Journal: Cell Death & Disease

Article Title: PRMT3 drives PD-L1-mediated immune escape through activating PDHK1-regulated glycolysis in hepatocellular carcinoma

doi: 10.1038/s41419-025-07482-7

Figure Lengend Snippet: A Schematic depiction of the HCC model induced by DEN-CCl 4 (upper). Representative images of livers derived from Prmt3 f/f and Prmt3 LKO mice at different time points (lower). Red arrows indicate tumor nodes. Scale bar, 1 cm. B Liver/body weight ratio in Prmt3 f/f and Prmt3 LKO mice at different time points. * P < 0.05. C Number and size of tumor nodules on the liver surface in both groups. * P < 0.05. D Tumor incidence scored at different time points in both groups. The number of mice recorded at different time points in B , C , and D was consistent with the number shown in D . E Statistical analysis of Ki67 and CD8 expression in tumor tissues of Prmt3 f/f and Prmt3 LKO mice at 26 weeks via immunohistochemistry. n = 7 in each group. * P < 0.05, ** P < 0.01. F Representative images of CD8, PD-1, and Granzyme B (GrB) detection via multiplex immunofluorescences in tumors of Prmt3 f/f and Prmt3 LKO mice. Green arrows indicate PD-1 + CD8 + cells and yellow arrows indicate GrB + CD8 + cells. Scale bar, 25 μm. The data are presented as mean ± SD, and the statistical tests were all two-tailed.

Article Snippet: Hepa1-6 cells with Prmt3 knockout were generated by Cyagen company (Shanghai, China) using CRISPR-Cas9 technology.

Techniques: Derivative Assay, Expressing, Immunohistochemistry, Multiplex Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PRMT3 drives PD-L1-mediated immune escape through activating PDHK1-regulated glycolysis in hepatocellular carcinoma

doi: 10.1038/s41419-025-07482-7

Figure Lengend Snippet: A Lactate levels, triglyceride (TG) levels, total cholesterol (TC) levels, and methionine levels were detected in tumors derived from DEN-CCL 4 -induced HCC. n = 6 in each group. ** P < 0.01, n.s, no significant difference. B Lactate levels were detected in subcutaneous Hepa1-6 tumors derived from nude mice and C57BL/6 mice. n = 5 or 4 in each group. Each point represents data from one individual mouse. * P < 0.05. C Cell extracts from Huh7 cells with PRMT3 overexpression were subjected to immunoprecipitation using an anti-PRMT3 antibody, followed by immunoblotting to detect PDHK1. D The binding of PRMT3 and PDHK1 in Huh7 cells transfected with HA-PRMT3 and FLAG-PDHK1 were verified by reciprocal immunoprecipitation assays. E The colocalization of PRMT3 and PDHK1 in Huh7 cells was measured by immunofluorescence assay. Scale bar, 20 μm. Pearson’s correlation coefficient (PCC) for evaluating the colocalization of PRMT3 and PDHK1 was analyzed using ImageJ software . The mean PCC R value obtained was 0.82 (R = 0.82). F Representative images of PLA performed in Huh7 cells using antibodies against PDHK1 and PRMT3 were shown. Scale bar, 20 μm. The green dots indicated PDHK1 interacting with PRMT3. G PDHK1 was immunoprecipitated from Huh7 cells treated as indicated, and ADMA levels were subsequently detected by immunoblot analysis. H Representative images of PLA performed in Huh7 cells with PRMT3 overexpression using antibodies against PDHK1 and ADMA were shown. Scale bar, 20 μm. The green dots, as indicated by the white arrows, represent PDHK1 that has undergone ADMA modification. I Wildtype PDHK1, R363K mutant, R368K mutant, R363/ R368K mutant, and their corresponding vector control were transfected into Huh7 cells with PRMT3 overexpression. Subsequently, these PDHK1 variants were immunoprecipitated using Anti-FLAG beads and subjected to immunoblot analysis to detect the level of ADMA. * indicated heavy chain. J The amino acid sequences containing the R363 and R368 sites in various species. K Wildtype PDHK1 and R363/R368K were transfected in Huh7 cells with or without PRMT3 overexpression for 48 hours. Subsequently, PDHK1 and R363/R368K mutations were immunoprecipitated with Anti-FLAG beads and subjected to immunoblot analysis to detect the p-PDHA level. L Wildtype PDHK1, R363/R368K, and their corresponding vector control were transfected in Huh7 cells with PRMT3 overexpression for 48 hours. Then the levels of PDHA, p-PDHA and lactate were detected. M Wildtype PDHK1, R363/R368K, and their corresponding vector control were transfected in Huh7 cells with PRMT3 overexpression for 12 hours. Subsequently, the cells were seeded into a 96-well plate at a density of 5000 cells per well to assess proliferation at the indicated time points. The data are presented as mean ± SD, and the statistical tests were all two-tailed.

