Silencing OTUB1 promotes pyroptosis and M1 macrophages polarization to inhibit HepG2 cell proliferation

Technical Summary: Silencing OTUB1 Promotes Pyroptosis and M1 Macrophage Polarization to Inhibit HepG2 Cell Proliferation

Problem Statement
Liver cancer remains a highly malignant tumor with significant global mortality. The tumor microenvironment (TME), particularly the polarization status of tumor-associated macrophages (TAMs), critically influences cancer progression. While M1-like macrophages exhibit anti-tumor characteristics, M2-like macrophages are generally immunosuppressive and pro-tumorigenic. Additionally, pyroptosis, a form of inflammasome-dependent programmed cell death, plays a dual role in tumor survival and immune regulation. The deubiquitinating enzyme OTUB1 is known to regulate various tumor processes, including cell proliferation and immune evasion; however, its specific role in regulating pyroptosis and its subsequent impact on macrophage polarization within the liver cancer microenvironment remains unclear. This study investigates whether silencing OTUB1 can modulate pyroptosis in HepG2 liver cancer cells and THP-1-derived macrophages, and how these changes influence macrophage polarization and tumor cell proliferation.

Methodology
The study utilized a co-culture system involving HepG2 liver cancer cells and THP-1-derived M0 macrophages.

• Cell Models and Transfection: HepG2 cells and THP-1 cells (differentiated into M0 macrophages using PMA) were transfected with OTUB1-specific siRNA. Transfection efficiency was verified via immunofluorescence and Western blot.
• Co-culture System: A Transwell system was employed to co-culture HepG2 cells and M0 macrophages, allowing for the study of paracrine interactions.
• Functional Assays:
  • Proliferation: Cell viability was assessed using the CCK-8 assay at 24, 48, and 72 hours.
  • Morphology: Transmission electron microscopy (TEM) was used to observe pyroptotic morphological changes (e.g., cell swelling, membrane rupture).
  • Protein Expression: Western blotting quantified the expression of OTUB1, pyroptosis-related markers (Cleaved-Caspase-1, Cleaved-Caspase-4, GSDMD), inflammatory cytokines (IL-1β, IL-18), and macrophage polarization markers (CD86 for M1, CD206 for M2).
  • Interaction Network: Co-immunoprecipitation (Co-IP) coupled with mass spectrometry was performed to identify OTUB1-interacting proteins and construct a protein-protein interaction (PPI) network.
• Statistical Analysis: Data were analyzed using Student's t-test and one-way ANOVA via GraphPad Prism.

Key Results

1. OTUB1 Silencing Efficiency: OTUB1 was effectively silenced in both HepG2 cells and M0 macrophages at 72 hours post-transfection.
2. Impact on Cell Proliferation:
   • OTUB1 silencing significantly inhibited the proliferation of both HepG2 cells and M0 macrophages.
   • In co-culture, the presence of macrophages generally promoted HepG2 proliferation. However, when OTUB1 was silenced in the co-culture system, this pro-proliferative effect was reversed, resulting in a significant decrease in HepG2 viability compared to the non-silenced co-culture group.
3. Induction of Pyroptosis:
   • TEM revealed typical pyroptotic morphology (nuclear pyknosis, membrane rupture, vacuolation) in both HepG2 and M0 cells following OTUB1 silencing.
   • Western blot analysis showed that OTUB1 silencing increased the levels of Cleaved-Caspase-1 and Cleaved-Caspase-4 in both cell types.
   • Interestingly, intracellular levels of GSDMD, IL-1β, and IL-18 decreased in the silencing groups. The authors attribute this to the endpoint detection time, suggesting these proteins may be cleaved, consumed, or released extracellularly during late-stage pyroptosis.
   • In the co-culture group without silencing, HepG2 pyroptosis was inhibited (lower Cleaved-Caspase-1/4, higher GSDMD/cytokines). OTUB1 silencing reversed this trend, promoting pyroptosis activation in HepG2 cells.
4. Macrophage Polarization:
   • Co-culture alone induced M0 macrophages to polarize toward the M2 phenotype (increased CD206, decreased CD86), correlating with enhanced HepG2 proliferation.
   • In the OTUB1-silenced co-culture group, macrophages exhibited a shift toward the M1 phenotype (increased CD86), correlating with the inhibition of HepG2 proliferation.
5. Protein Interaction Network: Co-IP and mass spectrometry identified a network of OTUB1-interacting proteins, including PSMC6, DDX17, LUC7L2, TUFM, RPS14/RPS24, ALDH2, ERP70, ACAA2, and PRDX3. GO and KEGG analyses indicated enrichment in redox processes, translation, and hepatocellular carcinoma-related pathways.

Significance and Claims
The study concludes that silencing OTUB1 acts as a key regulatory mechanism in the liver cancer microenvironment by:

1. Promoting Pyroptosis: Directly inducing pyroptosis in both liver cancer cells and macrophages.
2. Reprogramming Immunity: Driving macrophage polarization from a pro-tumorigenic M2 phenotype to an anti-tumorigenic M1 phenotype.
3. Inhibiting Tumor Growth: The combined effect of induced pyroptosis and M1 polarization results in the suppression of HepG2 cell proliferation.

The authors posit that OTUB1 is a critical regulator of the "pyroptosis–macrophage polarization–liver cancer progression" axis. They suggest that targeting OTUB1 could offer a theoretical basis for new immunotherapeutic strategies aimed at reshaping the tumor microenvironment. The study acknowledges limitations, including the reliance on a single cell line (HepG2), the need for further validation of the specific molecular mechanisms linking OTUB1 to pyroptosis, and the necessity of in vivo animal models to confirm these findings.