MiR-140 and miR-328 regulate in vitro lung development and Fgf9 expression.
(A-B) Validation of tiny LNA antagomers ability to block the activity of miR-140 and miR-328. Repression of the Fgf9 3’ UTR by 10 nM miR-140 (A) or 10 nM miR-328 (B) transfected into HEK293 cells with a luciferase reporter construct containing a wild type mouse Fgf9 3’ UTR was blocked by adding 10 nM of the corresponding tiny LNAs to the culture medium. The control LNA (LNA-con) contains a single mismatch in the LNA-140 sequence. (C-F) E12.5 lung explants were cultured in the presence of 100 nM tiny LNA oligonucleotides (S2 Table) for 48 hr. (C) Control LNA (100 nM), (D) LNA-328, E) LNA-140, and (F) 50 nM of LNA-140 and LNA-328 (total concentration 100 nM). Red lines indicate mesenchymal thickness. (G and H) Quantification of mesenchymal thickness (G) and the epithelial bud number (H) of lung explants in response to treatment with tiny LNA antagomers (n = 4–5 explants per condition). (I and J) Whole mount in situ hybridization showing expression of Fgf9 in E12.5 wild type lung explants cultured in the presence of 100 nM control LNA (I) or LNA-140 (J). Images shown are representative of at least three independent experiments. *P<0.05, **P<0.01, *** P<0.001. Scale bars: 200 μm.