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Widespread 8-oxoguanine modifications of miRNA seeds differentially regulate redox-dependent cancer development

Nature Cell Biology volume 25, pages 1369–1383 (2023)Cite this article

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Abstract

Oxidative stress contributes to tumourigenesis by altering gene expression. One accompanying modification, 8-oxoguanine (o8G) can change RNA–RNA interactions via o8G•A base pairing, but its regulatory roles remain elusive. Here, on the basis of o8G-induced guanine-to-thymine (o8G > T) variations featured in sequencing, we discovered widespread position-specific o8Gs in tumour microRNAs, preferentially oxidized towards 5′ end seed regions (positions 2–8) with clustered sequence patterns and clinically associated with patients in lower-grade gliomas and liver hepatocellular carcinoma. We validated that o8G at position 4 of miR-124 (4o8G-miR-124) and 4o8G-let-7 suppress lower-grade gliomas, whereas 3o8G-miR-122 and 4o8G-let-7 promote malignancy of liver hepatocellular carcinoma by redirecting the target transcriptome to oncogenic regulatory pathways. Stepwise oxidation from tumour-promoting 3o8G-miR-122 to tumour-suppressing 2,3o8G-miR-122 occurs and its specific modulation in mouse liver effectively attenuates diethylnitrosamine-induced hepatocarcinogenesis. These findings provide resources and insights into epitranscriptional o8G regulation of microRNA functions, reprogrammed by redox changes, implicating its control for cancer treatment.

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Fig. 1: Widespread o8G > T sequences in miRNA seed positionally clustered in patients with tumours.
Fig. 2: G > T variants in miRNAs are associated with the survival of patients with LGG and LIHC.
Fig. 3: Sequencing and functional studies of o8G-miRNAs in LGG cells.
Fig. 4: Transcriptome-wide target repression by 4o8G-miR-124 and 4o8G-let-7 in LGG.
Fig. 5: Sequencing of o8G-miRNAs in DEN-treated rat models and patients with LIHC.
Fig. 6: Sequencing and functional analyses of o8G-miR-122 and o8G-let-7 in LIHC cells.
Fig. 7: Transcriptome-wide changes of miRNA target regulation by o8G in LIHC.
Fig. 8: 3o8G- and 2,3o8G-miR-122 critically regulate LIHC development in vivo.

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Data availability

All raw sequencing data from in vitro oxidized RNA (SRP391033 and SRP391030), small RNA-seq (SRP353704), o8G-miSeq (SRP353708, SRP353711, SRP353713, SRP353717 and SRP353718), RNA-seq (SRP353641, SRP353644, SRP353643, SRP353687, SRP353639 and SRP391054) and WGS (SRP390848) were deposited in the SRA. All FASTQ files are available at http://clip.korea.ac.kr/o8g_cancer. Previously published sequencing data that were re-analysed here are available under accession code (GSE106810 (ref. 33), GSE52084 (ref. 34), GSE97056 (ref. 43) and GSE73059 (ref. 55)). The human LIHC and LGG data were derived from the TCGA Research Network (http://cancergenome.nih.gov/). The dataset derived from this resource that supports the findings of this study is available in Supplementary Table 1. Supplementary tables including lists of TCGA data analysed in the study are available in the figshare repository (https://doi.org/10.6084/m9.figshare.22351609). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

The Python scripts used in this work are available at http://clip.korea.ac.kr/oxog/.

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Acknowledgements

We thank all members of the Chi laboratory for their help and discussions. We are especially grateful to the late S.-D. Yoo and the Y.S. Lee for motivating us to strive to understand liver cancer. We thank J.-W. Nam for his help in initiating the TCGA data analysis, S.-H. Koo for providing the albumin promoter-containing vector, M.-S. Lee for providing SK-HEP-1, J. Hwang for help with arranging clinical samples, J. H. Kim for his help in preliminary studies on glioblastoma, Y. Koh for help in analysing RNA-seq data, and Y. Jeong, D.-N. Lee, Y. Kang and K.-H. Lee for their initial assistance in preliminary studies. This work was supported by the Samsung Research Funding & Incubation Center of Samsung Electronics under project number SRFC-MA1801-10, grants from the NRF funded by the Ministry of Science, ICT & Future Planning (NRF-2023R1A2C3005802, to S.W.C.; NRF-2021R1I1A1A01045508, to S.E.), Korea University grants, the KU-KIST Graduate School of Converging Science and Technology Program, and Korea Institute of Science and Technology (KIST) Institutional Program (2V09840). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Sangkyeong Eom, Jongjin Peak, Jongyeun Park, Seung Hyun Ahn.

