Journal: Cell Death & Disease

Article Title: TRIM31 triggers colorectal carcinogenesis and progression by maintaining YBX1 protein stability through ubiquitination modification

doi: 10.1038/s41419-025-07922-4

Figure Lengend Snippet: A The distribution of 85 tripartite motif containing (TRIM) gene family protein-coding genes among all chromosomes. B Heatmap analysis of differential expression patterns of the TRIM protein family in colorectal tumor tissue (orange) and adjacent normal tissue (blue) in two independent datasets ( GSE134525 [left] and GSE103512 [right]). C Box plot depicting TRIM31 expression (log2 TPM values) across four tissue cohorts. D Activation of TRIM31 were associated with shorter OS in TCGA-COAD, and worse OS/DFS in Rectal_MSK2022 cohort. E Demonstration of TRIM31 immunohistochemical staining differences between normal colon tissue and three colorectal tumors (Tumor-1/2/3) at 10X/20X magnification.Scale bar, 100 µm. F The histogram showed that tumor tissue had significantly higher TRIM31 IHC scores than normal tissue, n = 24 per group. G Survival curves confirmed that patients in the TRIM31 high expression group ( n = 57) had a lower overall survival rate than those in the low expression group ( n = 19). TRIM31 expression in normal colorectal cells (NCM460) and colorectal cancer cells, assessed by qRT-PCR ( H ) and Western Blot ( I ), n = 3 per group. ** P < 0.01; *** p < 0.001.

Article Snippet: Sections were blocked with 5% BSA and incubated overnight at 4 °C with a primary antibody against TRIM31 (Proteintech, UK) at a 1:200 dilution.

Techniques: Quantitative Proteomics, Expressing, Activation Assay, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: TRIM31 triggers colorectal carcinogenesis and progression by maintaining YBX1 protein stability through ubiquitination modification

doi: 10.1038/s41419-025-07922-4

Figure Lengend Snippet: A qRT-PCR experiments were performed to detect the efficiency of siRNA to knock down TRIM31 in CRC cells. n = 3 per group. B Western Blot was performed to detect TRIM31 protein levels after siRNA treatment of DLD-1, HT-29, LOVO cells and after overexpression of TRIM31 in SW480 cells. n = 3 per group. C Cell viability was determined by CCK-8 assay after knockdown of TRIM31 in HT-29, DLD-1, LOVO cells. n = 3 per group. D Clone formation assay after knockdown of TRIM31 in HT-29, DLD-1, LOVO cells. n = 3 per group. E Cell viability was determined by CCK-8 assay after overexpression of TRIM31 in SW480 cells. n = 3 per group. F Clone formation assay after overexpression of TRIM31 in SW480 cells. n = 3 per group. CDX experiments were performed after knockdown of TRIM31 in HT-29 cells, subcutaneous tumors were removed after surgery ( G ) and weighed ( I ), and tumor volumes were measured every 2 days ( H ) n = 5 per group. CDX experiments were performed after overexpression of TRIM31 in SW480 cells, subcutaneous tumors were removed after surgery ( J ) and weighed ( L ), and tumor volume was measured every 2 days ( K ) n = 5 per group.

Article Snippet: Sections were blocked with 5% BSA and incubated overnight at 4 °C with a primary antibody against TRIM31 (Proteintech, UK) at a 1:200 dilution.

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Over Expression, CCK-8 Assay, Tube Formation Assay

Journal: Cell Death & Disease

Article Title: TRIM31 triggers colorectal carcinogenesis and progression by maintaining YBX1 protein stability through ubiquitination modification

doi: 10.1038/s41419-025-07922-4

Figure Lengend Snippet: A Scratch assay to detect the migration ability of cells after knockdown of TRIM31 in DLD1, HT-29, LOVO cells. n = 3 per group. Scale bar, 200 µm. B Transwell assay to detect the migration ability of cells after knockdown of TRIM31 in DLD1, HT-29, LOVO cells. n = 3 per group. Scale bar, 200 µm. C Scratch assay to detect the migration ability of cells after overexpression of TRIM31 in SW480 cells. n = 3 per group. D Transwell assay to detect cell migration after overexpression of TRIM31 in SW480 cells. n = 3 per group. Scale bar, 200 µm. E Tail Vein Injection for Lung Metastasis Model to study the metastatic ability of tumor cells in mice after knockdown of TRIM31. n = 5 per group. F H&E staining of tissues in ( E ). Scale bar, 20 µm. G The number of metastatic foci was counted in ( E ). H Tail Vein Injection for Lung Metastasis Model to study the metastatic ability of tumor cells in mice after overexpression of TRIM31. n = 5 per group. I H&E staining of tissues in ( H ). Scale bar, 20 µm. J The number of metastatic foci was counted in ( H ). * P < 0.05; ** P < 0.01; *** p < 0.001.

