Polyubiquitination and accumulation of PEG10 regulated by SIAH1/2 affiliates the progression of hepatocellular carcinoma

Abstract

Background

SIAH1 and SIAH2 are key regulators of hepatocellular carcinoma (HCC) initiation and progression, but their precise molecular mechanisms remain unclear. Although both interact with the oncoprotein PEG10, it is unknown whether PEG10 mediates their regulatory effects on HCC or what the underlying mechanisms are.

Methods

The effects of SIAH1/2 on HCC cell proliferation and metastasis were assessed in vitro and in vivo by EdU, colony formation, transwell assays, and tumor xenograft models. Co-immunoprecipitation, ubiquitination assays, and site-directed mutagenesis were applied to investigate the interaction with PEG10 and the specific ubiquitination mechanisms. Clinical samples were used for analyzing the correlation between SIAH1/2 and PEG10 protein expression in HCC patients.

Results

We demonstrated that SIAH1 and SIAH2 played opposing roles in HCC: SIAH1 suppressed cell proliferation and metastasis, whereas SIAH2 promoted these processes. Mechanistically, both proteins bound to the PAIR domain of PEG10 and promoted its polyubiquitination via K48/63-linked chains. However, they differed in lysine specificity: SIAH1 mainly mediated K48-linked ubiquitination at Lys 36 and K63-linked ubiquitination at Lys 170 of PEG10, whereas SIAH2 primarily mediated K48-linked ubiquitination at Lys 36, and K63-linked ubiquitination at Lys 19 and Lys 155. In vivo analyses confirmed that SIAH1 expression negatively correlated with PEG10 protein levels, while SIAH2 showed a positive correlation.

Conclusions

Our study reveals that SIAH1 and SIAH2 differentially regulate HCC progression by modulating PEG10 stability through distinct ubiquitination site targeting. These findings elucidate a key molecular mechanism in HCC and highlight the SIAH-PEG10 axis as a potential therapeutic target.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-026-02958-1.

Background

Hepatocellular carcinoma (HCC) represents a significant malignant neoplasm within the digestive system. In recent years, there have been considerable advancements in therapeutic strategies for HCC. Particularly in the realm of targeted therapy, multiple effective molecular targets have been identified and are currently undergoing preliminary evaluation. However, to date, the regulatory mechanisms underlying many potential targets remain inadequately characterized, posing a critical bottleneck for the progression of targeted therapies in HCC.

Seven in absentia homologs (SIAH) is a highly conserved RING-type E3 ubiquitin ligase, with three members identified in the human family: SIAH1, SIAH2, and SIAH3. Accumulating evidence indicates that SIAH1 and SIAH2 are ubiquitously expressed across all human epithelial cell lines examined thus far. In contrast, SIAH3 does not contain the canonical RING domain and exhibits a restricted expression pattern, limited to only a small subset of cancer cell lines [1]. Our previous research findings indicate that, in HCC cells, SIAH1/2 is involved in the regulation of ubiquitination across a range of substrates, such as CTR9 [2], HBx [3], FASN [4], and others. As a result, SIAH1/2 contributes to the modulation of fundamental cellular biological processes and plays a critical role in promoting HCC-related phenotypes, including cell proliferation, invasion, and migration. However, a significant number of SIAH1/2 substrates have yet to be identified, thereby limiting the full elucidation of their functional roles and molecular mechanisms in HCC.

Paternally expressed gene 10 (PEG10), also known as EDR, HB-1, Mar2, MEF3L, Mart2, and RGAG3, has been indicated to play a critical role in the initiation and progression of HCC [5–7]. It is specifically and highly expressed in HCC, where it plays a critical regulatory role in key biological processes including cell proliferation, migration, invasion, cell cycle progression, epithelial-mesenchymal transition (EMT), and the development of drug resistance in HCC cells [8–11]. Our previous research has also confirmed that the transcriptional regulator CTR9 facilitates HCC progression and metastasis through the enhancement of PEG10 transcriptional activity [12]; The tumour susceptibility gene 101 (TSG101) enhances the proliferation, migration, and invasion capabilities of HCC cells by modulating PEG10 expression [13]. However, as of now, no conclusive studies have been published to clarify the molecular mechanisms driving the overexpression of PEG10 in HCC.