Authors and Affiliations

  1. Department of Life Sciences, Korea University, Seoul, Korea

    Sangkyeong Eom, Jongjin Peak, Jongyeun Park, Seung Hyun Ahn, You Kyung Cho, Yeahji Jeong, Hye-Sook Lee, Elizaveta Ignatova, Sung Eun Lee, Yunji Hong, Dowoon Gu, Geun-Woo D. Kim, Dong Chan Lee, Ja Young Hahm, Eun-Sook Jang & Sung Wook Chi

  2. KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, Korea

    Jung Lee, Geun-Woo D. Kim & Sung Wook Chi

  3. Division of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea

    Sung Eun Lee & Sung Wook Chi

  4. Department of Surgery, Hanyang University College of Medicine, Seoul, Korea

    Jaemin Jeong & Dongho Choi

  5. Medicinal Materials Research Center, Biomedical Research Division, Korea Institute of Science and Technology (KIST), Seoul, Korea

    Sung Wook Chi

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Contributions

S.E., J. Peak and J. Park performed the major experiments. S.H.A., J. Park, Y.K.C. and J.L. performed major bioinformatics analyses. S.H.A. and Y.K.C. analysed the TCGA data, and E.I. and S.H.A. constructed the related web data server. S.E., J. Peak and J. Park performed o8G-miSeq and sequencing of in vitro oxidized RNAs. S.E., J. Peak, Y.K.C., Y.J., H.-S.L., Y.H. and J.Y.H. performed RNA-seq. S.H.A., J. Park, J.L., G.-W.D.K. and S.W.C. performed bioinformatics and data analyses. S.E., J. Peak, Y.J., J. Park, H.-S.L., D.G. and Y. H. performed molecular and cell biology experiments. S.E., J. Peak, Y.J., Y.H, D.C.L and S.E.L. performed qPCR. S.H.A., J. Park and E.I. analysed the AGO CLIP, AGO RIP and CLEAR-CLIP data. J.L. and J. Park analysed WGS data. S.E., J. Peak, Y.K.C., Y.J. and H.-S.L. conducted luciferase reporter assays. S.E., J. Peak and Y.J. conducted flow cytometry analyses. S.E. and J. Park performed mouse and rat experiments. J.J. and D.C. prepared the clinical samples and analysed the data, and S.E. and J. Park performed the experiments with clinical samples. E.-S.J. and S.W.C. conceived and designed the study. S.W.C. supervised the study and wrote the manuscript.

Corresponding author

Correspondence to Sung Wook Chi.

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Competing interests

S.E. and S.W.C. are inventors of a related patent on o8G-miRNAs described herein. All other authors declare no competing interests.

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Nature Cell Biology thanks Marino Resendiz, Maite Huarte and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Widespread positional o8G > T clusters in tumor miRNAs.

