的研发提供了重要参考。