The ubiquitin-proteasome system (UPS) is widely acknowledged as a central regulatory mechanism for protein stability [14]. Based on this established functional role, it is scientifically plausible to propose that the upregulated expression of PEG10 observed in HCC may be subject to modulation via the UPS pathway. Research findings indicate that SIAH1/2 can directly interact with PEG10 [15]. Moreover, SIAH1 has been found to downregulate the expression of PEG10 at the protein level [16]. However, the precise molecular mechanism by which SIAH1/2 regulates PEG10 remains to be fully elucidated, and it has not yet been established whether this regulatory interaction plays a role in the overexpression of PEG10 in HCC.

In this study, we conducted a detailed investigation into the roles of SIAH1/2 in the proliferation, invasion, and migration of HCC cells, revealing that PEG10 plays a critical role in these processes. Moreover, our findings demonstrated that SIAH1 mediated the K48/63-linked ubiquitination-dependent degradation of PEG10, while SIAH2 promotes the stabilization of PEG10 through K48/63-linked ubiquitination. These results further elucidate the regulatory mechanisms by which SIAH1/2 modulate PEG10, while also affirming the critical role of the SIAH1/2-PEG10 axis in the progression of HCC, thereby establishing a basis for future investigations.

Methods

Tissues

Clinical tissue specimens were obtained from the Department of Hepatobiliary and Pancreatic Surgery at the Affiliated Hospital of Xuzhou Medical University. HCC tissues were verified via histopathological examination, while normal liver tissues were collected from patients who underwent partial hepatectomy as a result of traumatic liver injury. All samples were promptly stored at -80 °C immediately after collection to preserve tissue integrity and ensure the reliability of subsequent analyses.

Cell lines

The 293T cells and HCC cell lines Huh7 and MHCC97H were sourced from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Cultures were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibico) medium supplemented with 10% fetal bovine serum (FBS, Gibico), under controlled conditions of 37 °C and 5% carbon dioxide.

Cell transfection and establishment of lentiviral-transduced cell lines

293T cells in the logarithmic growth phase were seeded into six-well plates. The target plasmid and helper plasmids pMD2.G and psPAX2 were co-transfected at a molar ratio of 2:1:1, with a transfection reagent to plasmid mass ratio of 2.5:1 (µL:µg). Six hours later, the medium was replaced with fresh complete medium. Viral supernatant was harvested after 48 h and stored at -80 °C. Liver cancer cells were seeded at ~ 50% confluency and infected with the lentivirus-containing supernatant after attachment. Polybrene was added to enhance infection efficiency. Medium was refreshed 24 h post-infection. Fluorescence was assessed after 72 h, and cells were selected with puromycin when needed.

EdU assay

HCC cells in the logarithmic growth phase were seeded into 96-well plates at 10⁴ cells per well. After 24 h, the cells were treated with 50 µmol/L EdU (Ribobio, Guangzhou, China) and incubated at 37 °C for 2 h. The cells were then washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100, each for 20 min. Following three PBS washes, the cells were stained with 100 µL of 1× Apollo^® solution in the dark at room temperature for 30 min. Finally, nuclei were stained using 100 µL of Hoechst 33,342 (5 µg/mL) for 20 min, and images were captured using a fluorescence microscope (IX73; Olympus, Tokyo, Japan).

Cell colony assay

Cells in the logarithmic growth phase were resuspended to prepare a single-cell solution. Five hundred cells were seeded into each 6-cm dish and cultured under standard conditions for two weeks. The experiment was stopped when visible colonies formed. After removing the medium, the cells were rinsed twice with PBS. Colonies were fixed with 4% paraformaldehyde for 20 min and washed once with PBS. Then, they were stained with 1% crystal violet for 20 min, washed twice with PBS, and air-dried.