Article Snippet: Sections were blocked with 5% BSA and incubated overnight at 4 °C with a primary antibody against TRIM31 (Proteintech, UK) at a 1:200 dilution.

Techniques: Wound Healing Assay, Migration, Knockdown, Transwell Assay, Over Expression, Injection, Staining

Journal: Cell Death & Disease

Article Title: TRIM31 triggers colorectal carcinogenesis and progression by maintaining YBX1 protein stability through ubiquitination modification

doi: 10.1038/s41419-025-07922-4

Figure Lengend Snippet: A An Immunoprecipitation in HT-29 cells using TRIM31 antibody followed by Caulmers Brilliant Blue staining. B Immunoprecipitation assay to verify the interaction between YBX1 and TRIM31. n = 3 per group. C Western Blot analysis of changes in YBX1 ‌protein‌ expression levels, ‌combined with qRT-PCR analysis of YBX1 mRNA levels‌, was performed after knockdown or overexpression of TRIM31 in CRC cells. n = 3 per group. D CHX digestion assay to examine the effect of knockdown or overexpression of TRIM31 on YBX1 stability. n = 3 per group. E MG132 treatment to examine the effect of knockdown or overexpression of TRIM31 on YBX1 ubiquitination levels. n = 3 per group. F Immunoprecipitation and Western Blot to detect TRIM31-mediated ubiquitination of YBX1 species. n = 3 per group. G The ubiquitination level of each lysine of TRIM31 was detected after mutation and overexpression of each lysine to confirm the specific site of ubiquitination of TRIM31. n = 3 per group.

Article Snippet: Sections were blocked with 5% BSA and incubated overnight at 4 °C with a primary antibody against TRIM31 (Proteintech, UK) at a 1:200 dilution.

Techniques: Immunoprecipitation, Staining, Western Blot, Expressing, Quantitative RT-PCR, Knockdown, Over Expression, Ubiquitin Proteomics, Mutagenesis

Journal: Cell Death & Disease

Article Title: TRIM31 triggers colorectal carcinogenesis and progression by maintaining YBX1 protein stability through ubiquitination modification

doi: 10.1038/s41419-025-07922-4

Figure Lengend Snippet: A Cell viability was determined by CCK-8 assay after knockdown of YBX1 in HT-29 and DLD-1 cells. n = 3 per group. B Clone formation assay after knockdown of YBX1 in HT-29 and DLD-1 cells. n = 3 per group. C Transwell assay to detect the migration ability of cells after knockdown of TRIM31 in DLD-1 cells. n = 3 per group. Scale bar, 200 µm. D Overexpression of YBX1 restored the decreased levels of TRIM31 in HT-29 cells caused by TRIM31 knockdown. n = 3 per group. Overexpression of YBX1 restored the reduced proliferation ( E ) and metastatic ( F ) capacity of HT-29 cells caused by TRIM31 knockdown. n = 3 per group. Scale bar, 200 µm. ns not significant, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Sections were blocked with 5% BSA and incubated overnight at 4 °C with a primary antibody against TRIM31 (Proteintech, UK) at a 1:200 dilution.

Techniques: CCK-8 Assay, Knockdown, Tube Formation Assay, Transwell Assay, Migration, Over Expression