a, miRNA sequencing data in tumors, derived from TCGA (n = 8,253) and GEO (n = 434) databases; tissue distribution of samples in TCGA. b, The positions of SNVs in miRNAs were displayed as a heatmap, sorted depending on average read-count (avr) in total patient tumor samples and compared depending on the frequency (top 100 versus bottom 100; left panel); Top 100 SNVs were separately analyzed for each base that was mutated (A > , T > , G> and C > ; right panel). c, The fractions of positional SNVs in tumor miRNAs (top 10,000 versus bottom 10,000). d, Same results from analyses of base composition (A, T, G, and C) and mutation types (G > T, G > C, and G > A) as in Fig. 1c except highlighting SNVs in let-7a, miR-122, and miR-124. e, f, Hierarchical clustering of G > T SNVs in expressed miRNAs (n = 85) for LGG (n = 512, e) and LIHC patients (n = 372, f) as analyzed in Fig. 1d (G > T SNV frequency; %); Positional G > T clusters (clusters 3-1, 3-2, 2-1, 2-2, 4-1, and 4-2) and subgroups in the G > T clusters are highlighted (dotted line or labelled); G > T clusters in other positions are represented as colored boxes in the left. g-i, Consensus sequence patterns of the identified G > T clusters in seed regions (g, h) and remaining clusters located in other positions (clusters 11 and 12, i), displayed with information contents (bits score, intensity in the sequence alignment). j, Schematic of inducing in vitro oxidation (Fenton reaction) of random fragments from artificial transcripts (n = 5), which contain all possible combinatorial sequences (3 nucleotides) interspaced with one G. Of note, we designed these artificial transcripts to avoid any continuous stretch of Gs to examine the positional bias of oxidation while excluding the reported effect from GG on the redox potential of nucleic acids19,20.

Source data

Extended Data Fig. 2 Positional G > T variants in miRNA sequencing associate with LGG and LIHC patients.

a, 4G > T miR-124 rate (%) and miR-124 expression (RPM; miRNA-Seq data) in LGG patients (n = 512); High (upper quartile; red box) and low 4G > T ratio patients (lower quartile; blue circles) with miR-124 expression (RPM > 100) were selected for survival analysis (Fig. 2b, right panel). b, In let-7 family members (left panel; seed region, red line), frequency of SNVs at G of position 2 and 4 in LGG patients are separately analyzed (RPM in box plots, right panel); the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima, and ‘x’ marks the mean value. c, Hierarchical clustering of patients with LGG depending on SNV rates (SNV read/corresponding miRNA read, %; heatmap) in let-7a sequence (position 1-18); Positions 2 to 4 are zoomed-in (right panel). 4G > T and 2G > T clusters are highlighted (dashed line boxes). d, Kaplan-Meier survival analysis for the LGG patients in the 2G > T let-7a cluster, comparing high (upper quartile) versus low 4G > T ratio patients (lower quartile) with let-7a expression (RPM > 1000); P = 0.0014, two-sided log-rank test. e, Hierarchical clustering analysis as in c except for miR-122 in LIHC patients (n = 372); 2G > T miR-122 cluster, blue; 3G > T cluster, red. f, 3G > T miR-122 rate (%) and miR-122 expression (RPM; miRNA-Seq data) in LIHC patients, used to select high (upper quartile; red box) and low 3G > T patients (lower quartile; blue circles) with miR-122 expression (RPM > 10000). G, Entire clustering results of Fig. 2h (yellow boxed region), analyzed for the let-7 seed regions (position 2–8) in the matched normal-tumor pairs of LIHC patients (n = 47). H, Same analysis as in g except derived from all LIHC patients (n = 372). I, Distribution of 2G > T let-7 frequency (%) depending on patient survival (years, y), analyzed by box plots (upper graph, as displayed in b) and heatmap (lower panel). All survival analyses were derived from repeated data with biologically independent samples (TCGA).

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Extended Data Fig. 3 4o8G-miR-124 and 4o8G-let-7 function in glioma cells.

