Background: The discovery of water channel aquaporins (AQPs) has greatly expanded the understanding of the regulation of the water permeability of biological membranes. Aquaporin-1 (AQP1) may be involved in fluid transport in numerous pathological conditions. The objective of the present study was to examine whether AQP1 is present in cultured rat pleural mesothelial cells (PMCs) and to investigate the specific inhibitory effect of RNA interference (RNAi) on AQP1 expression in PMCs, which may provide a new method for the further studies on the relation between expression of AQP1 in PMCs and pleural fluid removal in vivo.
Methods: PMCs were isolated and cultured from rat pleura. The expression of AQP1 in PMCs was confirmed by immunocytochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR). Two eukaryotic expression plasmid vectors of short hairpin RNA (shRNA) specific for the AQP1 gene of rat sapien were designed and constructed. The recombinant plasmid vectors were transfected into cultured rat PMCs by cation liposomes. Flow cytometry was used to screen the most effective shRNA at 48 hours after transfection. The expressions of AQP1 mRNA and protein were detected by RT-PCR and Western blotting method at 48 hours after transfection.
Results: RT-PCR and immunostaining revealed that AQP1 mRNA and protein were present in cultured rat PMCs. Two effective eukaryotic expression plasmid vectors of shRNA specific for the AQP1 gene were constructed successfully. The levels of the expression of AQP1 were inhibited by 83.45%, 90.93%, respectively, at mRNA level and 41.24%, 67.60%, respectively at protein level by two recombinant plasmids at 48 hours after transfection. The expression of AQP1 in PMCs transfected with plasmid was significantly lower than that of the cells transfected with the control plasmid HK and that of the untransfected cells (P < 0.01). There was no significant difference in AQP1 expression between the control group and the group transfected with AQP1 nonspecific shRNAs (P < 0.05).
Conclusions: The expression of AQP1 was present in rat PMCs. The application of shRNA-AQP1 could markedly inhibit the expression of AQP1 in cultured rat PMCs. The use of RNAi is a promising tool for future research into the mechanisms of pleural fluid in vivo.