Loss of aquaporin-1 triggers epithelial-mesenchymal transition in human cholangiocarcinoma

Background and Aims: Epithelial-mesenchymal transition (EMT) is involved in metastatic dissemination and associated with the progression of cholangiocarcinoma (CCA). Aquaporin-1 (AQP1) is a water channel expressed by cholangiocytes described to modulate cell proliferation and invasion in several cancers. However, the role of AQP1 in CCA remains unknown. Our aim was to study the function of AQP1 in CCA. Material and methods: AQP1 expression was evaluated in 39 human intrahepatic CCA (iCCA) by transcriptomic analysis. AQP1-KO in CCA cells, HucCCT1 was achieved by CRISPR/Cas9. Incucyte live-cell imaging system was used for functional studies. Next-generation sequencing (RNAseq) was used to further study the consequences of AQP1 KO. EMT and signaling pathways were evaluated by RT-qPCR, western blot, and immunostaining. In vivo experiments were performed using a xenograft CCA model. Results: AQP1 expression correlated positively with overall survival in patients with iCCA and negatively with EMT, stemness, and proliferative signatures. In vitro, RNA-seq analysis of CCA cells depleted for AQP1 showed a downregulation of epithelial markers (CDH1 and KRT19; p< 0.001) and an upregulation of mesenchymal markers (VIM and FN1; p < 0.001). In these cells, we observed a phenotypic change with induction of cell scattering, a loss of cell-cell junction protein E-cadherin, and a higher expression of vimentin and ZEB1, an EMT-inducing transcription factor. Functionally, loss of AQP1 is associated with increased cell migration and proliferation. Moreover, we found an activation of the IGF2/IGF1R/IR pathway in AQP1-depleted CCA cells. In vivo, AQP1-depleted CCA cells displayed a higher tumorigenic potential and tumor burden than control cells. Conclusion: Our data suggest that AQP1 acts as a tumor suppressor in CCA by acting both on cell proliferation and migration through an EMT process. Therefore, AQP1 might have an important role in the regulation of CCA progression. Overall design: AQP1-KO in CCA cells, HucCCT1 was achieved by CRISPR/Cas9. Next-generation sequencing (RNAseq) was used to further study the consequences of AQP1 KO.

研究背景与目的：上皮间质转化（Epithelial-mesenchymal transition, EMT）参与肿瘤转移扩散，且与胆管癌（cholangiocarcinoma, CCA）的进展密切相关。水通道蛋白1（Aquaporin-1, AQP1）是胆管上皮细胞表达的水通道蛋白，在多种癌症中可调控细胞增殖与侵袭过程，但AQP1在胆管癌中的作用仍不明确。本研究旨在探究AQP1在胆管癌中的功能。
材料与方法：通过转录组分析，检测39例人肝内胆管癌（intrahepatic CCA, iCCA）样本中AQP1的表达水平。采用CRISPR/Cas9技术构建AQP1敲除（AQP1-KO）的胆管癌细胞系HucCCT1。使用Incucyte活细胞成像系统开展功能学实验，通过下一代测序（RNA测序, RNAseq）进一步探究AQP1敲除后的转录组变化。采用实时定量聚合酶链反应（RT-qPCR）、蛋白质印迹（western blot）及免疫荧光染色，检测上皮间质转化及相关信号通路的表达变化。采用胆管癌异种移植模型开展体内实验。
结果：肝内胆管癌患者中，AQP1的表达水平与总生存期呈正相关，而与上皮间质转化、干细胞特性及增殖相关基因特征呈负相关。体外实验中，对AQP1敲除的胆管癌细胞进行RNA测序分析发现，上皮标志物CDH1与KRT19的表达显著下调（p<0.001），而间质标志物VIM与FN1的表达显著上调（p<0.001）。在该细胞系中，我们观察到细胞表型发生改变：出现细胞散离现象、细胞间连接蛋白E-钙粘蛋白表达缺失，同时波形蛋白（vimentin）及上皮间质转化诱导转录因子ZEB1的表达水平升高。功能学实验显示，AQP1缺失可促进细胞迁移与增殖。此外，我们发现AQP1敲除的胆管癌细胞中，IGF2/IGF1R/IR信号通路被激活。体内实验中，与对照组细胞相比，AQP1敲除的胆管癌细胞具有更强的致瘤能力与更高的肿瘤负荷。
结论：本研究数据表明，AQP1可通过调控上皮间质转化过程影响细胞增殖与迁移，在胆管癌中发挥肿瘤抑制因子的作用。因此，AQP1可能在胆管癌进展的调控中具有重要价值。
整体实验设计：采用CRISPR/Cas9技术构建AQP1敲除的胆管癌细胞系HucCCT1，并通过下一代测序（RNAseq）进一步探究AQP1敲除后的转录组变化。