Journal: Cell Death & Disease

Article Title: TRIM31 triggers colorectal carcinogenesis and progression by maintaining YBX1 protein stability through ubiquitination modification

doi: 10.1038/s41419-025-07922-4

Figure Lengend Snippet: A The volcano plot displays the outcomes of RNA-seq analysis for genes that exhibit differential expression between KO-YBX1 and KO-NC CRC cells. B KEGG analysis identifies signaling pathways significantly affected by YBX1 knockdown in CRC cells. C Pie chart m5C peaks for percentage of YBX1 high confidence target genes. D A total of 132 genes were detected in the intersection of YBX1 post-KD down-regulation and gene fetching detected in YBX1 LACE-seq. E Changes in the expression levels of nine genes including FGF9 after knockdown of YBX1 in HT-29 and DLD-1 detected by qRT-PCR. n = 3 per group. F RIP assay to detect the enrichment of EREG, MAFG, GAS6 RNA on YBX1 protein. n = 3 per group. G Stability of EREG, MAFG and GAS6 detection after knockdown of YBX1 or TRIM31. n = 3 per group. H Schematic representation of m5C modification sites and sequencing in EREG mRNAs. Detection of EREG mRNA levels ( I ) and m5C modification levels ( J ) after knockdown of NSUN2. n = 3 per group. K , L IHC staining of TRIM31, YBX1, and EREG in colorectal cancer tissues from 24 patients ( K ). Scale bar, 20 µm. Pearson’s correlation analysis was performed to assess the association between TRIM31 expression and the expression levels of YBX1 and EREG, respectively ( L ). n = 24 per group. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Sections were blocked with 5% BSA and incubated overnight at 4 °C with a primary antibody against TRIM31 (Proteintech, UK) at a 1:200 dilution.

Techniques: RNA Sequencing, Quantitative Proteomics, Protein-Protein interactions, Knockdown, Expressing, Quantitative RT-PCR, Modification, Sequencing, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: TRIM31 triggers colorectal carcinogenesis and progression by maintaining YBX1 protein stability through ubiquitination modification

doi: 10.1038/s41419-025-07922-4

Figure Lengend Snippet: A Schematic showing potential transcription factor binding sites for TRIM31 analyzed by JASPAR. Protein levels of TRIM31 in HT-29 and DLD-1 cells after Betulinic Acid treatment in relation to Betulinic Acid dose ( B ) and treatment time ( C ). n = 3 per group. Changes in TRIM31 mRNA levels ( D ) and promoter activity ( E ) in HT-29 and DLD-1 cells after 8H treatment with Betulinic Acid. n = 3 per group. F Changes in protein levels of TRIM31 detected after different times of IL-1β (10 ng/mL) treatment. Changes in TRIM31 mRNA levels ( G ) and promoter activity ( H ) of TRIM31 in HT-29 and DLD-1 cells after IL-1β (10 ng/mL) treatment for 8H. n = 3 per group. I CHIP experiments show major regions of P65 binding to the TRIM31 promoter. J CHIP experiments show the effect of Betulinic Acid treatment on the binding of P65 to the TRIM31 promoter. n = 3 per group. Western Blot ( K ) and IHC ( L ) analysis of TRIM31 expression levels in normal intestine, intestinal polyps, intestinal ulcers and intestinal tumors. n = 3 per group. * P < 0.05; ** P < 0.01; *** P < 0.001. Scale bar, 10 µm.

Article Snippet: Sections were blocked with 5% BSA and incubated overnight at 4 °C with a primary antibody against TRIM31 (Proteintech, UK) at a 1:200 dilution.

Techniques: Binding Assay, Activity Assay, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: TRIM31 triggers colorectal carcinogenesis and progression by maintaining YBX1 protein stability through ubiquitination modification

doi: 10.1038/s41419-025-07922-4

Figure Lengend Snippet: TRIM31 directly interacts with YBX1 and catalyzes K63-linked polyubiquitination at YBX1 Lys81, stabilizing the YBX1 protein. Stabilized YBX1 enhances the mRNA stability of oncogenic targets (EREG, GAS6, MAFG) via both m5C-dependent and -independent recognition mechanisms. Furthermore, NF-κB activation promotes P65 binding to the TRIM31 promoter, increasing TRIM31 transcription. TRIM31, in turn, facilitates P65 nuclear translocation, establishing a TRIM31-NF-κB positive feedback loop. This loop perpetuates inflammation, promotes the inflammation-cancer transformation, and fuels colorectal carcinogenesis. TRIM31 thus acts as a central oncogenic driver linking ubiquitination, RNA stabilization, and inflammatory signaling in CRC. This schematic diagram was created using BioRender.com.

Article Snippet: Sections were blocked with 5% BSA and incubated overnight at 4 °C with a primary antibody against TRIM31 (Proteintech, UK) at a 1:200 dilution.

Techniques: Activation Assay, Binding Assay, Translocation Assay, Transformation Assay, Ubiquitin Proteomics