PRMT1-mediated modification of H4R3me2a promotes liver cancer progression by enhancing the transcriptional activity of SOX18

Abstract

Background: HCC is one of the most prevalent and deadliest malignancies worldwide, with a poor prognosis. Altered histone modifications have been shown to play a significant role in HCC. However, the biological roles and clinical relevance of specific histone modifications, such as the asymmetric dimethylation on arginine 3 of histone H4 (H4R3me2a), remain poorly understood in HCC.

Methods: In this study, immunohistochemical staining was performed to assess histone H4R3me2a modification in 32 pairs of HCC tissues and corresponding adjacent nontumor liver tissues. Cellular-level experiments and subcutaneous xenograft models in nude mice were used to investigate the effects of silencing protein arginine methyltransferase 1 (PRMT1) with shRNA or pharmacologically blocking PRMT1 activity on HCC cell proliferation, migration, and invasion. RNA-seq analysis combined with Chip-qPCR validation was employed to explore the regulatory mechanism of PRMT1 on SOX18 expression. The downstream target of SOX18 was identified using the JASPAR database and a dual-luciferase reporter system.

Results: The level of histone H4R3me2a modification was significantly elevated in HCC tissues and closely associated with poor prognosis in patients with HCC. Silencing PRMT1 or pharmacologically inhibiting its activity effectively suppressed the proliferation, migration, and invasion of HCC cells. Mechanistically, PRMT1 was found to regulate SOX18 expression by modulating histone H4R3me2a modification in the SOX18 promoter region. LOXL1 was identified as a downstream target of the transcription factor SOX18.

Conclusions: This study revealed the clinical relevance of histone H4R3me2a modification in HCC and demonstrated that PRMT1 promotes malignant behavior in HCC cells by modulating H4R3me2a modification in the SOX18 promoter region. The findings elucidate the role and molecular mechanism of PRMT1-mediated histone H4R3me2a modification in HCC progression and highlight the potential clinical applications of PRMT1 inhibitors. These results may provide new insights into the treatment of HCC.

FIGURE 1

Upregulation of H4R3me2a and prognostic significance in HCC. (A) Representative images of immunohistochemical analysis for H4R3me2a from tumor tissues and adjacent nontumor normal tissues of 32 paired samples from patients with HCC. (B, C) Quantified H-scores of H4R3me2a and PRMT1 in the tumor tissues and adjacent nontumor normal tissues of these 32 paired HCC samples. The p value is calculated by paired 2-sided t test. (D) Representative images of immunohistochemistry for H4R3me2a and PRMT1 from the tumor tissues of these 32 paired HCC samples. (E) Correlation between H4R3me2a and PRMT1 in HCC tissues. Pearson correlation coefficient, r = 0.4524, p = 0.0053. (F) Representative images for high or low levels of H4R3me2a in HCC tissues. (G, H) Kaplan-Meier analyses of overall survival rates and progression-free survival rates for patients with HCC were performed according to H4R3me2a H-scores. High and low levels of H4R3me2a were defined according to the median value of H-scores. The p value was calculated by the log-rank (Mantel-Cox) test. ***p < 0.001. Abbreviations: H4R3me2a, histone H4 at arginine 3; PRMT1, protein arginine methyltransferase 1.

FIGURE 2

PRMT1-mediated H4R3me2a promotes the progression of liver cancer. (A) Analysis for PRMT1 and H4R3me2a expression level in liver cancer cell lines. (B) PRMT1 knockdown reduced the levels of methylation mark H4R3me2a in HCC cells. (C) Long-term cell proliferation assay for HCC cells with PRMT1 knockdown. (D, E) Effects of HCC cells with PRMT1 knockdown on migration and invasion. (F, G) PRMT1 knockdown restrained tumor growth in the subcutaneous xenograft tumor model. Representative images of subcutaneous tumors (F); mice body weight, tumor weight, and tumor volume (G). (H, I) Immunohistochemical analysis for PRMT1 and Ki67 in tumor tissues from the subcutaneous xenograft tumors. (J) The plasmid of PRMT1 mutation (PRMT1-Mut), which mutated the arginine methylation active site (GSGTG, amino acids 86–90) of PRMT1 was employed to explore the effects of PRMT1 on the levels of methylation mark H4R3me2a. (K, L) Effects of HCC cells with enhanced H4R3me2a deposition on colony formation and migration. (M, N) Overexpression of PRMT1 effectively enhanced the H4R3me2a deposition and promoted tumor growth in the subcutaneous xenograft tumor model. Representative images of subcutaneous tumors (M); mouse weight, tumor weight, and tumor growth curve (N); (O, P) Immunohistochemical analysis for PRMT1 and Ki67 in tumor tissues from the subcutaneous xenograft tumors. n.s: no significance. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: H4R3me2a, histone H4 at arginine 3; PRMT1, protein arginine methyltransferase 1.