a, b, Luciferase reporter assays of 4oxo sites for 4o8G-miR-124 and miR-124; IC50 values (n ≥ 5; a); 100 μM H2O2 treatment (for 4 hours) and/or miR-124 inhibitor transfection in SH-SY5Y (b); *P = 1.28 × 10−9, relative to no site (‘-‘); n ≥ 13. c, Positional SNVs of miR-124 sequences (mismatch; %); serum starvation (SS; 48 hours); ROS dye (CM-H2DCFDA; inner set); two-sided Fisher’s exact test (n ≥ 2); *P = 5.79 × 10−9. d, Luciferase reporter assays as in b except including miR-124 seed sites, SS, and HS683 cell line; *P = 0.0004 and 0.0002 (upper to lower graph), relative to mock (n = 6). e, ROS in HS683 cells (DHE; flow cytometry); antioxidant treatment or SS (n = 8,000 cells); *P = 0.001, 2.6 ×10−7, 1.5 × 10−5 and 1.8 × 10−5, respectively; Blue, ROS−; orange, ROS+; red, ROS++. f-h, o8G-miSeq results in HS683 cells; o8G-miRNA (log2[o8G-IP]) versus miRNA expression (log2[input]), depending on antioxidant treatment (f); o8G-miRNA (g); positional SNVs of miR-124 (h); two-sided Fisher’s exact test (n = 2); *P = 1.43 × 10−10. i-k, Morphology of HS683 after transfection (72 hr) of 4o8G- or 4U-miR-124 (left panel), or 4o8G- or 4U-let-7a (right panel), compared with NT (i); n = 3; scale bar, 200 μm; cell cycle analyses (PI staining; j); *P = 4.7 × 10−5, 3.6 ×10−5, 0.008 and 0.038, respectively (n ≥ 3); invasion assays using Matrigel inserts in transwell (H&E; k, left panel); quantitation results from the zoomed-in replicates (mean ± s.d.; k, right panel); *P = 0.03, 0.004, 0.015, and 0.005, respectively (n ≥ 3 biologically independent samples). l, Transfection of miR-124 or let-7 into HS683; morphology (first panel); cell cycle analyses (second panel); *P = 8.5 × 10−7 and 0.022, respectively; cell death analyses (third panel); *P = 4.4 × 10−12; Ki67 analyses (fourth panel); *P = 7.0 × 10−6 and 0.033, respectively. m, Transfection of miR-124, 4o8G-miR-124 and 4U-miR-124 into U87MG; morphology (first panel); cell cycle analyses (second panel); *P = 8.4 × 10−4, 0.037, and 0.007, respectively; cell death analyses (third panel); *P = 1.1 × 10−6, 1.1 × 10−5, and 4.5 × 10−6, respectively; Ki67 analyses (fourth panel); *P = 2.0 × 10−4, 4.4 × 10−4, and 3.3 × 10−5, respectively. n, let-7a-, 4U-let-7a- and 4o8G-let-7a-transfected U87MG cells; cell death analyses (upper panel); *P = 1.6 ×10−6 and 2.1 × 10−5, respectively; Ki67 analyses (lower panel); *P = 3.5 × 10−5 and 1.6 × 10−4, respectively. o, Luciferase reporter (4oxo site; 5x) assays for validating anti-4oxo (13x; pDual) against 4o8G-miR-124 (left panel) or 4o8G-let-7 in HS683 (right panel); no site (‘-‘); pDual vector with no insert (‘cont’); seed site (position 2–8, ‘seed’; 5x); *P = 0.0024 and 2.5 × 10−5, respectively (n = 6). p-t, Transfection of anti-4oxo (let-7 [4 sites, 4x RNA] or miR-124 [13x vector]) into HS683 (p, r, s) or U87MG (q, t); Ki67 analyses (p, q); *P = 1.2 × 10−4 (p); *P = 5.9 × 10−5 and 0.012, respectively (q); cell death analysis with TMZ treatment (r-t); *P = 3.8 × 10−4 and 0.009, respectively (r); *P = 9.2 × 10−11, 3.1 × 10−10, 3.2 × 10−10, and 2.8 × 10−9, respectively (s); *P = 3.0 × 10−7, 1.5 × 10−6, 1.0 × 10−9, and 1.0 × 10−7, respectively (t). All scale bars are 200 μm; all P values are obtained from two-sided t-tests; repeated with biologically independent samples; n ≥ 3; data are mean ± s.d. unless otherwise indicated.

