Zhihong Chen1, Ziqiang Zhang, Yutong Gu, Chunxue Bai. 1. Department of Respiratory Medicine, Research Institute of Respiratory Disease, Fudan University, Zhongshan Hospital, Shanghai 200032, PR China.
Abstract
BACKGROUND: Aquaporin 5 (AQP5) is widely expressed in various organ and tissues. In light of the novel oncogenic properties of aquaporins (AQPs), here we investigated the effect of AQP5 knockdown by RNAi on transmembrane osmotic water permeability, cell migration potential and cell volume regulation ability. METHODS: AQP5 expression was inhibited by short hairpin RNA in SPC-A1 cells, a lung adenocarcinoma cell line. Cells loaded with a fluoroprobe (calcein-AM) were immersed in either isosmotic, hyperosmotic or hyposmotic solutions, and fluorescence intensity was recorded using confocal microscopy. These measurements were used to calculate osmotic water permeability coefficients (Pf) and to monitor regulated volume decrease (RVD). Tumor cell migration and invasion assays were performed in a modified Boyden chamber. Wound healing and colony forming ability were also tested. RESULTS: Although self-quenching was not found in SPC-A1 cells, we observed a linear relationship between fluorescence intensity and cell water volume, suggesting that this method is a sensitive and reproducible way to measure single-cell transmembrane water permeability. Cells in which the AQP5 gene was silenced showed a 49.4% decrease in osmotic water permeability, a 55.3% decrease in migration and a 28.4% decrease in invasion potential. In addition, RVD decreased remarkably with reduced osmotic water permeability. CONCLUSION: Our results suggest that AQP5, which mediates water permeability and thus regulates cell shape and volume, is a potentially important determinant in cell migration.
BACKGROUND:Aquaporin 5 (AQP5) is widely expressed in various organ and tissues. In light of the novel oncogenic properties of aquaporins (AQPs), here we investigated the effect of AQP5 knockdown by RNAi on transmembrane osmotic water permeability, cell migration potential and cell volume regulation ability. METHODS:AQP5 expression was inhibited by short hairpin RNA in SPC-A1 cells, a lung adenocarcinoma cell line. Cells loaded with a fluoroprobe (calcein-AM) were immersed in either isosmotic, hyperosmotic or hyposmotic solutions, and fluorescence intensity was recorded using confocal microscopy. These measurements were used to calculate osmotic water permeability coefficients (Pf) and to monitor regulated volume decrease (RVD). Tumor cell migration and invasion assays were performed in a modified Boyden chamber. Wound healing and colony forming ability were also tested. RESULTS: Although self-quenching was not found in SPC-A1 cells, we observed a linear relationship between fluorescence intensity and cell water volume, suggesting that this method is a sensitive and reproducible way to measure single-cell transmembrane water permeability. Cells in which the AQP5 gene was silenced showed a 49.4% decrease in osmotic water permeability, a 55.3% decrease in migration and a 28.4% decrease in invasion potential. In addition, RVD decreased remarkably with reduced osmotic water permeability. CONCLUSION: Our results suggest that AQP5, which mediates water permeability and thus regulates cell shape and volume, is a potentially important determinant in cell migration.
Authors: Brian R Haas; Vishnu A Cuddapah; Stacey Watkins; Katie Jo Rohn; Tiffany E Dy; Harald Sontheimer Journal: Am J Physiol Cell Physiol Date: 2011-08-03 Impact factor: 4.249
Authors: Jing Ge; David K Wood; David M Weingeist; Somsak Prasongtanakij; Panida Navasumrit; Mathuros Ruchirawat; Bevin P Engelward Journal: Cytometry A Date: 2013-05-06 Impact factor: 4.355