Article Snippet: Hepa1-6 cells with Prmt3 knockout were generated by Cyagen company (Shanghai, China) using CRISPR-Cas9 technology.

Techniques: Derivative Assay, Over Expression, Immunoprecipitation, Western Blot, Binding Assay, Transfection, Immunofluorescence, Software, Modification, Mutagenesis, Plasmid Preparation, Control, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PRMT3 drives PD-L1-mediated immune escape through activating PDHK1-regulated glycolysis in hepatocellular carcinoma

doi: 10.1038/s41419-025-07482-7

Figure Lengend Snippet: A Immunoblot analysis of the PDHA and phosphorylated PDHA levels in Huh7 cells with indicated treatments. The proliferation capacity ( B ), and migration and invasion capacity ( C ) of the indicated cells were evaluated. Huh7 cells were infected with Lenti-PRMT3 or Lenti-Ctrl and then treated with JX06 (200 nM) or DMSO. The representative images of stained transwell chamber were shown at the right of Fig. 3C. Scale bar, 200 μm. ** P < 0.01, *** P < 0.001. The tumor growth curve ( D ), tumor images ( E ), and tumor weight ( F ) of Huh7 xenografts were shown. n = 5 in each group. Huh7 cells infected with Lenti-PRMT3 or Lenti-Ctrl were inoculated into nude mice. Tumor-bearing mice were then administered JX06 or vehicle via intraperitoneal injection every 2 days at a dose of 30 mg/kg. * P < 0.05, *** P < 0.001. G Immunoblot analysis of PRMT3, PDHK1, and p-PDHA (phosphorylated PDHA) levels in Huh7 xenografts derived from nude mice. H Lactate production was measured in Huh7 xenografts obtained from nude mice. n = 5 in each group. * P < 0.05, ** P < 0.01. The data are presented as mean ± SD, and the statistical tests were all two-tailed.

Article Snippet: Hepa1-6 cells with Prmt3 knockout were generated by Cyagen company (Shanghai, China) using CRISPR-Cas9 technology.

Techniques: Western Blot, Migration, Infection, Staining, Injection, Derivative Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PRMT3 drives PD-L1-mediated immune escape through activating PDHK1-regulated glycolysis in hepatocellular carcinoma

doi: 10.1038/s41419-025-07482-7

Figure Lengend Snippet: A KEGG analysis revealed the top 30 signaling pathways by utilizing RNA-seq data obtained from tumor tissues of Prmt3 f/f and Prmt3 LKO mice. n = 3 in each group for RNA-seq analysis. Differentially expressed genes were selected using a significance q value ( q < 0.05), with a fold-change cut-off of 2 for upregulation and 0.5 for downregulation. B GSEA was performed for PD-1 Signaling and Co-stimulation by the CD28 family pathway using RNA-seq data. C A heatmap of tumor-associated immune checkpoint molecules expression profile based on GSEA. D Tumor-associated immune checkpoint molecules were detected by RT-PCR in tumor tissues of Prmt3 f/f and Prmt3 LKO mice. n = 7 in each group. ** P < 0.01, *** P < 0.001, n.s, no significant difference. E Expression levels of Pdl1 were examined by immunoblotting in tumor tissues of Prmt3 f/f and Prmt3 LKO mice. F Expression levels of Prmt3 and Pdl1 determined using immunohistochemical staining. Scale bars, 50 μm. Right panel: Semi-quantitative analysis of Pdl1 positive staining in tumors. n = 7 in each group. ** P < 0.01. G Expression of PD-L1 in Huh7 xenografts derived from nude mice treated with SGC707 was measured by RT-PCR (left) and immunoblotting (right). n = 7 in each group. ** P < 0.01. H Expression of Pdl1 in Hepa1-6 cells with Prmt3 deletion was detected by RT-PCR (left) and immunoblotting (right). ** P < 0.01. I Expression of Pdl1 in Hepa1-6 cells with Prmt3 overexpression were detected by RT-PCR (left) and immunoblotting (right). * P < 0.05, ** P < 0.01, *** P < 0.001. J Expression of PD-L1 in Huh7 cells with PRMT3 overexpression was detected by RT-PCR (left) and immunoblot analysis (right). * P < 0.05, ** P < 0.01. The data are presented as mean ± SD, and the statistical tests were all two-tailed.

