Extended Data Fig. 4: Oxidized miR-1 silences new target sites via o8G•A base pairing.
From: Position-specific oxidation of miR-1 encodes cardiac hypertrophy
a, dFP (dual fluorescent proteins) reporter with miR-1 seed sites in GFP (top) was validated to detect miR-1 dependent repression at the endogenous level of individual H9c2 cells; NT, non-targeting control; miR-1, synthesized miR-1 used as positive control; miR-1 inhibitor for blocking endogenous miR-1. b, Expression level of miR-1 in AC16 cells, H9c2 cells, rCMCs and mouse heart, measured by qPCR relatively to U6; error bars, s.e.m. Notably, heterogeneity of AC16 and H9c2 cells could result in low global level of miR-1 in total small RNAs, implicating the worth of examining miR-1 at individual cell level. c, Same dFP reporter analyses as performed in a except for 7oxo, 3oxo and 2oxo sites; 7U-miR-1, 3U-miR-1 or 2U-miR-1, used as positive control. Of note, every miR-1 oxo site (7oxo, 3oxo and 2oxo site) in the dFP reporters was endogenously suppressed with sensitivity to detect the repression mediated by the basal level of o8G-miR-1, confirmed by either transfecting miR-1 inhibitor or cognate miR-1 variants. d, Phenylephrine-dependent enlargement of H9c2 cells, measured by size (log10(FSC)) using flow cytometer. e, Distribution of GFP value (log10(GFP)) in dFP reporter, of which GFP contains miR-1 7oxo sites (left). Relative repression (log10(GFP/RFP)) was calculated by averaging the reporter fluorescence values (GFP-7oxo) with a similar range of control fluorescence values (RFP) in the cells (right). The relative repression was also examined in phenylephrine-treated H9c2 cells. Of note, flow cytometry in a, c–e used a blue laser for the excitation. f, g, Quantification of dFP reporter in H9c2 cells by flow cytometry. Based on scatter plot of GFP versus RFP (f), GFP value (log10(GFP)) with 7oxo sites (RFP:GFP-site; red) was examined by averaging GFP values derived from similar range of control RFP values in the cells (g), further normalized as relative fold change (Fig. 3g), normalized by dFP reporter with no site (RFP:GFP-no site; black); 7o8G-miR-1, used as positive control (purple). Vectors with no fluorescence protein gene (control) were used to measure level of autofluorescence. h, Same analyses as in f except for considering NAC treatment. Of note, 10 times greater amount of reporter vector was used in h than in f, resulting in fewer transfected cells in f. i–k, Flow cytometry analysis using switched dFP protein reporter, of which fluorescent proteins were interchanged in i, top, resulting in sensitive detection of the suppression of miR-1 7oxo site by the endogenous level of 7o8G-miR-1 (i, j); experiments with positive control, 7o8G-miR-1 (j). Repressive propensity of RFP with 7oxo site (GFP:RFP-site; i, middle) was shown in merge (i, right), relative to RFP with no site (GFP:RFP-no site; i, left), showing similar tendency of shifting; relative fold change, normalized RFP values denominated by dFP reporter with no site (RFP:GFP-no site; black). l, Same analysis as in i–k with switched dFP reporter except using phenylephrine (left) and miR-1 inhibitor (right). Of note, phenylephrine-induced repression of miR-1 7oxo sites became more substantial when only a limited cell population, for which reporter values (RFP) were in the lowest 25%, was considered (middle). Observation of the restored reporter activity (RFP) by introducing miR-1 inhibitor (right) further confirmed that phenylephrine-dependent repression of miR-1 7oxo sites was mediated by miR-1. The flow cytometer used in f–l was equipped with two separate lasers to maximize the excitation of both GFP and RFP, resulting in increased sensitivity of the detection. m, Luciferase reporter assays with miR-1 seed sites in the presence of 5 nM of control (cont; NT), 2o8G-miR-1, 3o8G-miR-1, 7o8G-miR-1 and miR-1; *P = 0.005, 0.4 × 10−3 and 0.1 × 10−4; error bars, s.e.m.; n = 6 biological independent samples. Notably, introduction of oxidized miR-1 (2o8G, 3o8G and 7o8G) could also suppress seed sites in luciferase reporter albeit less potent than unoxidized miR-1, presumably because of retained activity of o8G•C base pairing. All P values from two-sided t-test; *P < 0.05; n = 3 biological independent samples; data are mean ± s.d. unless otherwise indicated.
