TRIM21-mediated PRMT1 degradation attenuates colorectal cancer malignant progression

Abstract

Tripartite motif-containing 21 (TRIM21) plays a crucial role in antiviral responses and autoimmune diseases. While the impact of TRIM21 on cancer has been studied in various tumors, its role in colorectal cancer (CRC) remains unclear. In this study, we found that TRIM21 expression is reduced in primary CRC tissues. Low levels of TRIM21 in CRC are associated with unfavorable clinicopathological characteristics and shorter survival. Furthermore, we demonstrate that TRIM21 suppresses the proliferation, tumorigenesis, migration, and metastasis of CRC cells by promoting the ubiquitination-mediated degradation of PRMT1. These findings suggest that TRIM21 holds potential as a valuable predictive biomarker for assessing the prognosis of CRC patients.

Fig. 1. TRIM21 expression is decreased and correlates with poor survival in CRC patients.

A Protein expression level of TRIM21 in normal colon tissues and colon cancer tissues. Data were obtained from CPTAC database. B Representative immunohistochemistry images of TRIM21 protein expression in 427 paired adjacent non-cancerous tissue and CRC tissues. C, D Staining intensities (C) and immunohistochemistry score (D) of TRIM12 in CRC tissues compared with paired adjacent non-cancerous tissue. (N, paired adjacent non-cancerous tissues. T, tumor tissues). E, F The Kaplan–Meier survival curves of overall survival (E) and disease-specific survival (F) in patients with CRC stratified by Trim21 protein expression levels in CRC tissues. Data were expressed as the mean ± SD (**P < 0.01, *** P < 0.001).

Fig. 2. TRIM21 inhibits the proliferation of CRC cells in vitro.

A–D CCK-8 and colony formation assays were used to assess the effect of TRIM21 knockdown (A, B) or overexpression (C, D) on cell proliferation in HCT-116 and LoVo cell lines. E, F The impact of TRIM21 knockdown (E) or overexpression (F) on the major cyclins were detected by western blot analysis. Data are represented as mean ± SD of three independent experiments, and ***p < 0.001 (Student’s t-test).

Fig. 3. TRIM21 restricts the motility of CRC cells in vitro.

A Transwell migration assays and Matrigel invasion assays were used to detect the migration and invasion ability of CRC cells with TRIM21 deficiency (A, B) or TRIM21 overexpression (C, D). E, F Western blot analysis of EMT markers in TRIM21 knockdown (E) or TRIM21 overexpression CRC cells (F). Data are represented as mean ± SD of three independent experiments, and **p < 0.01, ***p < 0.001 (Student’s t-test).

Fig. 4. TRIM21 decreases the protein stability of PRMT1 in CRC cells.

A, B The mutual interaction between TRIM21 and PRMT1 were detected by Co-IP. C, D Western blot analysis was used to detect PRMT1 protein expression in TRIM21 deficiency (C) or TRIM21 overexpression (D) cells. E, F qPCR analysis was used to detect PRMT1 mRNA expression in TRIM21 deficiency (E) or TRIM21 overexpression (F) cells. G Effect of TRIM21 on PRMT1 protein half-life in HCT-116 cells overexpressing TRIM21 and treated with CHX. Data are represented as mean ± SD of three independent experiments, and ns (no significance), **p < 0.01 (Student’s t-test).

Fig. 5. TRIM21mediates K48-linked ubiquitination degradation of PRMT1 in CRC cells.

A TRIM21 mediated ubiquitination degradation of PRMT1 in HCT-116 and LoVo cells. B Western blot analysis showing the effect of the proteasome inhibitor MG132 (10 μM for 6 h) treatment on PRMT1 protein accumulation in HCT-116 and LoVo cells. The relative intensity of PRMT1 proteins were quantified by software Image J. C Schematic representation of TRIM21 truncated mutations. D HCT-116 cells were transfected with wild-type TRIM21 or the indicated mutants to detected the potential domain that interacted with PRMT1. E K48-only and K63-only ubiquitin plasmids alone or co-transfected with TRIM21-HA plasmid into 293 T cells for 48 h, and the cells were then harvested and subjected to myc IP.

Fig. 6. PRMT1 is required for TRIM21-mediated CRC progression in vitro.

A Western blot was used to detect the overexpression efficiency of TRIM21 and PRMT1 in HCT-116 cells. The relative intensity of PRMT1 proteins were quantified by software Image J. B, C Clone formation assays (B) and CCK-8 assays (C) were used to assess the effect of TRIM21 overexpression, PRMT1 overexpression or both on HCT-116 cell proliferation. D Detection of p16 and p21 expression in TRIM21 overexpression HCT116 cells after over-expressing PRMT1 or not. E, F Representative images (E) and statistical results (F) of transwell migration assays and Matrigel invasion assays to assess the migration and invasion ability of HCT-116 cells with TRIM21 overexpression, PRMT1 overexpression or both.Data are represented as mean ± SD of three independent experiments, and *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Fig. 7. TRIM21 affects the CRC progression in a PRMT11-dependent manner in vivo.

A, B Tumor tissue images (A), tumor volume statistical curves (B) of HCT116 tumor-bearing mice in indicated groups (n = 6 for each group). C IHC staining was performed on the subcutaneous tumor tissue to analyze the expression of TRIM21, PRMT1, and Ki-67. D Lung metastatic nodules were examined macroscopically after nude mice were sacrificed. The red arrows denote the metastatic nodules. E Bouin staining and HE staining of lung metastases after tail vein injection of BALB/C nude mice (Left panel). statistical analysis of lung nodule numbers (Right panel). Data were expressed as the mean ± SD (*P < 0.05, **P < 0.01, *** P < 0.001). F A proposed working model for this study. TRIM21-mediated PRMT1 K48-linked ubiquitination decreases PRMT1 expression, which suppresses the proliferation, tumorigenesis, migration, and metastasis of CRC cells.