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Extended Data Fig. 4 Global target repression mediated by 4o8G-miR-124 and 4o8G-let-7 in LGG.

a, RNA-Seq analysis of 4o8G-miR-124-transfected HS683 cells; cumulative distribution function (CDF) plot (left panel) and MA plot (right panel), relative to NT expression; 4oxo site targets (miR-124), 6mer match (position 2-7) in 3’UTRs; P-value from two-sided Kolmogorov–Smirnov (KS) test, relative to total mRNAs (left panel); validated targets (red with the label, right panel). b, RNA-Seq analysis as in a except for 4o8G-let-7a transfection. c, d, RNA-Seq analysis as in a except inhibiting endogenous 4o8G-miR-124 (13x; pDual plasmid; c) or -let-7 (5x; d) by anti-4oxo. e, f, miR-124 4oxo sites in 3’UTRs of ATXN10, BCAT1, and IGF2BP3 (e) and let-7 4oxo sites in TIA1, HOXB3, and AZIN1 (f), overlapping with reproducible peaks from AGO RIP-Seq in patients with glioblastoma (biological complexity, BC ≥ 2; red) versus human brain (BA4); other peaks, grey; 6mer site, red; 7mer site, dark red. g, h, Luciferase reporter assays with miR-124 4oxo sites in MEOX2 and EGFR for their efficiency in target repression (g), measured as IC50; target sites inserted into luciferase reporters, green shade in Fig. 4m; luciferase assays for let-7 4oxo sites in CLTA, PUDP, and TIA1 (h); target sites, used for luciferase assay, were displayed as green shade in Fig. 4n (CLTA and PUDP) and f (TIA1); n ≥ 3 biological independent samples; Data are presented as mean values ± s.d. i, j, Schematic diagrams illustrating reprogramming of miR-124 and let-7 by increasing ROS, oxidized to tumor-suppressing 4o8G-miR-124 (i) and 4o8G-let-7 (j) during glioma development.

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Extended Data Fig. 5 Sequencing of o8G-miRNAs in liver cancer.

a, Quantification of proliferating cells (Ki67+) in DEN-treated liver tissue sections (relative to mock; n = 27); *P = 6.3 × 10−6 and 7.4 × 10−5, respectively; two-sided t-test. In box plots, the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima, and ‘x’ marks the mean value. b, Rat livers after co-injection of DEN and NAC. c-e, o8G-miSeq results in DEN-induced LIHC rat models (c, d) or patients (n = 2; e); o8G-miRNA (log2[o8G-IP]) versus miRNA expression (log2[input]; c, e); the dashed line, no change; o8G-miR-122 was significantly enriched (log2[o8G-IP/input]) despite the reduction in its level after DEN treatment; no enrichment of o8G-miR-21. Positional G > T frequency (%, heatmap intensity; d) of high-confident o8G-miRNAs (n = 56) for G > T clusters in LIHC patients (TCGA; Fig. 1e); individual replicate results in Fig. 5f (e, left panel); o8G enrichment versus o8G-miRNA (e, right panel) f, ROS levels (DHE; flow cytometry) in Huh7 cells after antioxidant or H2O2 treatment (for 6 hours; upper panels); Blue, ROS−; red, ROS+; *P = 0.0037; two-sided t-test (n ≥ 3); images of ROS fluorescence dye, CM-H2DCFDA (lower panels). g, qPCR results of miR-122 in Huh7, HepG2, and SK-HEP-1, relative to U6 (n = 3); mouse hepatocyte (mHC), positive control. h-j, o8G-miSeq results in antioxidant-treated Huh7; o8G-miRNA versus miRNA expression (h); volcano plot (relative enrichment versus statistical significance, -log10[P-value]; i); Positional o8G > T frequency (%), displayed as in Fig. 5e (j); IP, log2(o8G-IP); 2MM, two G > T mismatches in o8G-miSeq reads (log2). Of note, partial reduction of o8G > T at multiple positions of the oxidized miRNA was observed. k, miR-122 KO Huh7 cell line (using CRISPR/cas9); genomic PCR results of miR-122 locus (upper panel); qPCR results of miR-122, relatively to U6 level (n = 3; lower panel). l, Validation of luciferase reporters with miR-122 2oxo sites (left panel), 3oxo sites (middle panel), or 2,3oxo sites (right panel), responding to different concentrations of cognate o8G-miR-122 versus unoxidized miR-122; n = 6. All experiments were repeated with biologically independent samples; n ≥ 3; data are mean ± s.d. unless otherwise indicated.