Transwell assay

The Transwell assay used a polycarbonate filter membrane (6.5 mm diameter, 8 μm pore size; Invitrogen). To assess cell invasion, the membrane was coated with 10 µg Matrigel (BD). A serum-free suspension of 10⁴ cells was added to the upper chamber, and the lower chamber contained medium with 15% fetal bovine serum as a chemoattractant. After incubation at 37 °C for 48 h, non-invasive cells on the upper surface were removed with a cotton swab. The membrane was fixed with methanol (15 min) and stained with 1% crystal violet (10 min). Three random fields per well were imaged and counted under an inverted microscope. The migration assay followed the same procedure without Matrigel coating.

Wound healing assay

Cell migration was evaluated using the wound healing assay. Cells were seeded in 6-well plates and grown to full confluence. A scratch was made on the cell layer with a pipette tip. The cells were then washed with PBS to remove debris and incubated in serum-free medium for 48 h. Images of three random fields were captured at specified time points using an inverted microscope (Olympus).

Western blotting

Cells were lysed in RIPA buffer and centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant was used for Western blotting. Equal amounts of protein were separated by SDS-PAGE and transferred onto 0.45 μm PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skimmed milk and incubated overnight with primary antibody at 4 °C. After washing, they were incubated with secondary antibody for 1 h at room temperature. Protein bands were detected using ECL Plus substrate (Thermo Fisher, Waltham, MA, USA) and a chemiluminescence system (Tanon, Shanghai, China).

Immunoprecipitation

Cells were transiently transfected with the specified plasmid. Twenty-four hours later, they were lysed in ice-cold buffer (1% Triton X-100, 150 mM NaCl, 20 mM HEPES pH 7.4, 2 mM EDTA, 5 mM MgCl₂) containing protease inhibitors. Lysates were incubated on ice for 20 min and centrifuged to remove debris and nuclei. One milligram of total protein was immunoprecipitated using the designated antibody. The complexes were washed five times with lysis buffer, and proteins were eluted by boiling beads in SDS buffer. Proteins were then analyzed by western blotting.

Immunohistochemistry

Human clinical tissue specimens were deparaffinized with xylene, dehydrated through a graded ethanol series, and subjected to heat-induced antigen retrieval in citric acid buffer. Endogenous peroxidase activity was blocked and the sections were washed with 1× TBST buffer. The sections were incubated in 3% bovine serum albumin (BSA) and then probed with the corresponding primary antibody overnight at 4 °C. After another wash with 1× TBST buffer, the sections were incubated with the secondary antibody and washed again. The sections were stained with a DAB detection kit, counterstained with hematoxylin, dehydrated, and mounted. Images were captured using a light microscope (IX53; Olympus, Tokyo, Japan).

Animals and tumor xenograft model

Animal experiments followed the ethical guidelines of the Institutional Animal Protection and Utilization Committee at Xuzhou Medical University. Huh7 cells with stable SIAH1 knockdown (10⁷) or MHCC97H cells with stable SIAH2 knockdown (10⁷), along with control groups, were injected subcutaneously into the lateral abdomen of 4-week-old BALB/c nude mice (Vital River Laboratory Animal Technology, Beijing, China). After seven weeks, the mice were euthanized, and the subcutaneous tumors were removed, weighed, and analyzed using immunohistochemistry.

Statistical analysis

All experiments were repeated at least three times, and all image data were quantified by ImageJ or Image Pro Plus 6.0. All data were analyzed using GraphPad Prism 9.0. Differences between two groups were analyzed using Student’s t-test. P < 0.05 was statistically significant.

