Journal: Advanced Science

Article Title: NSUN3‐Associated m5C Modification of tRF5‐GlyTCC Suppresses Colorectal Cancer Progression via RPL23‐Linked Nucleolar Stress and p53‐Associated Autophagy and Enhances 5‐FU Sensitivity

doi: 10.1002/advs.78345

Figure Lengend Snippet: Effects of tRF5‐GlyTCC on tumor growth and response to 5‐FU. (A–B) CCK‐8‐based 5‐FU dose–response assays in HCT116 and DLD1 cells transfected with NC mimic or tRF5‐GlyTCC mimic and treated with increasing concentrations of 5‐FU in the presence or absence of chloroquine (CQ). (C) CCK‐8‐based 5‐FU dose‐response assay in SW1116 cells transfected with NC mimic or tRF5‐GlyTCC mimic. (D–G) Tumor growth curves and tumor weights in HCT116 xenograft models treated with agomir NC + PBS, tRF5‐GlyTCC agomir + PBS, agomir NC + 5‐FU, or tRF5‐GlyTCC agomir + 5‐FU. (H) Representative H&E staining and immunohistochemical staining for Ki‐67, PCNA, p53, and RPL23 in tumor tissue sections from each treatment group. Ki‐67 and PCNA show nuclear localization, with changes consistent with differences in tumor cell proliferation among the treatment groups. (I) RT‐qPCR analysis of tRF5‐GlyTCC abundance in xenograft tumor tissues following agomir treatment. Data are presented as mean ± SD. Statistical significance for the 5‐FU dose–response and CQ experiments was determined using two‐way ANOVA followed by Šídák's multiple‐comparisons test. P < 0.05, * P < 0.01, ** P < 0.001, *** P < 0.0001.

Article Snippet: Antibodies against GAPDH, RPL23, and p53 were obtained from Proteintech (China).

Techniques: CCK-8 Assay, Transfection, Xenograft Assay, Staining, Immunohistochemical staining, Tissue, Quantitative RT-PCR, Analysis

Journal: Advanced Science

Article Title: NSUN3‐Associated m5C Modification of tRF5‐GlyTCC Suppresses Colorectal Cancer Progression via RPL23‐Linked Nucleolar Stress and p53‐Associated Autophagy and Enhances 5‐FU Sensitivity

doi: 10.1002/advs.78345

Figure Lengend Snippet: Identification of RPL23 as a downstream target of tRF5‐GlyTCC. (A–B) Schematic overview of the transcriptomic analysis performed in tRF5‐GlyTCC‐overexpressing cells. (C) Venn diagram showing the overlap of candidate target genes identified by integrated bioinformatic analyses. (D) RT‐qPCR analysis of candidate genes following tRF5‐GlyTCC overexpression. (E) Schematic representation of the wild‐type (WT) and mutant (MUT) RPL23 3′UTR luciferase reporter constructs. (F–G) Dual‐luciferase reporter assays in SW1116 and HEK‐293T cells co‐transfected with tRF5‐GlyTCC mimic and WT or MUT RPL23 3′UTR reporters. (H) Western blotting analysis of RPL23 protein levels following tRF5‐GlyTCC overexpression or inhibition. (I) RPL23 expression levels in CRC tissues and matched adjacent non‐tumor tissues. (J) Pearson correlation analysis of tRF5‐GlyTCC and RPL23 expression in 97 paired CRC tissue samples. (K) Pathway enrichment analysis associated with RPL23 expression. P < 0.05, * P < 0.01, ** P < 0.001, *** P < 0.0001.

Article Snippet: Antibodies against GAPDH, RPL23, and p53 were obtained from Proteintech (China).

Techniques: Analysis, Quantitative RT-PCR, Over Expression, Mutagenesis, Luciferase, Construct, Transfection, Western Blot, Inhibition, Expressing, Tissue, Gene Expression

Journal: Advanced Science

Article Title: NSUN3‐Associated m5C Modification of tRF5‐GlyTCC Suppresses Colorectal Cancer Progression via RPL23‐Linked Nucleolar Stress and p53‐Associated Autophagy and Enhances 5‐FU Sensitivity

doi: 10.1002/advs.78345

Figure Lengend Snippet: Regulation of p53 signaling and autophagy‐associated responses by tRF5‐GlyTCC. (A–B) Gene set enrichment analysis (GSEA) showing enrichment of p53 signaling and autophagy‐related pathways in tRF5‐GlyTCC‐overexpressing cells. (C) Western blotting analysis of p53 and autophagy‐associated proteins following modulation of tRF5‐GlyTCC expression. (D) Representative Western blotting analysis of LC3‐II, p62, RPL23, and p53 in DLD1 and HCT116 cells transfected with a negative control (NC) mimic or tRF5‐GlyTCC mimic (tRF5‐M), with or without chloroquine (CQ) treatment. (E) Quantification of LC3‐II and p62 protein levels normalized to GAPDH. Data are presented as mean ± SD from independent experiments. Statistical significance was determined using one‐way ANOVA followed by Šídák's multiple‐comparisons test. (F) Representative transmission electron microscopy images showing autophagosome formation in CRC cells following gain‐ or loss‐of‐function of tRF5‐GlyTCC. (G–H) Western blotting analysis of p53 protein levels following RPL23 silencing. P < 0.05, * P < 0.01, ** P < 0.001.

Article Snippet: Antibodies against GAPDH, RPL23, and p53 were obtained from Proteintech (China).

