Purpose: The purpose of this work is to examine the impact of AQP8 on the proliferation and development of human glioma cell lines A172 and U251 and to determine if aquaporin 8 (AQP8) is associated with GSK-3β phosphorylation and nuclear transport of β-catenin in the Wnt signaling pathway.
Methods: AQP8 knockdown cell lines were constructed using a CRISPR/Cas9 double vector lentivirus infection. SAM/dCas9 was used to construct AQP8 overexpression cell lines and the CV084 lentivirus vector was used to construct AQP8 rescue cell lines. AQP8 and its mRNA, and phosphorylated GSK-3β, β-catenin, and other related proteins, were detected using western blot and qRT-PCR. Glioma cell apoptosis was detected using Hoechst 33342 dye. The migration of glioma cells was discovered using a wound healing assay. β-catenin localization in cells was detected using immunofluorescence staining.
Results: The proliferative and migratory capacities of A172 and U251 cells were significantly enhanced after AQP8 overexpression. The Wnt signaling pathways appeared to have higher levels of phosphorylated GSK-3β and β-catenin, and a rise in the fluorescence intensity ratio of β-catenin in the nucleus and cytoplasm, which suggests that β-catenin translocated into the nucleus, while AQP8 knockdown produced the opposite effect. Further, overexpression of AQP8 in AQP8 knockdown cell lines rescued the reduction of related protein levels caused by AQP8 knockdown.
Conclusion: High AQP8 expression promotes proliferation and growth of glioma cells, a process associated with phosphorylation of GSK-3β and nuclear translocation of β-catenin.
Keywords: AQP8; Wnt signaling pathways; glioma; phosphorylated GSK-3β; β-catenin.
© 2023 International Society for Developmental Neuroscience.