Repeated siRNA application is a precondition for successful mRNA gammaENaC knockdown in the murine airways.
The volume of the airway surface liquid is regulated by Na(+) absorption and Cl(-) secretion by the respiratory epithelium. In cystic fibrosis, Na(+) hyperabsorption caused by the absence of functional CFTR protein leads to an altered airway surface liquid composition and finally to a deteriorated mucociliary clearance. It has been suggested that down regulation or inhibition of the amiloride-sensitive epithelial Na(+) channel (ENaC) could restore the disrupted airway hydration. Therefore, targeting ENaC by RNA interference could be of therapeutic relevance. In this context, we investigated whether RNAi could lead to a reduction in gammaENaC expression in epithelia in vitro and in vivo in mice. Transfection of cells with specific siRNA sequences for gammaENaC subunit reduced expression to approximately 10% relative to control. For in vivo experiments, siRNA sequences specific for the gammaENaC subunit were administered to the murine nasal cavity and, 72h later the animals were killed. In the first approach, only a single application of naked siRNA was given. In the second approach, repeated siRNA applications were performed. The single application of siRNA sequences had no effect on mRNA content of the targeted gammaENaC subunit, whereas repeated siRNA application resulted in a significant reduction in gammaENaC mRNA in the respiratory tissue. We conclude that repeated siRNA application is necessary for gammaENaC knockdown in the murine airways.
Yueksekdag
Guelnihal
G
Department of Pediatrics, Ludwig-Maximilians University, Munich, Germany.
Drechsel
Marei
M
Rössner
Michaela
M
Schmidt
Christa
C
Kormann
Michael
M
Illenyi
Marta C
MC
Rudolph
Carsten
C
Rosenecker
Joseph
J
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers; D053503:Epithelial Sodium Channels / Q000235:genetics*; D055785:Gene Knockdown Techniques; D051379:Mice; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D014132:Trachea / Q000378:metabolism*
D017931:DNA Primers; D053503:Epithelial Sodium Channels; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C507053:Scnn1g protein, mouse