Extended Data Fig. 10: in vivo function of 3o8G-miR-122 and 2,3o8G-miR-122 in DEN-induced hepatocarcinogenesis.
a-b, Replicate results from hepatocyte-specific transgenic mice (TG; albumin enhancer/promoter) expressing 2,3U-miR-122 in Fig. 8i, j (a, b); WT (n = 3) versus TG mice (n = 6) in response to DEN-induced hepatocarcinogenesis (a); red arrows, tumor tissues; scale bars, 100 μm; replicate experiments with a different TG line from Fig. 8j (with qPCR, relative to albumin mRNA; b). c, replicate experiments with a different 3U-miR-122 TG line from Fig. 8k (n = 1). d, Derepression of 3oxo target (AKT1S1; left panel) or 2,3oxo target mRNA (ASPH1; right panel) by hepatocyte-specific expression of anti-3oxo (left panel, albumin::anti-3oxo) or anti-2,3oxo (right panel, albumin::anti-2,3oxo) in Huh7; qPCR (relative to GAPDH mRNA); P = 0.06 and 0.025, respectively; two-sided t-tests; n ≥ 3 biologically independent samples; data are mean ± s.d. e, RNA-Seq analyses (CDF) of miR-122 3oxo or 2,3oxo targets in TG expressing anti-3oxo (TG+++; Fig. 8p, right panel) or anti-2,3oxo (TG+++; Fig. 8o, right panel) in liver; putative miR-122 3oxo targets, selected by showing anti-3oxo-dependent derepression (log2 ratio <0; Fig. 8f, left panel) and containing 3oxo sites (6mer, position 2-7) in their 3’UTRs; P-values from two-sided KS test. f-i, TG expressing anti-2,3oxo (n = 5, 2; f, g) or anti-3oxo (n = 4, 1; h, i) as in Fig. 8m, n; anti-2,3oxo TG (g) and anti-3oxo TG (i) were sacrificed at 10 months. j, A database web server for putative o8G-miRNA in tumor based on G > T analysis (http://clip.korea.ac.kr/gt_db/). k, Analyses of SNV frequency within miRNA sequences (miRNA-Seq, Fig. 5d, e; lower right panel) and their genomic regions (whole genome sequencing, WGS; lower left panel) in DEN-induced liver cancer tissues (DEN-tumor; the same samples in Fig. 5a), relative to control samples (normal rat liver; Mock-normal); variation ratios (%) were calculated for WGS reads on miRNA regions. l, Luciferase reporter assays with miR-122 seed sites (5x; position 1-9), responding to transfection of miR-122 (blue) and 3o8G-miR-122 (grey) into SK-HEP-1 cells (IC50,); n ≥ 3 biologically independent samples. m, Proliferation assays of HS683 cells (Ki67 staining) after 100 μM H2O2 (for 6 hours) or antioxidant treatment (antioxidant supplement for a week, followed by additional 10 mM NAC treatment for 24 hr); blue, Ki67−; red, Ki67+; *P = 3.0 × 10−7 and 3.0 × 10−6, respectively; n = 3, *P < 0.05. All P values are obtained from two-sided t-tests; repeated with biologically independent samples; data are mean ± s.d. unless otherwise indicated.