Results

Upregulation of SIAH1 and downregulation of SIAH2 inhibit the proliferation and migration of HCC cells through regulating PEG10

Our previous study showed that SIAH1 inhibited the proliferation and migration of HCC cells, while SIAH2 got the opposite effect [2–4, 17]. To verify the role of SIAH1//2 on HCC progression, we replicated the relevant findings by employing two distinct combinations of HCC cell lines up/down-regulated SIAH1/2. Briefly, overexpression of SIAH1 significantly suppressed the proliferation, invasion, and migration of HCC cells, whereas silencing SIAH1 markedly enhanced these processes (Figs. 1A-E, Supplementary Figs. 1 A-E). Likewise, silencing SIAH2 effectively inhibited the proliferation, invasion, and migration of HCC cells, whereas overexpression of SIAH2 promoted these processes to a substantial extent (Figs. 1F-J, Supplementary Figs. 1 F-J). This further refines our previous understanding by indicating that the low expression of SIAH1 in conjunction with the high expression of SIAH2 in HCC is associated with the malignant progression of the disease.

Fig. 1.

Upregulation of SIAH1 and downregulation of SIAH2 inhibit the proliferation and migration of HCC cells. A Representative bolts and quantification to show the protein levels of SIAH1. B Image and quantification of the EdU incorporation assay performed in HCC cells upon upregulation of SIAH1. C Image and quantification of the colony formation assay with SIAH1 upregulated Huh7 and MHCC97H cells. D Image and quantification of the transwell migration and invasion assay with SIAH1 upregulated Huh7 and MHCC97H cells. E Image and quantification of the wound healing assay performed in Huh7 and MHCC97H cells upon upregulation of SIAH1. F Representative bolts and quantification to show the protein levels of SIAH2. G Image and quantification of the EdU incorporation assay performed in HCC cells upon knocking down of SIAH2. H Image and quantification of the colony formation assay with SIAH2 downregulated Huh7 and MHCC97H cells. I Image and quantification of the transwell migration and invasion assay with SIAH2 downregulated Huh7 and MHCC97H cells. J Image and quantification of the wound healing assay performed in Huh7 and MHCC97H cells upon knocking down of SIAH2. *P < 0.05, **P < 0.01, ***P < 0.001

Research findings demonstrate that SIAH1/2 is capable of directly interacting with PEG10 [15]. Furthermore, SIAH1 has been demonstrated to downregulate PEG10 expression at the protein level [16]. Therefore, we proposed that SIAH1 and SIAH2 may regulate the proliferation and metastatic potential of HCC cells by modulating PEG10 protein expression. As anticipated, SIAH1 suppressed the expression of PEG10 at the protein level (Figs. 2A and B), while SIAH2 promoted its accumulation (Figs. 2C and D). Subsequently, we further manipulated the expression levels of PEG10—either overexpressing or silencing it—in HCC cells that had previously undergone overexpression or knockdown of SIAH1/2, respectively, in order to perform a rescue analysis. The findings revealed that PEG10 overexpression effectively counteracted the suppression of HCC cell proliferation and metastatic potential caused by SIAH1 overexpression, while PEG10 knockdown significantly diminished the elevated proliferative and metastatic activities associated with SIAH1 silencing (Figs. 2E-H, Supplementary Fig. 2). Similarly, the suppression of PEG10 expression inhibited the proliferation and metastasis of HCC cells induced by SIAH2 overexpression, whereas increased PEG10 expression counteracted the reduction in proliferative and metastatic potential resulting from SIAH2 knockdown (Figs. 2I-L, Supplementary Fig. 3). These findings provide evidence that both SIAH1 and SIAH2 play regulatory roles in modulating the proliferation and metastatic potential of HCC cells, primarily through their influence on PEG10 protein expression.

Fig. 2.

