Fig. 1

A cis-eQTL variant in NSUN2 locus promotes esophageal squamous-cell carcinoma progression and radiochemotherapy resistance by mRNA-m⁵C modification of cancer-related genes. a Immunohistochemistry (IHC) analysis of NSUN2 protein levels in paired human esophageal squamous-cell carcinoma (ESCC) and adjacent normal tissues. Left panel shows representative IHC pictures of tissue arrays (scale bar, 100 μm) and right panel shows the statistics of IHC score (IRS) indicating that 85.3% (168/197) of ESCCs had significantly higher NSUN2 level compared with normal tissues. ****P < 0.0001 of Mann-Whitney test. IRS, immunoreactive score. b Kaplan-Meier survival curves for patients with high (IRS ≥ 6) or low (IRS < 6) NSUN2 level in ESCC. HR (95% CI) was computed with Cox hazard proportion model. c Comparison of NSUN2 mRNA levels in ESCCs as function of the rs10076470 genotype, showing that the A allele (GA and AA genotypes) had significantly higher levels compared with the G allele. **P < 0.01 and ns, not significant of Mann-Whitney test. d The rs10076470 A allele resides within a potential STAT1 binding motif. e The effect of STAT1 knockdown on luciferase activity of constructs containing the rs10076470 G or rs10076470 A allele in KYSE150 cells. Data are mean ± S.E.M. from 3 replicate experiments. *P < 0.05 and ***P < 0.001 of Student’s t-test. f Super-shift EMSA competition assay with STAT1 antibody and KYSE150 cell nuclear extract. I points the rs10076470A -specific band and II points a super-shifted band by STAT1. g Chromatin immunoprecipitation coupled qPCR analysis shows that STAT1 binds to NSUN2 promoter in an allele-specific manner. Data are mean ± S.E.M. from 3 replicate experiments. Anti-S, anti STAT1 antibody. *** and ###P < 0.001; **** and ####P < 0.0001 and ns, not significant of Student’s t-test between siSTAT1 and siControl and among different genotypes. h Venn diagram displays 1348 overlapped genes among 2750 and 2780 apparently downregulated genes in cells with NSUN2 knockout (KO) #1 and NSUN2 KO #2, respectively. Among 1348 overlapped genes, 509 differentially expressed genes (DEGs) had higher mRNA levels in ESCCs than adjacent normal tissues (fold change >1.4, FDR < 0.05). i Metascape gene enrichment analysis of the 509 DEGs (http://metascape.org/gp/index.html#/main/step1). j Western blot analysis of NSUN2 and TIGAR protein levels in KYSE150 cells with NSUN2 overexpression (OE) or KO. k Spearman correlation between NSUN2 and TIGAR protein levels expressed as IRS in ESCC. l The effect of NSUN2 OE on the TIGAR mRNA stability in KYSE150 cells determined by RT-qPCR at indicated time points after treatment with 6 μM actinomycin D. Data are mean ± S.E.M. In most data points, the error bars are within the symbols. ****P < 0.0001 of Student’s t-test. m NSUN2 OE significantly inhibited ROS production in KYSE150 cells, which could be rescued by silencing TIGAR. Data are mean ± S.E.M. from 3 experiments and each had 3 replications. **P < 0.01 and ****P < 0.0001 of Student’s t-test. n The effect of NSUN2 KO on colony formation ability of KYSE150 cells with or without ionizing radiation (IR, 4 Gy) and TIGAR OE. Shown are mean ± S.E.M. from 3 experiments and each had 3 replications. In some data points, the error bars are within the symbols. *P < 0.05; **P < 0.01; ***P < 0.001 and P < 0.01; ****P < 0.0001 of Student’s t-test. o Extreme limiting dilution assays show survival fractions in KYSE150 cells with NSUN2 KO caused by IR (4 Gy) and TIGAR OE. *P < 0.05 and **P < 0.01 of ELDA analysis program. p The effect of NSUN2 OE on DNA double-strand breaks detected by γ-H2AX in KYSE150 cells with or without IR (4 Gy) and TIGAR knockdown. q The effect of NSUN2 OE on DNA double-strand breaks detected by comet assays in KYSE150 cells with or without IR (4 Gy) and TIGAR knockdown. Left panels show fluorescence images of comet assays (scale bars, 100 μm) and right panels show the statistics. Data are mean ± S.E.M. from 3 replicates and 3 fields were randomly selected from each experiment. siCtrl, siControl; siT, siTIGAR. r Representative images showing NSUN2 and TIGAR immunohistochemical staining in ESCC establishing PDXs with differential radiosensitivity. Scale bars, 100 μm. s Box and bar plots comparing the NSUN2 protein levels in PDXs with differential radiosensitivity. Sens, sensitive and Resist, Resistance. **P < 0.01 of Mann-Whitney test. t Representative images showing NSUN2 and TIGAR immunohistochemical staining in ESCC biopsy specimens taken before adjuvant radiochemotherapy in patients with differential sensitivity. Scale bars, 100 μm. u Box and bar plots comparing the NSUN2 protein levels in ESCC biopsy specimens with differential sensitivity to adjuvant radiochemotherapy. **P < 0.01 of Mann-Whitney test. v The schematic illustration for the possible mechanisms of aberrant NSUN2 expression and NSUN2-mediated radiochemo-resistance in ESCC. Part of the schematic illustration is generated from BioRender (https://biorender.com/).