Extended Data Fig. 11: Loss-of-function study of 7o8G-miR-1 in vivo by establishing an anti-7oxo transgenic mouse.
From: Position-specific oxidation of miR-1 encodes cardiac hypertrophy
a, The ISO treatment schedule for anti-7oxo TG, cardiomyocyte-specific transgenic mice with expression of anti-7oxo (a specific sponge inhibitor against 7o8G-miR-1); intraperitoneal (IP) injection. b, Heart size of anti-7oxo TG(−) versus TG(+) in the presence of ISO administration (three independent F0 lines, #69, #68, #44; left); HW/BW; error bars, s.d.; *P = 0.014; n ≥ 3 (right). c, Genotyping results of F1, generated from two different F0 lines (#71, top; #77, bottom). d, Section of hearts from littermates of two different lines (#71, left; #77, right); TG(−) versus TG(+) under ISO treatment. e, Heart size in d was quantified as HW/TL (bottom) or HW/BW (top); box plots with median line, first and third quartile; whiskers, minima and maxima; x, mean; *P = 0.007 and 0.013. Of note, ISO-induced hypertrophy is prevented in anti-7oxo TG (F1). f, H&E stained tissue sections of interventricular septum (IS) of anti-7oxo TG (F0); n = 3; each experiment was repeated independently with similar results. g, Immunofluorescence staining of cardiomyocytes in IS of anti-7oxo TG (F0); wheat germ agglutinin (WGA) for refining the cell border; MF20 for cardiomyocytes; DAPI for nuclear staining; scale bar, 100 μm. h, Relative cell size of MF(+) in g was quantified (n = 200, ImageJ; *P = 3.2 × 10−59); box plots with median line, first and third quartile; whiskers, minima and maxima; “x”, mean. Notably, size of cardiomyocyte was maintained in anti-7oxo TG even in the presence of ISO treatment. i, A schematic model of the position-specific oxidation of miR-1; 7o8G-miR-1 targets identified in the adrenergic receptor (AR) pathway, red; others, blue. j, Quantification of o8G-miR-1 in ISO-injected mice in time-dependent manner. After IP injection of ISO, hearts were collected at different time points (0, 1, 5 and 7 days; top), of which small RNA was used to measure relative amount of o8G-miR-1 to o8G spike-in control by performing o8G IP and qPCR (miR-1; bottom). Notably, sustained oxidation of miR-1 was observed up to 7 days from ISO treatment; *P = 0.01, 0.03 and 0.01, respectively; n = 4 biologically independent mice; error bars, s.e.m. k, Sequence conservation of o8G-miR-1 target sites in 3′UTR. Conservation rates for miR-1 oxo sites (2oxo, 3oxo and 7oxo sites) including seed site were indicated in cumulative graph (left) and distribution (right) of seed sites of conserved miRNAs (conserved; blue line) and all heptamers (total; black line). Notably, sequences of miR-1 2oxo, 3oxo and 7oxo sites were evolutionally conserved in 3′UTR (11.3, 13.0 and 12.3%, respectively) as much as seed sites of conserved miRNAs in mammals—higher than median conservation rate (10.5%, 6mers) but lower than miR-1 seed site (14.5%). All P values from two-sided t-test, two-sided; n ≥ 3 biological independent samples; data show the mean unless otherwise indicated.