Upregulation of SIAH1 and downregulation of SIAH2 inhibit the proliferation and migration of HCC cells through regulating PEG10. A Representative bolts and quantification to show the protein levels of PEG10 in HCC cells overexpression of SIAH1. B Representative bolts and quantification to show the protein levels of PEG10 in HCC cells silencing of SIAH1. C Representative bolts and quantification to show the protein levels of PEG10 in HCC cells overexpression of SIAH2. D Representative bolts and quantification to show the protein levels of PEG10 in HCC cells silencing of SIAH2. E Colony formation assay showed overexpression of PEG10 effectively restored the cell proliferation induced by upregulation of SIAH1. F Colony formation assay showed knocking down of PEG10 effectively inhibited the cell proliferation induced by downregulation of SIAH1. G Transwell migration and invasion assay showed overexpression of PEG10 effectively restored the cell invasion and migration induced by upregulation of SIAH1. H Transwell migration and invasion assay showed knocking down of PEG10 effectively inhibited the cell invasion and migration induced by downregulation of SIAH1. I Colony formation assay showed knocking down of PEG10 effectively inhibited the cell proliferation induced by upregulation of SIAH2. J Colony formation assay showed upregulation of PEG10 effectively restored the cell proliferation induced by downregulation of SIAH2. K Transwell migration and invasion assay showed knocking down of PEG10 effectively inhibited the cell invasion and migration induced by upregulation of SIAH2. L Transwell migration and invasion assay showed upregulation of PEG10 effectively restored the cell invasion and migration induced by downregulation of SIAH1. *P < 0.05, **P < 0.01, ***P < 0.001

SIAH1/2 interacts with PEG10, thereby modulating the proteasomal degradation of PEG10 through a direct protein-protein interaction

To explore the mechanism of SIAH1/2 regulated PEG10, we analyzed the interaction of them by co-IP assay. The results showed that SIAH1/2 interacted with PEG10 in both 293T and HCC cells (Fig. 3A). The subsequent GST pull-down assay further substantiated the direct and specific interaction between SIAH1/2 and PEG10, providing additional evidence for their functional association (Fig. 3B). Subsequently, we constructed truncated mutants corresponding to the PEG10 domain and further examined the interaction between SIAH1/2 and PEG10 (Fig. 3C). Our findings provided evidence that both SIAH1 and SIAH2 specifically interacted with the PAIR domain of PEG10 (Fig. 3D). More importantly, SIAH1 significantly reduced the protein stability of PEG10, while SIAH2 exhibited a stabilizing effect (Figs. 3E and F). Furthermore, HCC cells overexpressing SIAH1 or silenced for SIAH2 were treated with the lysosomal inhibitor chloroquine (CHL) and the proteasome inhibitor MG132, respectively, to elucidate the specific pathway by which SIAH1/2 regulates the PEG10 protein level. The results indicated that MG132, but not chloroquine, effectively restored the decreased expression of PEG10 induced by SIAH1 overexpression or SIAH2 silencing (Figs. 3G and H). These findings suggest that SIAH1/2 interacts with PEG10, thereby modulating PEG10 proteasomal degradation through direct protein–protein interactions.

Fig. 3.

SIAH1/2 interacts with PEG10, thereby modulating the proteasomal degradation of PEG10 through a direct protein-protein interaction. A Co-immunoprecipitation assay showed that His tagged SIAH1/2 interacted with Flag tagged PEG10 in 293T and HCC cells. B GST Pull-down assay showed SIAH1/2 directly interacted with PEG10. C Diagram of the truncation pattern of PEG10. D Co-immunoprecipitation assay showed that His-SIAH1/2 interacted with the PAIR domain of Flag-PEG10 in HCC cells. E Representative bolts and quantification showed upregulation of SIAH1 decreased the stability of PEG10. F Representative bolts and quantification showed upregulation of SIAH2 increased the stability of PEG10. G Representative bolts and quantification showed proteasome inhibitor MG132 could block the degradation of PEG10 in SIAH1 up-regulated cells. H Representative bolts and quantification showed proteasome inhibitor MG132 could block the degradation of PEG10 in SIAH2 down-regulated cells

SIAH1/2 facilitates the polyubiquitination of PEG10 through the K48/K63-linked ubiquitin chain

