srGAP1 mediates the migration inhibition effect of Slit2-Robo1 in colorectal cancer.
srGAP1 mediates the migration inhibition effect of Slit2-Robo1 in colorectal cancer.
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srGAP1 介导 Slit2-Robo1 在结直肠癌中的迁移抑制作用。
DOI：
10.1186/s13046-016-0469-x
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发表时间：
2016-12-07
期刊：
影响因子：
--
通讯作者：
Huang Z
中科院分区：
文献类型：
--
作者：
Feng Y;Feng L;Yu D;Zou J;Huang Z
神经引导分子Slit2在肿瘤发生发展中起抑制作用。我们之前的研究表明，Slit2-Robo1抑制结直肠癌（CRC）的细胞迁移。然而，对其在CRC中的下游效应物知之甚少。本研究试图确定slit2 - robo Rho GTPase激活蛋白1 （srGAP1）是否可以介导slit2 - rob1对结直肠癌细胞迁移的抑制作用。免疫组化染色检测srGAP1蛋白在临床结直肠癌组织中的表达。条件培养基由稳定表达Slit2-myc、Robo1- ha或RoboN （Robo1的可溶胞外结构域）的HEK293细胞制备。采用免疫沉淀法（Immunoprecipitation， IP）检测Robo1与srGAP1的相互作用，免疫荧光法（immunofluorescence， IF）检测Robo1与srGAP1的亚细胞定位。采用小GTPase下拉法测定Cdc42的活性。采用改良的伤口愈合试验检测细胞迁移。与癌旁非癌组织相比，47.5%的结直肠癌组织中srGAP1蛋白表达明显降低，srGAP1表达降低与淋巴浸润、肿瘤分化差、TNM分期高、生存率差相关（P < 0.05）。IP和IF实验显示srGAP1是一个与robo1相互作用的蛋白，在Slit2处理后，srGAP1在CRC细胞中表现出类似的动态亚细胞分布。Small GTPase pull-down实验和迁移实验表明，Slit2-Robo1信号通过srGAP1抑制Cdc42活性和CRC细胞运动。srGAP1在结直肠癌中的下调与肿瘤进展和不良预后相关。srGAP1是CRC中Slit2信号传导的重要下游分子，通过抑制Cdc42介导Slit2的抗迁移功能。
The neuronal guidance molecule Slit2 plays suppressive role in tumorigenesis and progression. We previously showed that Slit2-Robo1 inhibit cell migration in colorectal cancer (CRC). However, little is known about its downstream effectors in CRC. This study tries to identify whether the Slit-Robo Rho GTPase activating protein 1 (srGAP1) could mediate the inhibitory effect of Slit2-Robo1 on CRC cell migration. The protein expression of srGAP1 in clinical CRC tissues was tested by immunohistochemistry staining. Conditioned medium was prepared from HEK293 cells stably expressing Slit2-myc, Robo1-HA or RoboN (a soluble extracellular domain of Robo1). Immunoprecipitation (IP) was applied to check the interaction between Robo1 and srGAP1, and immunofluorescence (IF) was used to observe the subcellular localization of Robo1 and srGAP1. Small GTPase pull-down assay was used to determine the activity of Cdc42. A modified wound healing assay was performed to detect cell migration. The protein expression of srGAP1 was remarkably decreased in 47.5% of CRC tissues compared with adjacent noncancerous tissues, and the decreased srGAP1 expression was associated with lymphatic invasion, poor tumor differentiation, high TNM stage, and poor survival (P < 0.05). IP and IF assays revealed that srGAP1 was a Robo1-interacting protein and exhibited similar dynamic subcellular distribution after Slit2 treatment in CRC cells. Small GTPase pull-down assay and migration assay indicated that Slit2-Robo1 signaling inhibited Cdc42 activity and CRC cell motility through srGAP1. Downregulation of srGAP1 in CRC was associated with tumor progression and poor prognosis. srGAP1 is an important downstream molecule of Slit2 signalling in CRC, and mediates the anti-migration function of Slit2 by inhibiting Cdc42.
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