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Extended Data Fig. 6 Functional validation of 3o8G- and 2,3o8G-miR-122 in LIHC cells.

a, b, Cytometric analyses of cell cycle using PI staining after transfecting 3o8G- or 2,3o8G-miR-122 into HepG2 cells, relative to NT-transfected cells (a); cells in G0/G1 (%, red, vertical value); *P = 0.001 and 0.01, respectively; 3o8G-, 2,3o8G-, 3U- and 2,3U-miR-122-transfected SK-HEP-1 cells (b); *P = 0.037, 0.014, 0.069, and 3.1 × 10−3, respectively. c, Wound-healing assays, of which images (left panel) were quantified for wound areas using ImageJ program, relative to NT-transfected cells (right panel); *P = 0.012 and 0.032, respectively (n = 3). d, Wound-healing assays as in c except transfecting 3o8G-, 2,3o8G-, 3U- or 2,3U-miR-122 into SK-HEP-1 cells; *P = 0.014, 3.2 × 10−3, 5.7 × 10−4, and 1.5 × 10−3, respectively (n = 3). e, Cytometric measurement of proliferation by Ki67 staining in 3U-miR-122- or 2,3U-miR-122-transfected HepG2 cells; blue, Ki67−; red, Ki67+; quantification results (right panel); *P = 8.4 × 10−6 and 4.3 × 10−4, respectively (n ≥ 3). f, Wound-healing assays of 3U-miR-122- or 2,3U-miR-122-transfected HepG2 cells as performed in c; *P = 3.4 × 10−3 and 8.1 × 10−3, respectively (n = 3). g, Measurement of proliferating cells as in e except for 3o8G-miR-122- or 2,3o8G-miR-122-transfected NCTC clone 1469 cells; *P = 0.043. Notably, proliferation was enhanced by 3o8G-miR-122, but no difference was detected by the tumor-suppressive 2,3o8G-miR-122 presumably because NCTC clone 1469 cells are from normal liver cell. h, Same cell cycle analysis as in a except transfecting anti-3oxo or anti-2,3oxo into Huh7 cells; *P = 0.006 and 0.003, respectively. i, Same wound-healing analysis as in c except transfecting anti-3oxo or anti-2,3oxo into Huh7 cells; *P = 0.006 and 0.03, respectively (n ≥ 3). All experiments were repeated with biologically independent samples; n ≥ 3; all P values are obtained from two-sided t-tests; data are mean ± s.d. unless otherwise indicated.

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Extended Data Fig. 7 Induction of 2,3o8G-miR-122 by expressing miR-122 precursor and functions of o8G-miRNAs in LIHC cells.