As classic ubiquitin ligases, SIAH1 and SIAH2 are hypothesized to regulate the polyubiquitination of PEG10. To test this hypothesis, we generated exogenous plasmids devoid of ubiquitin ligase activity by mutating cysteine 44 of SIAH1 to serine. In parallel [4], both histidine 99 and cysteine 102 of SIAH2 were substituted with alanine to construct the corresponding mutant variants [3]. First, we transfected cells with plasmids encoded His-tagged wide-type SIAH1, the E3 ligase-dead mutant SIAH1-C44S, wide-type SIAH2 and the E3 ligase-dead mutant SIAH2-H99A/C102A. PEG10 expression was then examined by Western blotting. The results showed that both SIAH1-C44S and SIAH2-H99A/C102A lose the ability to regulate PEG10 expression (Supplementary Figs. 4A and B). Next, we performed the ubiquitination assays, the results demonstrated that overexpression of wild-type SIAH1 significantly enhanced the polyubiquitination of PEG10, whereas the mutant form lacking ubiquitin ligase activity failed to exert this effect (Fig. 4A). Similarly, when compared to wild-type SIAH2, the corresponding mutant devoid of ubiquitin ligase activity was also incapable of promoting PEG10 polyubiquitination (Fig. 4B). Further, we also assessed the effects of E3 ligase dead mutants of SIAH1/2 on HCC cell proliferation, migration, and invasion. The results showed that both SIAH1-C44S and SIAH2-H99A/C102A abolished the effects of SIAH1/2 on cell proliferation, invasion, and migration (Supplementary Figs. 4 C-F). These findings demonstrate that PEG10 is regulated via the SIAH1/2 dependent ubiquitination-proteasome pathway, and the regulatory of SIAH1/2 on HCC cells proliferation, invasion and migration mainly dependent on their ligase activity.

Fig. 4.

SIAH1/2 facilitates the polyubiquitination of PEG10 through the K48/K63-linked ubiquitin chain. A Representative blots of ubiquitinated PEG10 in HCC cells with a SIAH1 mutation. B Representative blots of ubiquitinated PEG10 in HCC cells with a SIAH2 mutation. C SIAH1 ubiquitinated PEG10 through Lys48/63-linked ubiquitin chains in HCC cells. D SIAH2 ubiquitinated PEG10 through Lys48/63-linked ubiquitin chains in HCC cells

It has been reported that ubiquitin contains seven lysine residues: K6, K11, K27, K29, K33, K48, and K63 [18, 19]. These residues play a critical role in facilitating the assembly of structurally distinct polyubiquitin chains on target substrates, thereby contributing to a spectrum of functional outcomes [20, 21]. To elucidate the molecular mechanism underlying SIAH1/2-mediated PEG10 ubiquitination, we co-transfected wild-type or mutant HA-Ub together with His-SIAH1/2 into HCC cells and quantitatively assessed the extent of PEG10 ubiquitination. Our findings demonstrated that the K48/63 mutation markedly enhanced PEG10 ubiquitination in HCC cells, thereby supporting the conclusion that Lys48/63-linked polyubiquitin chains were critical mediators of SIAH1/2-induced PEG10 ubiquitination (Figs. 4C and D).