Techniques: Analysis, Western Blot, Expressing, Transfection, Negative Control, Transmission Assay, Electron Microscopy

Journal: Advanced Science

Article Title: NSUN3‐Associated m5C Modification of tRF5‐GlyTCC Suppresses Colorectal Cancer Progression via RPL23‐Linked Nucleolar Stress and p53‐Associated Autophagy and Enhances 5‐FU Sensitivity

doi: 10.1002/advs.78345

Figure Lengend Snippet: Further mechanistic insights into RPL23‐associated nucleolar stress and p53 signaling in CRC cells. (A)Immunofluorescence analysis of nucleophosmin (NPM1) in DLD1 and HCT116 cells transfected with NC, tRF5‐GlyTCC inhibitor, siRPL23, or siRPL23 + tRF5‐GlyTCC inhibitor. Scale bars: 10 µm. (B) Western blotting analysis of RPL23 and p53 in DLD1 and HCT116 cells following the indicated genetic manipulations. (C) SUnSET assay assessing global protein synthesis in DLD1 cells transfected with tRF5‐GlyTCC mimic or NC mimic. Puromycin incorporation indicates nascent protein synthesis. (D) Representative images of p53‐GFP reporter fluorescence in DLD1 cells following tRF5‐GlyTCC overexpression. (E–F) Quantification of puromycin incorporation (E) and p53‐GFP fluorescence intensity (F). (G–H) Western blotting analysis of RPL23, p53, LC3II, and p62/SQSTM1 expression in HCT116 cells following tRF5‐GlyTCC overexpression (G) or RPL23 knockdown (H). (I) Quantification of RPL23 and p53 protein levels shown in (B). Data are presented as mean ± SEM. ns, not significant; P < 0.05, * P < 0.01, ** P < 0.001.

Article Snippet: Antibodies against GAPDH, RPL23, and p53 were obtained from Proteintech (China).

Techniques: Immunofluorescence, Analysis, Transfection, Western Blot, Fluorescence, Over Expression, Expressing, Knockdown

Journal: Advanced Science

Article Title: NSUN3‐Associated m5C Modification of tRF5‐GlyTCC Suppresses Colorectal Cancer Progression via RPL23‐Linked Nucleolar Stress and p53‐Associated Autophagy and Enhances 5‐FU Sensitivity

doi: 10.1002/advs.78345

Figure Lengend Snippet: NSUN3‐associated m5C modification contributes to the stability of tRF5‐GlyTCC. (A–B) Prediction and MeRIP‐qPCR validation of m5C modification in the annotated precursor tRNA‐Gly‐TCC region encompassing the predicted C66 site. (C–D) NSUN3 expression in CRC tissues ( n = 97) and CRC cell lines. (E) Knockdown efficiency of NSUN3 siRNA in CRC cells. (F) Effects of NSUN3 silencing on tRF5‐GlyTCC expression. (G) Effects of NSUN3 silencing on the abundance of the annotated precursor tRNA‐Gly‐TCC. (H) MeRIP‐qPCR analysis of m5C enrichment in the precursor tRNA‐Gly‐TCC region encompassing C66 following NSUN3 knockdown. (I) Pearson correlation analysis between NSUN3 and tRF5‐GlyTCC expression in 97 paired CRC tissue samples ( n = 97). (J–K) Actinomycin D chase assays showing tRF5‐GlyTCC decay in DLD1 and HCT116 cells following NSUN3 knockdown. tRF5‐GlyTCC levels were measured by RT‐qPCR at 0, 2, 4, and 8 h after Actinomycin D treatment and normalized to the corresponding 0 h level. Decay curves were fitted by nonlinear regression, and statistical significance was determined using two‐way ANOVA followed by Šídák's multiple‐comparisons test. (L–M) Kaplan–Meier survival analysis of the prognostic relevance of NSUN3 stratified by TP53 mutation status using the Kaplan–Meier Plotter platform. (L) TP53‐mutant; (M) TP53‐wild‐type. (N) Western blotting analysis showing that NSUN3 knockdown increases RPL23 expression and is accompanied by decreased p53 and LC3II levels and increased p62/SQSTM1 levels. (O) Gene set enrichment analysis (GSEA) showing positive associations between NSUN3 expression and p53‐related signaling pathways. P < 0.05, * P < 0.01, ** P < 0.001, *** P < 0.0001.

Article Snippet: Antibodies against GAPDH, RPL23, and p53 were obtained from Proteintech (China).

Techniques: Modification, Stability, Biomarker Discovery, Expressing, Knockdown, Analysis, Tissue, Quantitative RT-PCR, Mutagenesis, Western Blot, Protein-Protein interactions, Gene Expression

Journal: Advanced Science

Article Title: NSUN3‐Associated m5C Modification of tRF5‐GlyTCC Suppresses Colorectal Cancer Progression via RPL23‐Linked Nucleolar Stress and p53‐Associated Autophagy and Enhances 5‐FU Sensitivity

doi: 10.1002/advs.78345

Figure Lengend Snippet: Proposed working model of NSUN3‐associated regulation of tRF5‐GlyTCC in colorectal cancer. The proposed model summarizes the relationships supported by the present study. NSUN3‐associated m5C modification in the annotated precursor tRNA‐Gly‐TCC region, encompassing C66, is associated with maintenance of tRF5‐GlyTCC stability. Increased tRF5‐GlyTCC expression is associated with reduced RPL23 expression, which is accompanied by NPM1 redistribution and a nucleolar stress phenotype, together with increased p53 signaling and autophagy‐associated responses. These changes are associated with tumor‐suppressive effects and enhanced sensitivity to 5‐fluorouracil (5‐FU). The model is intended to represent the experimentally supported relationships and does not imply a strictly linear causal pathway.

Article Snippet: Antibodies against GAPDH, RPL23, and p53 were obtained from Proteintech (China).

Techniques: Modification, Stability, Expressing, Sensitivity