Silencing OTUB1 induces pyroptosis to inhibit the growth of liver cancer HepG2 cells via the NLRP3/caspase/GSDM signaling pathway

Introduction

Liver cancer ranks among the most prevalent types of malignant neoplasms globally and has been a continual concern among both basic and clinical researchers. The occurrence and development of cancer cannot be separated from cell proliferation and cell death. Studying the regulatory mechanisms and signaling pathways involved in the cell death process is helpful for understanding the operational mechanism of cells and provides a theoretical basis for solving clinical medical problems such as cancer (1). Pyroptosis is a novel form of programmed cell death that differs from apoptosis and is considered to be closely related to tumors (2,3). It is currently a hotspot in cancer research. Pyroptosis is regulated by β-catenin (4), STAT3 (5), TGF-β/Smad (6) and other signaling pathways and plays important roles in the occurrence and development of liver cancer (7).

Pyroptosis is a novel form of programmed cell death that plays an important role in the occurrence and development of liver cancer. Inflammasomes are essential for most pyroptosis pathways, and NLRP3 inflammasomes play important roles in pyroptosis. Researches have shown that activation of NLRP3 and its related signaling pathways are closely associated with pyroptosis in liver cancer and other tumors (3,8-11). NLRP3 inflammasome-induced pyroptosis has specific effects on different tumors. For example, the pyroptosis-induced cellular inflammatory response promotes tumor development in gastric cancer (10), whereas the pyroptosis-induced cellular inflammatory response promotes tumor cell death and inhibits tumor development in liver cancer (11). Because of the dual nature of pyroptosis in tumors, specific studies are needed in different tumors. In addition, pyroptosis signaling pathways [including the NLRP3, caspase, gasdermin, interleukin (IL)-1β, and IL-18 pathways] are regulated by a variety of deubiquitinases (8,12).

OTUB1, a member of the deubiquitinase family, plays an important biological role in the regulation of the ubiquitin-proteasome pathway, and its abnormality is also closely related to cancer (13-21). Previous studies have shown that OTUB1 is highly expressed in liver cancer tissue and is involved in the invasion and metastasis of liver cancer. Bioinformatics statistics show that OTUB1 can be used as a molecular marker for prognosis in digestive cancers (15). OTUB1 accelerates hepatocellular carcinoma by stabilizing receptor for activated C kinase 1 via its noncanonical ubiquitination (22). Thus, it can be inferred that OTUB1 is closely related to the occurrence and development of liver cancer. OTUB1 is known to be involved in the regulation of tumor cell proliferation, apoptosis (14,15), autophagy (15), and ferroptosis (16,17), among others, but its role in pyroptosis has not been studied. Recent studies have shown that the interaction between OTUB1 and tumor necrosis factor receptor-associated factor 3 mediates NLRP3 inflammasome activity to regulate transforming growth factor beta 1 (TGF-β1)-induced BEAS-2B cell injury (23); thus, it is hypothesized that OTUB1 may be involved in regulating pyroptosis. To verify this effect, we silenced OTUB1 expression in HepG2 liver cancer cells and observed changes in pyroptosis and relevant factors to explore the role and mechanism of OTUB1 in pyroptosis. We present this article in accordance with the MDAR reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-aw-822/rc).

Methods

Materials

The HepG2 cell line was purchased from Abcell (AC111, RRID: CVCL_0027). Lipofectamine™ 8000 was bought from Beyotime Biotechnology (C0533FT, Shanghai, China). Small interfering RNA (siRNA) targeting OTUB1 was meticulously designed and synthesized by Shanghai GenePharma Co., Ltd. (TY-siRNA02, Shanghai, China). The siRNA sequences used were as follows: sense, 5'-GCGACUCCGAAGGUGUUAATT-3'; and antisense, 5'-UUAACACCUUCGGAGUCGCTT-3'. Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose content was procured from Shanghai Hecoly Automatic Control Equipment Co., Ltd., located in Shanghai, China. Additionally, fresh fetal calf serum (FCS) was obtained from TBD Biotechnology Corp. based in Tianjin, China. The primary antibodies used were rabbit anti-OTUB1 (Immunoway, Beijing, China, YT5679, RRID: AB_3663004), anti-β-actin (Abways, Shanghai, China, AB0035, RRID：AB_2904142), anti-NLRP3 (Abways, CY5651, RRID: AB_3663006), anti-cleaved-caspase-1 (Immunoway, YC0002, RRID: AB_3663002), anti-cleaved-caspase-3 (Abways, CY5501, RRID: AB_3663003), anti-cleaved-caspase-4 (Immunoway, YC0028, RRID: AB_3662999), anti-cleaved-caspase-8 (Abways, CY5750-20, RRID: AB_3663007), anti-gasdermin D (GSDMD) (Immunoway, YT7991, RRID: AB_3663000), anti-gasdermin E (GSDME) (Immunoway, YT7990, RRID: AB_3663001), anti-IL-1β (Immunoway, YM4682, RRID: AB_3662998), anti-IL-18 (Abways, YN1926, RRID: AB_2921216), anti-β-catenin (Abways, CY3523, RRID: AB_3663008), anti-STAT3 (Abways, CY5165, RRID: AB_3663009), anti-TGF-β1 (Abways, CY6608, RRID: AB_3663010) and anti-Smad4 (Abways, CY5014, RRID: AB_3663011). The secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies (RS0002, 100 µL) were purchased from Immunoway (Shanghai, China).