a, b, Wound-healing assays of HepG2 (left panel) and SK-HEP-1 cells (right panel), transfected with miR-122 vector (expressing miR-122 precursor; n = 3, 6; a) or synthetic miR-122 duplex (miR-122; n = 3, 5; b); wound area (%); *P = 0.02 and 6.5 × 10−4, respectively (a); relative to control vector (cont); transfection of the miR-122 duplex showed no change (b). c-e, Various miR-122 duplex forms, derived from human miR-122 locus (hsa-miR-122; c); wound-healing assays in SK-HEP-1 (n = 3, 3; d,e); no significant change was observed. f, o8G-miSeq results from miR-122 vector transfection (HepG2); o8G-miRNAs versus miRNA expression (left panel); grey line, no change; positional SNV frequencies (%) in the seed region of miR-122 (right panel). g, Wound-healing assays of Huh7 after transfection of miR-122 inhibitor; *P = 0.02 (n = 3). h-j, Cell death analyses with Sorafenib treatment in 3o8G- or 2,3o8G-miR-122-transfected HepG2 (n ≥ 2; h) or miR-122 KO Huh7 (n ≥ 3; i) and 3U- or 2,3U-miR-122-transfected HepG2 (n = 3; j); *P = 4.0 × 10−3 (3o8G-miR-122) and 0.034 (2,3o8G-miR-122), relative to NT (h); *P = 0.031 (3o8G-miR-122) and 4.4 × 10−3 (2,3o8G-miR-122), relative to NT+Sorafenib; *P = 0.019 (3o8G-miR-122) and 8.0 × 10−3 (2,3o8G-miR-122), relative to NT (i); *P = 1.1 × 10−3 (3o8G-miR-122) and 0.016 (2,3o8G-miR-122), relative to NT+Sorafenib; *P = 0.003 (3U-miR-122) and 0.001 (2,3U-miR-122), relative to NT+Sorafenib (j). k, l, Cell cycle analyses (PI staining) in 4o8G-let-7a-transfected HepG2 (k) or 4o8G- or 4U-let-7a-transfected SK-HEP-1 (l), relative to NT-transfected cells; cells in G0/G1 (%, red, vertical value); *P = 9.7 × 10−3 (n = 3; k); *P = 6.6 × 10−6 and 7.4 × 10−6, respectively (n = 3; l). m, Wound-healing assays in 4o8G- or 4U-let-7a-transfected SK-HEP-1; *P = 3.9 × 10−4 and 0.053, respectively (n = 3). n, Cell death analyses as in h-j except for 4o8G-let-7a; *P = 0.009, relative to NT+Sorafenib (n = 3). o, Cell cycle analysis after transfecting anti-4oxo RNA (let-7a) into Huh7; *P = 0.028 (n = 3). All experiments were repeated with biologically independent samples; n ≥ 3; all P values are obtained from two-sided t-tests; data are mean ± s.d. unless otherwise indicated.

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Extended Data Fig. 8 Global target repression mediated by 3o8G-miR-122, 2,3o8G-miR-122, and 4o8G-let-7 in LIHC.

a, RNA-Seq analyses of 3o8G-miR-122-transfected HepG2 cells; CDF (left panel) and volcano plot (right panel), relative to control NT expression; 3oxo site targets (miR-122), 7mer match (position 2–8) in 3’UTRs; P-value from two-sided KS test, compared with total mRNAs (left panel); downregulated differentially expressed genes (DEGs) with the 3oxo site (right panel, orange shade), selected by cutoffs (red dotted lines) in statistical significance (>1.3, y-axis; P < 0.05, two-sided t-test) and fold change (log2 ratio <−0.2, x-axis); critical target genes are highlighted in red with labels. b, c, Same RNA-Seq analyses as in a except for 2,3o8G-miR-122 (b) and 4o8G-let-7 (c). d, RNA-Seq analysis as in a except using 3o8G-miR-122-transfected SK-HEP-1 cells, e, RNA-Seq analysis for inhibiting endogenous 3o8G-miR-122 by anti-3oxo RNA transfection in Huh7; same CDF analysis as in a except for examining derepression of putative 4oxo targets (left panel), which contain 3oxo sites in 3’UTRs and showed 3o8G-miR-122-dependent downregulation (log2 ratio < 0) in a; fold change cutoff (right panel; log2 ratio > 0.2); derepressed 4oxo targets in volcano plot, highlighted with orange shade. f, g, Same RNA-Seq analyses as in e except using anti-2,3oxo RNA (miR-122; f), and anti-4oxo (let-7; 5x sites; g). h, Validation of miR-122 3oxo targets in 3o8G-miR-122-transfected SK-HEP-1, as conducted in Fig. 7b; *P = 2.1 × 10−4 and 0.016, respectively (n ≥ 3); P values are obtained from two-sided t-tests. i, Positional G > T variants of Ago-associated miR-122 sequences (AGO CLIP data in LIHC patients) in the seed region (position 2–8), analyzed between matched normal-tumor pairs in LIHC patients; mismatch rate (%) in the seed is represented as the individual rate (%) for each position. j-l, miR-122 3oxo sites in 3’UTRs of NHLRC2 and TMEM170B (j), overlapping with mRNA peaks from AGO CLIP sequencing in patients with LIHC; AGO-miR-122 2,3oxo sites in 3’UTRs of ADAM9, FAM168B, STEAP2, LRP10 and SMOC1 (k) and let-7 4oxo sites in 3’UTRs of CD164 and PAWR (l) are also displayed with AGO CLIP peaks in LIHC patients. All P values were obtained from two-sided KS test or two-sided t-test; repeated with biologically independent samples; n ≥ 3; data are mean ± s.d. unless otherwise indicated.

