Extended Data Fig. 8: Transcriptome-wide analysis of o8G-miR-1 transfected H9c2 cells.
From: Position-specific oxidation of miR-1 encodes cardiac hypertrophy
a, d, CDF analysis as performed in Fig. 5a except for 7o8G-miR-1-transfected H9c2 cells; all 7oxo sites (a), 7mer 7oxo site (positions 2–8; b), 6mer seed site (positions 2–7; c) and 7mer seed site (positions 2–8; d). Kolmogorov–Smirnov test (two sided) results were displayed in the table (b, c, d, top right table); mean. Control in c, d denotes mismatched site at position 6 of miR-1 in 3′UTRs but no seed site in the transcript. e, Gene Ontology analysis results of 7o8G-miR-1 targets identified in H9c2 cells (Fig. 5c); intensity of bubble colour, P value (derived from DAVID); bubble size, frequency of the Gene Ontology term. Highly similar Gene Ontology terms are linked by edges in the graph, where the line width indicates the degree of similarity. Of note, only Gene Ontology terms in biological process were used in this analysis. f–h, CDF analysis as in a–d except for 2o8G-miR-1 (f) or 3o8G-miR-1 (h) by using RNA-seq results (Supplementary Table 2c, d); Kolmogorov–Smirnov test (two sided) results in h. i, j, Gene Ontology analysis results as performed in e except for 2o8G-miR-1 (i) and 3o8G-miR-1 (j); P < 0.05 (derived from DAVID), n = 176 (2o8G-miR-1), n = 34 (3o8G-miR-1). 2o8G-miR-1 targets and 3o8G-miR-1 targets were selected as containing 6mer sites (position 2–9) in 3′UTR and showing log2 ratio <−0.8 in f, g. The biggest cluster was displayed as representative (i, j). k, Validation of putative 7o8G-miR-1 targets by qPCR in 7o8G-miR-1-transfected rCMC as performed in Fig. 5d, f. As control, GAPDH (for FBXO32 and PTEN) or ACTB (for USP18) was also measured for normalization. FBXO32, PTEN and USP18, of which 3′UTRs contain 7oxo sites (Supplementary Table 4c) were examined; t-test, two-sided; *P = 0.003, 5.4 × 10−4, 2.1 × 10−4 and 0.044, respectively; n ≥ 3 biological independent samples; data are mean ± s.e.m. Notably, PTEN also contains a miR-1 seed site in its 3′UTR. l, m, CDF analyses as conducted in Fig. 5g except for all o8G-miR-1 targets (2oxo, 3oxo or 7oxo sites; l) or only for 2o8G-miR-1 (m, left panel) or 3o8G-miR-1 targets (m, right). n, GSEA results performed as in Fig. 5h except using 2o8G-miR-1 (top) and 3o8G-miR-1 (bottom) dependent changes of putative cognate mRNA targets (6mers, 3′UTR) in H9c2 cells (Supplementary Table 2c, d) for the enrichment of ISO-dependent downregulation (log ratio < 0); NES, normalized enrichment score; P value, adjusted P value derived from GSEA.
