接头蛋白CRKL调控SLC7A5基因转录并介导胃癌细胞转移的机制研究

Tumor metastasis is an important prognostic factor for gastric cancer, related with complex molecular mechanisms. Our previous research found that CRKL aberrant expression in gastric cancer is significantly related with the malignant behaviors of invasion and metastasis. By knocking down CRKL in gastric cancer cells, the ability of invasion and migration of tumor cells was significantly depressed. However the mechanism of how CRKL drives the metastasis in gastric cancer is unknown. Our preliminary work discovered that the expression of SLC7A5 is correlated with CRKL. And the depletion of CRKL in gastric cancer could significantly induce the decreasing of SLC7A5 expression. Moreover, we found two potential binding site of SLC7A5 interacting with HIF2α, who is an important transcription factor involving in tumorigensis. Therefore, we assume that there could be a potential regulating relationship between these three molecules. The aim of this study is to investigate the pathway of CRKL regulating SLC7A5 through HIF2α, and to provide pre-clinical data for the target therapeutic treatment. In details: (1) we will observe the consequent cellular biological changes by modifying the gene expression of CRKL, HIF2α and SLC7A5 in gastric cancer cells; (2) we will study the relationship and interaction among CRKL, SLC7A5 and HIF2α in gastric cancer metastatic process; (3) we will validate the mechanism of the CRKL-HIF2α-SLC7A5 pathway by applying bio-techniques including luciferase reporter gene assay and chromatin immunoprecipitation.

肿瘤转移是影响胃癌患者预后的重要因素。本课题组前期发现接头蛋白CRKL与胃癌浸润、转移等恶性表型密切相关，敲低CRKL能显著抑制胃癌侵袭迁移，但具体机制不明。通过特征基因数据挖掘发现，SLC7A5与CRKL表达呈正相关。进一步实验表明，敲低CRKL，SLC7A5表达下调。此外，SLC7A5基因具有转录因子HIF2α潜在结合位点，而HIF2α表达与CRKL相关。据此，我们推测CRKL、HIF2α、SLC7A5间存在调控关系。本课题拟阐明上述CRKL通路影响胃癌转移的分子机制，为后续探索相关分子靶向治疗奠定基础，具体内容包括：1）调节胃癌细胞CRKL、HIF2α、SLC7A5表达，观察转移相关生物学行为变化；2）探讨CRKL、HIF2α、SLC7A5间作用关系，结合临床明确它们在胃癌转移中的意义；3）通过荧光素酶报告基因、ChIP等技术，明确CRKL经HIF2α调控SLC7A5表达的作用机制。

肿瘤转移是影响胃癌患者预后的重要因素。本课题申报基础为前期研究证实接头蛋白CRKL与胃癌浸润、转移等恶性表型相关；同时，生物信息数据挖掘结果显示，SLC7A5与CRKL表达呈正相关，且SLC7A5基因具有转录因子HIF2α潜在结合位点，据此推测CRKL经HIF2α调控SLC7A5表达，进而介导胃癌细胞转录。在本课题研究过程中，我们证实转录因子HIF2α作为CRKL的下游因子，参与调控SLC7A5表达，并验证了CRKL作用下，SLC7A5表达上调促进胃癌细胞侵袭转移的细胞生物学功能。在对CRKL的研究中，我们还发现其上游miR-126通过转录后调控机制分别影响胃癌细胞中RGS3、ADAM9等促癌基因表达，协同CRKL影响胃癌转移的重要机制。对于以上发现，我们已撰写相关论文并发表。此外，为配合外科亚专科研究转型和发展需要，课题组同时在胃癌和肝细胞癌中进行肿瘤转移相关的转录后调控机制的探索，通过结合生物信息学和分子生物学等技术，找到了ROBO1、AKR1B10、SPP1等特征基因，拓展了消化道肿瘤细胞增殖、转移等恶性生物学行为分子机制的认识，为进一步改善消化道肿瘤患者，尤其是胃癌和肝细胞癌患者的预后和整体生存率提供了可靠的理论基础。