Kaplan-Meier survival analysis

Survival data of 364 liver cancer patients were obtained from The Cancer Genome Atlas (TCGA) database. The expression levels of OTUB1 gene were analyzed, and the patients were categorized into high and low expression groups. Kaplan-Meier survival curves were then plotted to compare the survival rates between the two groups. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Cell culture

HepG2 cells were seeded into culture flasks and subsequently cultured in DMEM enriched with 10% (volume/volume) of fresh FCS. This was conducted at a temperature of 37 ℃ within a humidified environment containing 5% carbon dioxide (CO₂). Upon achieving confluency, the cells were transferred to new culture vessels and subjected to a 0.25% trypsin solution for a duration of 1 minute to facilitate the propagation of the cell line. The cells were cultured three times for the purpose of conducting repeated experiments.

Transient transfection

A total of 3×10⁵ cells were seeded into six-well plates and cultured until they reached 30–50% confluence. For the transfection procedure, 125 µL of siRNA was combined with Lipofectamine 8000 and subsequently introduced into each well containing cells, along with 2 mL of DMEM devoid of FCS. The plates were then carefully agitated to ensure even distribution. Following a 6-hour transfection period, the medium was exchanged for DMEM supplemented with 10% (v/v) FCS. The cells were subsequently collected after a 72-hour incubation period, and proteins were extracted for subsequent analytical procedures. Control groups, which included cells not subjected to transfection reagent and those transfected with negative siRNA, were also incorporated into this study. All experimental procedures were executed in triplicate, and the outcomes presented are representative of the collective results.

Utilization of the Cell Counting Kit-8 (CCK-8) assay for the detection of HepG2 cell growth

The obtained samples were subdivided into following groups: A, control group without transfection reagent; B, negative control siRNA transfection group; C, the OTUB1 siRNA transfection group.

A total of 2.0×10³ cells were plated in 96-well plates in triplicate and cultured to approximately 30–50% confluence at the time of transfection. A total of 12.5 µL of siRNA-Lipofectamine™ 8000 complexes were applied to each well containing cells and 100 µL of DMEM without FCS, and the plates were gently shaken back and forth. The medium was replaced with DMEM containing 10% vol/vol FCS after 6 h of transfection. At 24, 48, and 72 h after transfection, 10 µL of CCK-8 solution was added to the culture medium. After a subsequent 2 h incubation, the absorbance was quantified employing a microplate reader (Model MK3, Thermo Labsystems Co., Shanghai, China) at a wavelength of 450 nm to ascertain the quantity of viable cells. Cell viability was calculated on the basis of the measured absorbance values and compared with that of the control groups to evaluate the effect of transfection on cell growth. All the experiments were repeated three times. The data were normalized to their respective controls and are presented as a line chart.

Observation of cell morphology and structure

At 72 h after transfection, the cells in the six-well plates were washed 3 times with PBS and then digested with 0.25% trypsin. The digested cells were gently and evenly blown, placed in a centrifuge tube, and centrifuged at 3,000 r and 4 ℃ for 10 min. The supernatant was discarded, and 1 mL of 2.5% glutaraldehyde was added to the cell mixture to fix their morphology and structure. The fixed cells were then observed by transmission electron microscopy (TEM) to evaluate pyroptosis.

Microscope image acquisition: HITACHI-7650, Japan; type: TEM; magnification: 5,000×; numerical aperture of the objective lenses: 0.2–1.4; temperature: 4 ℃; imaging medium: electron beam; fluorochromes: lead citrate; camera make and model: Charge-Coupled device; acquisition software: digital micrograph.

