Figure 3

Assessment of miRNA inhibition in cultured cells and in vivo. (A) Upper panel. Relative luciferase activity of the miR-21 reporter containing a perfect match miR-21 target site co-transfected into HeLa cells with 1 or 5 nM tiny LNA-antimiR-21, or 5 nM 8-mer 2'-O-Me antimiR-21, LNA mismatch (mm) or LNA scramble (scr) control oligonucleotides, respectively. Error bars represent s.e.m. Lower panel. Northern blot analysis of miR-21 in HeLa cells transfected with 5 nM antimiR-21 or LNA scramble control. U6 is shown as control. (B) Relative luciferase activity of the miR-21 reporter co-transfected into HeLa cells, with 5 nM tiny seed-targeting LNAs harbouring single or two adjacent mismatches at all possible nucleotide positions in the antimiR-21 sequence (highlighted in red). (C) Relative luciferase activity of a miR-122 reporter containing a perfect match miR-122 target site co-transfected into HeLa cells with pre-miR-122 and tiny 8-mer antimiR-122 or 15-mer antimiR-122. Error bars represent s.e.m. (D) Northern blot analysis of liver RNAs from mice after treatment with three intravenous doses of 20 mg/kg 8-mer antimiR-122, 15-mer antimiR-122 or LNA scramble control or with saline. The Northern blot was probed for miR-122 and U6. (E) Quantification of the AldoA and Bckdk target mRNAs (same samples as in D, normalized to GAPDH; error bars, s.e.m.; n = 5). (F) Sylamer analyses performed on microarray data from mouse liver RNAs after treatment with three intravenous doses of 20 mg/kg 8-mer antimiR-122 or 15-mer antimiR-122. Shown are Sylamer enrichment landscape plots for 7 nt sequence words. The highlighted words in the plots correspond to canonical miR-122 seed match sites and to perfect match binding sites for the 8-mer antimiR-122. (G) Total plasma cholesterol levels in mice treated with three intravenous injections of 8-mer antimiR-122, 15-mer antimiR-122 or LNA scramble control or with saline (error bars, s.e.m.; n = 5). Adapted from Obad et al. [103].