FIGURE 3

TC-E 5003, as a PRMT1 inhibitor, suppresses the HCC growth. (A) Long-term cell proliferation assays for TC-E 5003 (PRMT1 inhibitor) in liver cancer cell lines. Cells were treated with indicated concentrations of TC-E 5003. Long-term cell proliferation assays were measured by colony formation. (B, C) In short-term cell proliferation assays, cells were treated with indicated concentrations of TC-E 5003 for 3 days and detected by CCK8. IC₅₀ values of TC-E 5003 were determined. (D–G) TC-E 5003 restrained tumor growth in the subcutaneous xenograft tumor model. Representative images of subcutaneous tumors (D). Mice body weight and tumor volume (E). Tumor volume growth curve (F). Immunohistochemical analysis for Ki67 in tumor tissues from the subcutaneous xenograft tumors (G). n.s., no significance. ***p < 0.001. Abbreviations: H4R3me2a, histone H4 at arginine 3; IC₅₀, half maximal inhibitory concentration; PRMT1, protein arginine methyltransferase 1.

FIGURE 4

Enhanced H4R3me2a methylation at the promoter region of SOX18 stimulates HCC proliferation. (A, B) Volcano plots depicting differentially expressed genes identified by RNA-seq analysis in MHCC97H cells after knockdown of PRMT1 or treatment with PRMT1 inhibitor (TC-E 5003). The x-axis represented Log₂ (Fold change), and the y-axis represented −Log₁₀ (p value). Significant genes were defined as Log₂ (Fold change) ≥1 or ≤−1, and p < 0.05. (C) Heatmap displaying the overall clustering feature of the 4 groups (NC, shPRMT1, DMSO, and TC-E 5003) based on RNA-seq data. (D) GO enrichment analysis of significantly changed genes from the shPRMT1 versus NC RNA-seq data. (E) Dynamic Venn diagram identified 15 changed genes between the shPRMT1 versus NC and TC-E 5003 versus DMSO group based on RNA-seq data. (F) Heatmap showing the 9 changed genes with consistent trends and their expression differences among groups. (G, H) Downregulation of SOX18 expression was validated by western blotting and q-PCR in MHCC97H cells with PRMT1 knockdown. (I) Representative images of immunohistochemical analysis for H4R3me2a, PRMT1, and SOX18 in 32 paired patients with HCC. (J) Correlation between H4R3me2a and SOX18 normalized H-scores in HCC tissues. Pearson correlation coefficient, r = 0.4513, p < 0.0001. (K) Differential levels of H4R3me2a binding to the promoter region of SOX18 were analyzed by Chip-qPCR in MHCC97H cells treated with NC or shPRMT1. (L–N) Long-term cell proliferation assay and western blotting of PRMT1 and SOX18 in MHCC97H cells treated with NC or shPRMT1 and oe-SOX18. n.s., no significance. **p < 0.01, ***p < 0.001. Abbreviations: Chip, chromatin immunoprecipitation; GO, gene ontology; H4R3me2a, histone H4 at arginine 3; PRMT1, protein arginine methyltransferase 1; q-PCR, quantitative polymerase chain reaction.

FIGURE 5

PRMT1-H4R3me2a-SOX18 promotes liver cancer proliferation through the LOXL1/STAT3 signaling. (A) Correlation between SOX18 and LOXL1 mRNA levels in TCGA-LIHC HCC tissues. (B) Downregulation of LOXL1 expression was validated by q-PCR in MHCC97H cells with SOX18 knockdown. (C) Diagram of predicted transcription factor SOX18 binding sites to the LOXL1 promoter region. The activities of serially truncated LOXL1 promoter reporter vectors in the 293T cells cotransfected with PGMLV-CMV-SOX18. (D, E) Protein levels of PRMT1, SOX18, LOXL1, p-STAT3, and STAT3 in the indicated MHCC97H cells and Hep3B cells. (F) Long-term cell proliferation assay in indicated MHCC97H cells treated with DMSO or STAT3 inhibitor (WP1066). (G) Schematic diagram of this study. n.s., no significance. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: H4R3me2a, histone H4 at arginine 3; PRMT1, protein arginine methyltransferase 1; q-PCR, quantitative polymerase chain reaction.