Western blot analysis

The cells underwent lysis in RIPA buffer (9806 S, Cell Signaling Technology, Shanghai, China), which was augmented with 1 mM PMSF (ST506; Beyotime Biotech, Shanghai, China). Subsequent to the lysis, the lysates were subjected to centrifugation at a rate of 12,000 rpm/min for a duration of 15 minutes at a temperature of 4 ℃, after which the supernatants were harvested. The protein concentrations were quantified utilizing an enhanced bicinchoninic acid (BCA) assay protein assay kit (P0010, Beyotime, China). Protein specimens were subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (OTUB1, β-catenin, NLRP3, β-actin, GSDMD, GSDME, cleaved-caspase-1, cleaved-caspase-3, cleaved-caspase-4, cleaved-caspase-8, Smad4, TGF-β1, STAT3, IL-1β, and IL-18) and subsequently transferred onto 0.45-mm polyvinylidene difluoride (PVDF) membranes. The membranes were blocked by incubation in Tris-buffered saline with Tween (TBST) solution, 50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, and 0.1% Tween-20) at 37 ℃ for 2 h and then incubated with specific primary antibodies (1:2,000 dilution) at 4 ℃ overnight. On the subsequent day, the PVDF membranes were washed thrice with TBST. Subsequently, they were incubated with goat anti-rabbit IgG H&L (HRP) (1:10,000; SA00001–2, Proteintech Group) at 25 ℃ for 1 hour. Thereafter, the membranes were washed three times with TBST again and subsequently developed using 3,3'-diaminobenzidine.

Upon the emergence of protein bands on the membranes, the color development process was terminated, and the membranes were subsequently rinsed using distilled water. The images were obtained utilizing a gel imaging system (Tanon Science and Technology Co., Shanghai, China), and the subsequent analysis was conducted with the aid of Quantity One software (Bio-Rad Laboratories, Berkeley, CA, USA). The statistical analysis was performed using the quantitative outcomes derived from the grayscale analysis. The above process was performed three times.

Statistical analysis

The dataset underwent comparison through the application of one-way analysis of variance (ANOVA). P<0.05 was deemed indicative of a statistically significant variance. In this study, the dataset required comparison of differences in the mean values of the dependent variable among multiple independent groups (≥3 groups) under a single independent variable (such as different treatment groups, different time points, etc.). One-way ANOVA is suitable for this type of design.

Results

The evaluation of OTUB1 expression on the prognosis of patients with liver cancer by the Kaplan-Meier survival curves

The Kaplan-Meier survival curve indicated that patients with high OTUB1 expression (red line, N=182) had significantly lower survival rates compared to those with low expression (blue line, N=182) within 7 years (Figure 1). This suggested that high OTUB1 expression was associated with tumor progression and poorer prognosis, indicating that OTUB1 might serve as a biomarker for predicting the prognosis of liver cancer patient.

Figure 1 Patients with high expression of OTUB1 have lower survival rates for liver cancer. Survival data of liver cancer patients were obtained from TCGA database. The expression levels of OTUB1 gene were analyzed, and the patients were categorized into high and low expression groups. The Kaplan-Meier survival curve indicated that patients with high OTUB1 expression (red line, N=182) had significantly lower survival rates compared to those with low expression (blue line, N=182) within 7 years. This suggested that high OTUB1 expression was associated with tumor progression and poorer prognosis, indicating that OTUB1 might serve as a biomarker for predicting the prognosis of liver cancer patient. This correlation is not unique to liver cancer and may also occur in other tumors, such as esophageal cancer and colorectal cancer. TCGA, The Cancer Genome Atlas.

Effectiveness of silencing OTUB1

After the OTUB1 siRNA was transfected into HepG2 cells, the protein expression of OTUB1 was detected by Western blotting at 72 h and was significantly inhibited (P<0.0001) (Figure 2). These results indicated that the OTUB1 siRNA was effective in silencing the protein expression of OTUB1.

Figure 2 Protein expression of OTUB1 in HepG2 cells. A, the control group; B, the negative control siRNA transfection group; C, the OTUB1 siRNA-transfected group. Protein expression of OTUB1 in the C group was significantly inhibited (P<0.0001), which indicated that the OTUB1 siRNA was effective. The data were normalized to an internal control and are expressed as the mean ± standard deviation of three repetitions. P<0.05 compared with the control group. ****, P<0.0001.

