目的:为了研究尿皮质素-1(UCN-1)对生长的影响,迁移,结直肠癌(CRC)的体内外凋亡以及UCN-1在体外调节CRC细胞的机制。
方法:使用癌症基因组图谱(TCGA)数据库和组织微阵列评估UCN-1和CRC之间的相关性。使用定量实时聚合酶链反应(RT-qPCR)和蛋白质印迹法评估UCN-1在CRC细胞中的表达。体外,UCN-1对增殖的影响,凋亡,使用celigo细胞计数测定法或细胞计数试剂盒-8(CCK8)探索HT-29,HCT-116和RKO细胞的迁移,流式细胞术,伤口愈合或Transwell分析,分别。在体内,在裸鼠中评价UCN-1对CRC生长和进展的影响.使用RKO细胞中的磷酸激酶分析器阵列确定了UCN-1介导的CRC调控的下游途径。慢病毒用于敲低或上调细胞中的UCN-1表达。
结果:TCGA和组织微阵列结果显示,UCN-1在CRC患者组织中强烈表达。此外,组织芯片结果显示UCN-1在男性患者中的表达高于女性患者,UCN-1的高表达与淋巴结转移和病理分期较晚有关。UCN-1敲低导致CRC细胞增殖减少,迁移,和殖民地的形成,以及细胞凋亡的增加。在异种移植实验中,从具有UCN-1敲低的RKO细胞产生的肿瘤显示出体积和重量减少。异种移植肿瘤中Ki-67表达的减少表明UCN-1敲低抑制了肿瘤的生长。人磷酸激酶阵列显示p53信号通路参与UCN-1介导的CRC发生。p53信号通路抑制剂逆转了UCN-1敲低引起的迁移和增殖抑制,而细胞凋亡的增加受到抑制。另一方面,UCN-1过表达促进CRC细胞增殖和迁移,抑制凋亡。p53的过表达逆转了UCN-1过表达对CRC发展的影响。
结论:UCN-1通过抑制p53信号通路促进细胞迁移和增殖,抑制细胞凋亡。
OBJECTIVE: To investigate the effect of urocortin-1 (UCN-1) on growth, migration, and apoptosis in colorectal cancer (CRC) in vivo and vitro and the mechanism by which UCN-1 modulates CRC cells in vitro.
METHODS: The correlation between UCN-1 and CRC was evaluated using The Cancer Genome Atlas (TCGA) database and a tissue microarray. The expression of UCN-1 in CRC cells was assessed using quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting. In vitro, the influence of UCN-1 on the proliferation, apoptosis, and migration of HT-29, HCT-116, and RKO cells was explored using the celigo cell counting assay or cell counting kit-8 (CCK8), flow cytometry, and wound healing or Transwell assays, respectively. In vivo, the effect of UCN-1 on CRC growth and progression was evaluated in nude mice. The downstream pathway underlying UCN-1-mediated regulation of CRC was determined using the phospho-kinase profiler array in RKO cells. Lentiviruses were used to knockdown or upregulate UCN-1 expression in cells.
RESULTS: Both the TCGA and tissue microarray results showed that UCN-1 was strongly expressed in the tissues of patients with CRC. Furthermore, the tissue microarray results showed that the expression of UCN-1 was higher in male than in female patients, and high expression of UCN-1 was associated with higher risk of lymphatic metastasis and later pathological stage. UCN-1 knockdown caused a reduction in CRC cell proliferation, migration, and colony formation, as well as an increase in apoptosis. In xenograft experiments, tumors generated from RKO cells with UCN-1 knockdown exhibited reduced volumes and weights. A reduction in the expression of Ki-67 in xenograft tumors indicated that UCN-1 knockdown curbed tumor growth. The human phospho-kinase array showed that the p53 signaling pathway participated in UCN-1-mediated CRC development. The suppression in migration and proliferation caused by UCN-1 knockdown was reversed by inhibitors of p53 signal pathway, while the increase in cell apoptosis was suppressed. On the other hand, overexpression of UCN-1 promoted proliferation and migration and inhibited apoptosis in CRC cells. Overexpression of p53 reversed the effect of UCN-1 overexpression on CRC development.
CONCLUSIONS: UCN-1 promotes migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway.