Dataset Information

RNA interference for CFTR attenuates lung fluid absorption at birth in rats.

ABSTRACT:

Background

Small interfering RNA (siRNA) against alphaENaC (alpha-subunit of the epithelial Na channel) and CFTR (cystic fibrosis transmembrane conductance regulator) was used to explore ENaC and CTFR function in newborn rat lungs.

Methods

Twenty-four hours after trans-thoracic intrapulmonary (ttip) injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C2), we measured CFTR and ENaC expression, extravascular lung water, and mortality.

Results

alphaENaC and CFTR mRNA and protein decreased by approximately 80% and approximately 85%, respectively, following alphaENaC and CFTR silencing. Extravascular lung water and mortality increased after alphaENaC and CFTR-silencing. In pSi-C2-transfected isolated DLE cells there were attenuated CFTR mRNA and protein. In pSi-4-transfected DLE cells alphaENaC mRNA and protein were both reduced. Interestingly, CFTR-silencing also reduced alphaENaC mRNA and protein. alphaENaC silencing, on the other hand, only slightly reduced CFTR mRNA and protein.

Conclusion

Thus, ENaC and CFTR are both involved in the fluid secretion to absorption conversion around at birth.

SUBMITTER: Li T

PROVIDER: S-EPMC2515309 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Publications

RNA interference for CFTR attenuates lung fluid absorption at birth in rats.

Li Tianbo T Koshy Shyny S Folkesson Hans G HG

Respiratory research 20080724

<h4>Background</h4>Small interfering RNA (siRNA) against alphaENaC (alpha-subunit of the epithelial Na channel) and CFTR (cystic fibrosis transmembrane conductance regulator) was used to explore ENaC and CTFR function in newborn rat lungs.<h4>Methods</h4>Twenty-four hours after trans-thoracic intrapulmonary (ttip) injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C2), we measured CFTR and ENaC expression, extravascular lung water, and mortality.<h4>Results</h4>alphaENaC and CFTR mR ...[more]

PMID: 18652671

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Project description:The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199nm) with a slight positive charge (21.82mV); CLG-shAnx2 was of similar size (217nm) with decreased charge (12.11mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2h resulting in a 6-fold reduction in AnxA2 mRNA expression and 92% decreased protein expression. Fluorescence imaging confirmed targeting of tumors and lungs by DiR-CLG carriers with sustained localization up to 4h in mice. CLG-shAnxA2 treatment of mice significantly reduced the weights of lung tumors derived from H1650 SP cells and tumor burden was reduced to only 19% of controls. The loss in tumor weights in response to CLG-shAnxA2 was associated with a significant loss in the relative levels of AnxA2, SOX2, total β-catenin and S100A10, both at the RNA and protein levels. These results suggest the intriguing possibility that AnxA2 may directly or indirectly regulate relative levels of β-catenin, S100A10 and SOX2, and that the combination of these factors may contribute to the resistant phenotype of H1650 SP cells. Thus down-regulating AnxA2 using RNAi methods may provide a useful method for targeting cancer stem cells and help advance therapeutic efficacy against lung cancers.

Dataset Information

RNA interference for CFTR attenuates lung fluid absorption at birth in rats.

Background

Methods

Results

Conclusion

Publications

RNA interference for CFTR attenuates lung fluid absorption at birth in rats.

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