Effects of OTUB1 silencing on the growth of HepG2 cells according to the CCK-8 assay

After transfection of OTUB1 siRNA into HepG2 cells for 24, 48, and 72 hours, the results of CCK-8 assays are shown in Figure 2. There was no significant difference in cell viability between the control group and the negative control siRNA transfection group (P=0.9). The number of viable cells in the OTUB1 siRNA transfection group was significantly lower than that in the control group (P<0.0001) (Figure 3). The percentages of cell growth inhibition elicited by the siRNAs were 23% at 24 h, 30.5% at 48 h, and 42.1% at 72 h. These findings suggested that the growth of HepG2 cells was inhibited during the 24–72 h time period after OTUB1 siRNA transfection. In other words, short-term inhibition of cell growth was achieved by transient transfection of OTUB1 siRNA. Silencing OTUB1 may have a role in the treatment of liver cancer.

Figure 3 Effects of OTUB1 silencing on the growth of HepG2 cells. A CCK-8 assay was used to evaluate cell proliferation after OTUB1 knockdown. There was no significant difference in cell viability between the control group (Control) and the negative control siRNA transfection group (SiNC) (P=0.9). The number of viable cells in the OTUB1 siRNA transfection group was significantly lower than that in the control group (P<0.0001). These results indicated that the growth of HepG2 cells was inhibited during the 24–72 h time period after siRNA transfection. ****, P<0.0001. CCK-8, Cell Counting Kit-8.

Observation of cell morphology and structure

After the OTUB1 siRNA was transfected into HepG2 cells for 72 hours, TEM was used to observe the morphology and structure of the cells. Compared with those in the control group, nuclear pyretic contraction, chromatin condensation and mitochondrial swelling in the OTUB1 siRNA-transfected group were observed at 72 h after transfection. In addition, endoplasmic reticulum expansion, cytoplasmic vacuolation, cell membrane rupture, and cell content outflows were also observed (Figure 4). These results indicated that silencing OTUB1 could induce pyroptosis in HepG2 cells.

Figure 4 Effect of OTUB1 silencing on the pyroptosis of HepG2 cells. (A) Control group. (B) Negative control siRNA transfection group. (C) OTUB1 siRNA-transfected group. Transmission electron microscopy was used to observe the morphology and structure of the cells. Compared with those in the control group, nuclear pyretic contraction, chromatin condensation and mitochondrial swelling in the OTUB1 siRNA-transfected group were observed at 72 h after transfection. Endoplasmic reticulum expansion, cytoplasmic vacuolation, cell membrane rupture, and cell content outflows were also observed. These results indicated that silencing OTUB1 could induce pyroptosis in HepG2 cells.

Expression of pyroptosis-related proteins after silencing OTUB1

After transfection of OTUB1 siRNA into HepG2 cells for 72 hours, the expression levels of pyroptosis-related proteins (NLRP3, cleaved-caspase-1, -3, -4, -8, GSDMD, GSDME, IL-1β, and IL-18) were assessed via Western blotting at 72 h after transfection. The expression levels of NLRP3 protein (P<0.0001), as well as the cleaved forms of caspase-1 (P<0.0001), caspase-3 (P<0.0001), caspase-4 (P<0.0001) and caspase-8 (P=0.0017) were increased, whereas the protein expression levels of GSDMD (P<0.0001), GSDME (P<0.0001), IL-1β (P<0.0001) and IL-18 (P<0.0001) were decrease (Figure 5). These findings indicated that OTUB1 could regulate the expression of pyroptosis-related proteins, thereby influencing pyroptosis. The detailed underlying mechanisms remain unclear.

Figure 5 Expression of pyroptosis-related proteins after silencing OTUB1 in HepG2 cells. A, the control group; B, the negative control siRNA transfection group; C, the OTUB1 siRNA-transfected group. The expression levels of pyroptosis-related proteins (NLRP3, cleaved caspase-1, -3, -4, -8, GSDMD, GSDME, IL-1β, and IL-18) were assessed by Western blotting at 72 h after transfection. The expression levels of NLRP3 protein (P<0.0001), as well as the cleaved forms of caspase-1 (P<0.0001), caspase-3 (P<0.0001), caspase-4 (P<0.0001) and caspase-8 (P=0.002) were increased, whereas the protein expression levels of GSDMD (P<0.0001), GSDME (P<0.0001), IL-1β (P<0.0001) and IL-18 (P<0.0001) were decrease. These findings indicated that OTUB1 could regulate the expression of pyroptosis-related proteins, thereby influencing pyroptosis. The data were standardized relative to an internal control and are presented as the mean ± standard deviation derived from three replicates. P<0.05 was observed when compared to the control group. **, P<0.01; ****, P<0.0001. IL, interleukin.

Expression of signaling pathway-related proteins after silencing OTUB1

The expression levels of signaling pathway-related proteins (β-catenin, STAT3, TGF-β1 and Smad4) were assessed via Western blotting at 72 h after transfection. The protein expression levels of TGF-β1 (P<0.0001) and Smad4 (P<0.0001) were increased, whereas the protein expression levels of β-catenin (P<0.0001) and STAT3 (P<0.0001) were decrease (Figure 6). It is well known that signaling pathway-related proteins (β-catenin, STAT3, TGF-β1 and Smad4) may affect pyroptosis, so the findings suggested that silencing OTUB1 could induce pyroptosis by regulating the expression of signaling pathway-related proteins. The precise fundamental processes require further investigation.

Figure 6 Expression of signaling pathway-related proteins after silencing OTUB1 in HepG2 cells. A, the control group; B, the negative control siRNA transfection group; C, the OTUB1 siRNA-transfected group. The expression levels of signaling pathway-related proteins (β-catenin, STAT3, TGF-β1 and Smad4) were assessed by Western blotting at 72 h after transfection. The protein expression levels of TGF-β1 (P<0.0001) and Smad4 (P<0.0001) were increased, whereas the protein expression levels of β-catenin (P<0.0001) and STAT3 (P<0.0001) were decrease. It is well known that signaling pathway-related proteins (β-catenin, STAT3, TGF-β1 and Smad4) may affect pyroptosis, so the findings suggested that silencing OTUB1 could improve pyroptosis by regulating the expression of signaling pathway-related proteins. The data were normalized to an internal control (see Figure 5). ****, P<0.0001. TGF-β1, transforming growth factor beta 1.

Discussion

TCGA is a large public database supported by the National Cancer Institute of the United States, aimed at providing data support for early diagnosis of cancer, discovery of therapeutic targets, and personalized treatment through genomic analysis of various types of cancer. This study collected survival data of liver cancer patients from the TCGA database, categorized the patients into high-expression and low-expression groups according to the expression level of OTUB1, and plotted Kaplan-Meier survival curves to compare the survival rates of the two groups. This analysis helps to reveal whether OTUB1 might serve as a potential prognostic marker for liver cancer. From the survival curves, it was evident that patients exhibiting high expression levels of OTUB1 demonstrated a lower survival rate, indicating that OTUB1 might be associated with poor prognosis of liver cancer. This provided a potential prognostic evaluation indicator for clinical use. The survival rate of patients in the low-expression group showed a significant decline seven years later, which may be due to reduced sample size leading to data instability or an increase in non-tumor-related deaths among patients in the low-expression group.

siRNA interference technology, which was discovered in 1998, is an effective way to silence cytokines (24). In the present study, after OTUB1 siRNA was transfected into HepG2 cells for 72 h, Western blotting was used to detect the protein expression of OTUB1. The protein expression of OTUB1 decreased significantly, verifying the effectiveness of the OTUB1 siRNA.

In this experiment, cell viability was assessed using the CCK-8 assay. It was observed that the growth of HepG2 cells was inhibited from 24–72 hours after transfection with OTUB1 siRNA, suggesting a potential therapeutic effect on liver cancer. After 72 hours of transfection, increased pyroptosis in HepG2 cells was observed under TEM. These findings suggested that OTUB1 might regulate pyroptosis and thereby affected the growth of HepG2 cells. OTUB1 could become a new target for liver cancer treatment.