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Extended Data Fig. 9 Endogenous levels, target interactions, and oncogenic functions of o8G-miR-122 in LIHC.

a-c, Quantification of dFP (dual fluorescent proteins) reporter in Huh7 cells (flow cytometry); autofluorescence (a, left panel); miR-122 3oxo sites (a, right panel) versus no sites (a, middle panel); box plot results (b); the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima, and ‘x’ marks the mean value; log ratio of RFP/GFP, relative to antioxidant supplement (violin plots in Fig. 7r; n ≥ 1,000 cells); miR-122 KO Huh7 cells (b, right panel); average RFP value (log10[RFP]) with the 3oxo sites within similar ranges of control GFP values in the cells (c, left and middle panels), further normalized as relative log ratio by dFP reporter with no site (c, right panel). d, Analyses of AGO CLIP-Seq data in LIHC patients (n = 9); biological complexity (BC), degree of reproducibility; de novo tumor, AGO mRNA peaks only in tumor (subtracting the combined peaks in normal; n = 9); relative site frequency (%), normalized by average frequency of control sites (G•U, wobble pairing; G•G, no pairing; Exp, expected frequency); *P < 0.05, relative to control sites. e, Analyses of miR-122-target chimaera reads (left panel; Supplementary Table 23) in CLEAR-CLIP data (Huh-7.5; GSE73059), expanding the size of the sequences from the miR-122 ligated site (±0, 50 or 100 nt; right panel). Direct binding of 3o8G-miR-122 on 3oxo sites significantly increases in Huh-7.5; *P < 0.05, relative to control sites. f, Colony formation assays of 3o8G-miR-122- or 2,3o8G-miR-122-transfected SK-HEP-1; *P = 0.07 and 0.011, respectively; two-sided t-test; n = 3 biologically independent samples. g, Luciferase reporter assays for 3U-miR-122 and 2,3U-miR-122 expression vectors, transfected into miR-122 KO Huh7 cells and used for xenograft mouse models in Fig. 8b; *P = 1.7 × 10−8 and 3.8 × 10−9, respectively; two-sided t-test; n ≥ 3 biologically independent samples. h, Delivery of 2,3U-miR-122 to DEN-treated mouse liver tissue in Fig. 8c, estimated by qPCR; control NT was injected together to check the delivery; ‘Control’, 5×NT (100% NT); ‘2,3U-miR-122’, 1×NT (1:4; 25% NT, 75% 2,3U-miR-122); relative amount, normalized to U6, n = 3 biological independent mice; error bars, s.e.m.; *P = 0.056 and 0.131, respectively. i, 2,3U-miR-122 delivered mouse livers (10 M; n = 3) with DEN treatment; replicates of Fig. 8d; red arrows, tumor tissues. j, Anti-3oxo and anti-2,3oxo (miR-122) delivered mouse livers (10 M; n = 3) with DEN treatment; replicates of Fig. 8g. k, Same analysis of checking RNA delivery as in h except for 4U-let-7a used in Fig. 8h; *P = 0.019 and 0.029, respectively. l, Same as in j except for 4U-let-7a (n = 3); replicates of Fig. 8h. m, Schematic diagram illustrating oxidation of let-7 to tumor-suppressing 4o8G-let-7 during hepatocarcinogenesis. All P values are obtained from two-sided t-tests; repeated with biologically independent samples; data are mean ± s.d. unless otherwise indicated.

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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.

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Eom, S., Peak, J., Park, J. et al. Widespread 8-oxoguanine modifications of miRNA seeds differentially regulate redox-dependent cancer development. Nat Cell Biol 25, 1369–1383 (2023). https://doi.org/10.1038/s41556-023-01209-6

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