Extended Data Fig. 10: Specific inhibition of 7o8G-miR-1 by competitive inhibitors, anti-7oxo.
From: Position-specific oxidation of miR-1 encodes cardiac hypertrophy
a, Schematics of competitive miRNA inhibitors, which consist of tandem repeats of miRNA target sites (7mer, position 2–8) in RNA oligonucleotide; “Cont”, NT seed sites (2×); anti-seed, miR-1 seed sites (2×); Anti-7oxo, miR-1 7oxo sites (2×). Base pairing of miR-1 or 7o8G-miR-1 with cognate target site (seed or 7oxo site) was represented. b, c, Luciferase reporter assays used to validate activity and specificity of competitive miRNA inhibitors (anti-seed and anti-7oxo) in a. Luciferase reporter vector containing miR-1 seed sites or 7oxo sites was constructed and used to confirm derepression of the reporters by anti-seed (b) and by anti-7oxo (c); “Cont”, anti-NT (containing seed sites of NT); −, NT; *P = 1.87 × 10−11 and 5.08 × 10−12, respectively; n = 8 biologically independent samples (b); *P = 0.038; n ≥ 5 biologically independent samples (c); error bars, s.e.m. Of note, anti-7oxo significantly derepressed 7oxo site mediated target repression, albeit the extent was somewhat marginal. This is possibly because of containing only two repeats of target sites in the inhibitor. Notably, anti-7oxo showed no effect on seed site mediated target repression, confirming the specificity of anti-7oxo (c). d, CDF analysis to globally confirm derepression of 7oxo targets by anti-7oxo (9×); “7oxo site”, mRNAs with 7oxo site (7mer, position 2–8, in 3′UTR); “No site”, mRNAs with neither 7oxo site nor seed site (6mers, positions 2–8, in 3′UTR); “Cont”, transcripts with a mismatch site at position 6 of miR-1 (negative control; 7mer, positions 2–8, in 3′UTR) but without 7oxo (left) or seed site (right). Expressed transcripts (FPKM >1 in both samples, valid status from Cuffdiff, n = 7,958) from RNA-seq (Supplementary Table 6b) were used for the analysis. Notably, anti-7oxo (9×) significantly derepressed miR-1 7oxo sites (P = 0.038, relative to control; Kolmogorov–Smirnov test, two sided; left) but not miR-1 seed sites (P = 0.987, relative to control, Kolmogorov–Smirnov test, two sided; right). e, Time-lapse images of rCMC, transfected with either cont (anti-NT (4×); left) or anti-7oxo (4×; right) after phenylephrine treatment (with serum starvation); scale bar, 100 μm. Introduction of anti-7oxo to rCMCs attenuated phenylephrine-induced hypertrophy. f, Generation of cardiomyocyte-specific transgenic mice (TG) with expression of anti-7oxo (13×); transgene cassette, generated from α-MHC::anti-7oxo (13×) plasmid by BamHI. g, Genotyping results of four independent founders (F0; #65, #68, #69, #44). h, Mapping result of RNA-seq reads (Supplementary Table 6c) for the anti-7oxo transgene cassette, expressed in hearts of ISO-treated TG (Fo, #68). Notably, TG(+) showed sufficient expression of anti-7oxo (13×) sequences in heart tissue. i, CDF analyses of RNA-seq results (Supplementary Table 6c) to confirm anti-7oxo (13×) mediated derepression of 7o8G-miR-1 targets (7oxo sites) as analysed in d. Notably, significant derepression of 7o8G-miR-1 targets (as measured by log2 (TG(+)ISO/TG(−)ISO)) was observed (P = 0.021, relative to “Cont”, Kolmogorov–Smirnov test, two sided ; P = 4.1 × 10−5, relative to “no site”); by contrast there was no change for miR-1 seed targets (right). All P values from t-test, two-sided; *P < 0.05; n ≥ 3 biological independent samples; data are mean ± s.d. unless otherwise indicated.
