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Background The discovery of water channel aquaporins(AQPs)has greatly expanded the understanding of theregulation of the water permeability of biological membranes.Aquaporin-1(AQP1)may be involved in fluid transport innumerous pathological conditions.The objective of the present study was to examine whether AQP1 is present incultured rat pleural mesothelial cells(PMCs)and to investigate the specific inhibitory effect of RNA interference(RNAi)onAQP1 expression in PMCs,which may provide a new method for the further studies on the relation between expressionof 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 byimmunocytochemical staining and reverse transcriptase-polymerase chain reaction(RT-PCR).Two eukaryoticexpression plasmid vectors of short hairpin RNA(shRNA)specific for the AQP1 gene of rat sapien were designed andconstructed.The recombinant plasmid vectors were transfected into cultured rat PMCs by cation liposomes.Flowcytometry was used to screen the most effective shRNA at 48 hours after transfection.The expressions of AQP1 mRNAand 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.Twoeffective eukaryotic expression plasmid vectors of shRNA specific for the AQP1 gene were constructed successfully.Thelevels 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 inPMCs transfected with plasmid was significantly lower than that of the cells transfected with the control plasmid HK andthat of the untransfected cells(P<0.01).There was no significant difference in AQP1 expression between the controlgroup 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 markedlyinhibit the expression of AQP1 in cultured rat PMCs.The use of RNAi is a promising tool for future research into themechanisms of pleural fluid in vivo.
Background The discovery of water channel aquaporins (AQPs) has greatly expanded the understanding of thereulation of the water permeability of biological membranes. Aquaporin-1 (AQP1) may be involved in fluid transport innumerous pathological conditions. Objective of the present study was to examine whether AQP1 is present incultured in 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 byimmunocytochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR). Two eukaryoticexpression 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 cultur ed rat PMCs by cation liposomes. Flowcytometry 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 effector eukaryotic expression plasmid vectors of shRNA specific for the AQP1 gene were constructed successfully. These levels of 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. Expression of AQP1 inPMCs transfected with plasmid control significantly lower than that of the cells transfected with the control plasmid HK and of the untransfected cells (P < 0.01). There was no significant difference in AQP1 expression between the controlgroup 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 themechanisms of pleural fluid in vivo.