Article Snippet: Hepa1-6 cells with Prmt3 knockout were generated by Cyagen company (Shanghai, China) using CRISPR-Cas9 technology.

Techniques: Protein-Protein interactions, RNA Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Staining, Derivative Assay, Over Expression, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PRMT3 drives PD-L1-mediated immune escape through activating PDHK1-regulated glycolysis in hepatocellular carcinoma

doi: 10.1038/s41419-025-07482-7

Figure Lengend Snippet: A, B PD-L1 mRNA levels were measured in Huh7 and Hepa1-6 cells with the indicated treatments. Hepatoma cells were infected with Lenti-PRMT3 or Lenti-Ctrl and then treated with JX06 (200 nM) or DMSO. * P < 0.05, ** P < 0.01. C PD-L1 mRNA and protein levels were detected in Huh7 xenografts derived from nude mice with JX06 treatment. n = 5 in each group. * P < 0.05. D PD-L1 expression in Huh7 cells was detected by RT-PCR after 24 hours of lactate stimulation at indicated concentrations. * P < 0.05, ** P < 0.01. E PD-L1, pan-Kla, and H3K18la protein levels were detected by immunoblotting in Huh7 cells with lactate stimulation (left) or with PRMT3 overexpression (right). F Protein levels of PD-L1 and H3K18la were detected in Huh7 and Hepa1-6 cells with the indicated treatments. Briefly, Huh7 or Hepa1-6 cells pre-infected with lenti-PRMT3 or a control lentivirus (Ctrl) were seeded into 6-well plates at a concentration of 4 × 10 5 cells per well for overnight culture. Then, these cells were treated with DMSO or JX06 (200 nM) for 24 hours to analyze protein levels by immunoblot. G Schematic diagram showing the primer site of the PD-L1 promoter for chromatin immunoprecipitation assay (upper). Huh7 cells were treated with indicated lactate concentration for 24 hours and subsequently subject to a chromatin immunoprecipitation assay with H3K18la antibody. ChIP assay revealed that lactate increased H3K18la occupancy at the PD-L1 promoter. Immunoblot analysis confirmed the immunoprecipitation of H3K18la. * P < 0.05, ** P < 0.01, *** P < 0.001. H Enrichment of H3K18la at the PD-L1 promoter was detected in Huh7 cells with PRMT3 overexpression. * P < 0.05, ** P < 0.01. The data are presented as mean ± SD, and the statistical tests were all two-tailed.

Article Snippet: Hepa1-6 cells with Prmt3 knockout were generated by Cyagen company (Shanghai, China) using CRISPR-Cas9 technology.

Techniques: Infection, Derivative Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression, Control, Concentration Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PRMT3 drives PD-L1-mediated immune escape through activating PDHK1-regulated glycolysis in hepatocellular carcinoma

doi: 10.1038/s41419-025-07482-7

Figure Lengend Snippet: A PRMT3, p-PDHA, and PD-L1 were immunohistochemically analyzed using a tissue microarray (TMA) of HCC specimens (left). Scale bar, 50 μm. TMA analysis revealed a positive correlation between PRMT3 and p-PDHA expression, as well as with PD-L1 expression (right). B Timeline of the anti-PD-L1 therapy in Hepa1-6 cells of the subcutaneous mouse model. Hepa1-6 cells infected with Lenti-Prmt3 or Lenti-Ctrl were inoculated subcutaneously in the right flank of C57/BL/6 mice. Anti-PD-L1 was administered intraperitoneally on day 3 post-inoculation (twice per week, 100 μg/mouse). Tumor growth curve was shown. n = 7 in each group, * P < 0.05, ** P < 0.01. Tumor images ( C ) and tumor weight ( D ) were shown. n = 7 in each group, * P < 0.05, ** P < 0.01. E Semi-quantitative analysis of Ki67 and CD8 positive staining in the indicated tumor groups. n = 5 in each group, * P < 0.05, ** P < 0.01. F Lactate production was detected in the indicated tumor groups. n = 5 in each group, * P < 0.05. G PD-L1 expression levels in the indicated tumor groups were analyzed by immunoblotting. The data are presented as mean ± SD, and the statistical tests were all two-tailed.

Article Snippet: Hepa1-6 cells with Prmt3 knockout were generated by Cyagen company (Shanghai, China) using CRISPR-Cas9 technology.

Techniques: Microarray, Expressing, Infection, Staining, Western Blot, Two Tailed Test