It is now known that pyroptosis is regulated mainly by classical inflammasome signaling pathways (which are dependent on caspase-1) and nonclassical inflammasome signaling pathways (which are dependent on caspase-4, caspase-5, and caspase-11). The caspase-1-dependent signaling pathway activates the corresponding receptors, NLRP1b, NLRP3, NLRC4, AIM2 or Pyrin, by stimulating signals such as bacteria or viruses, which subsequently form macromolecular complexes with apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. Caspase-1 directly cleaves gasdermin D to initiate pyroptosis and can also split pro-IL-1β and pro-IL-18 precursors to convert IL-1β and IL-18 into mature functional proteins and subsequently release them into extracellular cells through necrotic membrane pores formed by gasdermin D-N such that inflammatory cells accumulate and expand the inflammatory response. The caspase-4-, 5- and 11-dependent signaling pathways are triggered by human caspase-4, caspase-5 or mouse caspase-11, and caspases can directly cleave gasdermin D to initiate pyroptosis. The N-terminal fragment of split gasdermin D can also activate the maturation of the NLRP3 inflammasome and caspase-1-dependent IL-1β and IL-18. In addition, there are other alternative pyroptosis pathways, including the cleavage of gasdermin D by caspase-8, the cleavage of gasdermin E by caspase-8 or granzyme B, and the cleavage of gasdermin B by caspase-1 or granzyme A, among others (25-27).

In this study, after OTUB1 was silenced for 72 h in HepG2 cells, protein expression of NLRP3 and caspases 1, 3, 4 and 8 increased, whereas protein expression of GSDMD, GSDME, IL-1β and IL-18 decreased (P<0.05). The expression of these proteins is related to the pyroptosis signaling pathway, so these results indicated that OTUB1 could regulate the expression of pyroptosis-related proteins and thus affect pyroptosis. The underlying mechanism might be as follows. Silencing OTUB1 can split GSDMD and GSDME through increases in the protein expression of NLRP3 and caspase-1, -3, -4, and -8 and subsequently reduce the protein expression of GSDMD and GSDME. IL-1β and IL-18 are converted into mature functional proteins and then released into the extracellular space, decreasing the levels of intracellular IL-1β and IL-18. The present results confirmed that OTUB1 could affect the pyroptosis of HepG2 cells through multiple pyroptosis pathways.

In the current study, after OTUB1 was silenced for 72 hours, protein expression of β-catenin and STAT3 decreased, whereas protein expression of TGF-β1 and Smad4 increased. These results indicated that OTUB1 could regulate the expression of these signaling pathway-related proteins. The regulation of STAT3 and Smad4 protein expression by OTUB1 is a novel finding. However, the regulation of β-catenin and TGF-β1 by OTUB1 is similar to previous studies in other tumors. For example, overexpression of OTUB1 exacerbates colorectal cancer malignancy by inhibiting protein degradation of β-catenin (21). Additionally, the deubiquitinase OTUB1 has been targeted as a therapeutic strategy for BLCA via the β-catenin/necroptosis signal pathway (28). Furthermore, OTUB1 mediates TGF-β1 to induce BEAS-2B cell injury (23).

Recent studies have shown that β-catenin, STAT3 and TGF-β1 signaling pathways are involved in pyroptosis. For example, TREM2/β-catenin attenuates NLRP3 inflammasome-mediated macrophage pyroptosis to promote bacterial clearance of pyogenic bacteria (4). FTO represses NLRP3-mediated pyroptosis and alleviates myocardial ischemia-reperfusion injury by inhibiting CBL-mediated ubiquitination and degradation of beta-catenin (29). Downregulating β-catenin signaling led to mitochondrial dysfunction, the activation of the NLRP3 inflammasome, and pyroptosis, ultimately resulting in hepatocyte injury. (30). STAT3 induces the transcription of tripartite motif-containing protein 21, triggering a cascade that activates gasdermin D, resulting in pyroptosis (31). The p-STAT3/annexin A2 axis promotes caspase-1-mediated hepatocyte pyroptosis in nonalcoholic steatohepatitis (32). STAT3 directly correlates with and positively regulates GSDME expression in atherosclerosis (5). Moreover, STAT3 regulates pyroptosis in colorectal cancer cells (33), neurons (34), and glioblastomas (35). TGF-β1 protects against LPC-induced cognitive deficits by attenuating pyroptosis in microglia (36). Macrophage pyroptosis promotes synovial fibrosis through the HMGB1/TGF-β1 axis (37). Therefore, OTUB1 may regulate pyroptosis through these signaling pathways. The specific mechanisms involved need to be unraveled in future studies.

Conclusions

Our study revealed that silencing OTUB1 induces pyroptosis via the NLRP3/caspase/GSDM signaling pathway, thereby inhibiting the growth of liver cancer HepG2 cells. The underlying mechanisms may be related to OTUB1 regulation of the β-catenin, STAT3 and TGF-β/Smad signaling pathways. These findings improve our understanding of the pathogenesis of liver cancer and reveal OTUB1 as a potential therapeutic target for liver cancer.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-aw-822/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-aw-822/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-aw-822/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-aw-822/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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