Objective: To investigate the role of Rho GTPase-activating protein 21 (ARHGAP21) in regulating the migration and metastasis of non-small cell lung cancer (NSCLC) cells.
Methods: TCGA, CPTAC database were used to analyze the correlation of ARHGAP21 expression level in NSCLC and the patients' prognosis. The expression of ARHGAP21 in clinical specimens of NSCLC tissues was examined using Western blotting and immunohistochemistry. The effect of ARHGAP21 knockdown on migration ability of lung cancer cell lines was examined using Transwell assay and wound healing assay. A nude mouse model with injection of lung cancer H1299 cells via the tail vein was used to examine the effect of ARHGAP21 knockdown on the metastatic ability of the tumor cells. The possible mechanism of ARHGAP21 was predicted by bioinformatics analysis and verified using Western blotting.
Results: A low ARHGAP21 expression was associated with poor prognosis of patients with NSCLC (P < 0.05). ARHGAP21 expression was significantly downregulated in lung cancer tissues as compared with the adjacent tissues (P < 0.001). In cultured lung cancer cells, ARHGAP21 knockdown obviously promoted the migration ability of the cells (P < 0.001). In the nude mouse models, injection of H1299 cells with ARHGAP21 knockdown, as compared with the negative control cells, resulted in a greater number of metastatic lung cancer nodules (P < 0.05), which expressed higher levels of N-cadherin and vimentin. Bioinformatic analysis showed a close correlation of ARHGAP21 with APC, GSK3β, and Axin (P < 0.001). Western blotting showed that ARHGAP21 knockdown significantly decreased ubiquitination of β-catenin, upregulated N-cadherin and activated the WNT signaling pathway in the lung cancer cells.
Conclusion: ARHGAP21 downregulation can significantly promote the migration and metastatic ability of NSCLC possibly as a result of WNT signaling pathway activation, which reduces the ubiquitination of β-catenin by affecting the expressions of APC, GSK3β, and Axin.
目的: 探究Rho GTPase激活蛋白21（ARHGAP21）对非小细胞肺癌（NSCLC）细胞迁移、转移的影响并探讨其作用机制。
方法: 通过TCGA、CPTAC数据库和临床资料确定ARHGAP21在NSCLC中的表达与患者预后的关系；通过免疫印迹（Western blot）和免疫组化验证ARHGAP21在NSCLC的基础表达；通过慢病毒稳转构建敲低ARHGAP21的NSCLC细胞系；通过Transwell实验和划痕愈合实验检测敲低ARHGAP21对肺癌细胞体外迁移能力的影响；通过裸鼠肺转移肿瘤模型的构建检测敲低ARHGAP21对肿瘤细胞体内转移能力的影响；通过生物信息学分析预测ARHGAP21的可能作用机制；通过Western blot实验验证其相关机制。
结果: ARHGAP21的低表达与不良预后相关（P < 0.05）；与正常组织相比，ARHGAP21在肺癌组织中表达下调（P < 0.001）；Transwell和划痕愈合实验结果显示敲低ARHGAP21促进了肺癌细胞迁移能力（P < 0.001）。裸鼠尾静脉肺转移模型结果显示与阴性对照组相比，敲低ARHGAP21组肺部肿瘤转移结节数量更多（P < 0.05）且具有更高表达水平的N-钙粘蛋白和波形蛋白。生物信息学分析表明，APC、GSK3β、Axin和ARHGAP21之间存在高度相关性（P < 0.001）。Western blot结果显示敲低ARHGAP21能够降低β-catenin的泛素化，上调N-钙黏蛋白并激活WNT信号通路。
结论: ARHGAP21的下调能够显著促进NSCLC的迁移、转移能力，这可能与ARHGAP21基因下调后激活WNT信号通路，影响APC、GSK3β、Axin的表达从而降低β- catenin的泛素化有关。
Keywords: Rho GTPase-activating protein 21; WNT signaling pathway; epithelial-mesenchymal transition; non-small cell lung cancer.