To further elucidate the specific modification sites through which SIAH1/2 regulate PEG10 polyubiquitination, we initially evaluated the influence of SIAH1 and SIAH2 on the ubiquitination levels of various truncated PEG10 mutants. The results demonstrated that the absence of either the Coiled-coil (Cc) domain or the PAIR domain in PEG10 abolished the ability of both SIAH1 and SIAH2 to modulate its ubiquitination levels (Figs. 5A and B). Furthermore, we utilized the CPLM database to predict the potential ubiquitination modification sites of PEG10 (Fig. 5C). Following the mutation of lysine residues at positions 19, 22, 36, 106, 136, 155, and 170 in PEG10 to arginine, we systematically evaluated the effects of SIAH1 and SIAH2 on the ubiquitination status of these mutant proteins. Our results demonstrated that SIAH1 predominantly mediated polyubiquitination at lysine residues 36 and 170 of PEG10, while SIAH2 primarily catalyzed ubiquitination at residues 19, 36, and 155 (Figs. 5D and E). These results indicate that although both SIAH1 and SIAH2 mediate K48/K63-linked ubiquitination of PEG10, they exert opposing regulatory effects on PEG10 protein levels. Then, we conducted further validation. We examined the regulation of K48/K63-linked ubiquitination of PEG10 mutants by SIAH1 and SIAH2, respectively. These findings showed that SIAH1 mainly mediated K48-linked ubiquitination at Lys 36 and K63-linked ubiquitination at Lys 170 of PEG10, whereas SIAH2 primarily mediated K48-linked ubiquitination at Lys 36, and K63-linked ubiquitination at Lys 19 and Lys 155 (Figs. 5F and G). We also found that SIAH1 regulated the protein levels of PEG10-K170R mutant but had no significant effect on the K36R mutant (Supplementary Fig. 5A). However, SIAH2 appeared to increase the protein levels of all three PEG10 mutants (K19R, K36R, and K155R) compared with wild-type (WT), while the fold change for the K36R mutant seemed more similar to that of the WT (Supplementary Fig. 5B). These results indicate that SIAH1 primarily promotes PEG10 degradation via K48-linked ubiquitination at Lys 36, whereas SIAH2 mainly stabilizes PEG10 through K63-linked ubiquitination at Lys 19 and Lys 155. Based on these results, we generated a PEG10-K19&155&170R mutant, and examined the protein stability of wild-type PEG10, K36R, and K19&155&170R mutant through CHX chase assays. The results showed that K36R was more stable, whereas the K19&155&170R mutant degraded faster, which further supporting our previous conclusions (Supplementary Fig. 5C). The PEG10-K36R mutant enhanced the ability to promote cell proliferation, invasion, and migration, while the promotion effect of the K19&155&170R mutant was diminished (Supplementary Figs. 5D and E). In summary, SIAH1 promotes PEG10 degradation via K48-linked ubiquitination at Lys 36, whereas SIAH2 enhances PEG10 stability through K63-linked ubiquitination at Lys19 and Lys 155.

Fig. 5.

SIAH1 facilitates the polyubiquitination of PEG10 at K36/107, SIAH2 facilitates the polyubiquitination of PEG10 at K19/36/155. A Representative blots showed that SIAH1 increased the Cc and PAIR domains ubiquitination level of PEG10 in HCC cells. B Representative blots showed that SIAH2 increased the Cc and PAIR domains ubiquitination level of PEG10 in HCC cells. C Predicted ubiquitination and ubiquitination sites for PEG10 according to the CPLM database (http://cplm.biocuckoo.org/). D Representative blots showed that SIAH1 increased the K36/107 sites ubiquitination level of PEG10 in HCC cells. E Representative blots showed that SIAH2 increased the K19/36/155 sites ubiquitination level of PEG10 in HCC cells. F Representative blots showed that SIAH1 did not increased K48-linked ubiquitination of PEG10-K36R and K63-linked ubiquitination of K170R. G Representative blots showed that SIAH2 did not increased K48-linked ubiquitination of PEG10-K36R and K63-linked ubiquitination of K19R and K155R

The protein expression of SIAH1/2 is correlated with PEG10 in vivo

To investigate whether SIAH1/2 also regulates the expression level of PEG10 at the protein level in vivo, we initially established a subcutaneous tumor model in nude mice by injecting Huh7 cells with stable overexpression of SIAH1. Our findings revealed that SIAH1 overexpression significantly suppressed tumor growth in vivo, which was accompanied by a notable decrease in PEG10 protein levels (Figs. 6A-C). To further validate this regulatory relationship, we generated an identical in vivo model using MHCC97H cells with SIAH2 knockdown. The results demonstrated that silencing SIAH2 also led to a significant inhibition of tumor growth and a concomitant reduction in PEG10 protein expression (Figs. 6D-F), thereby supporting the hypothesis that SIAH1/2 may jointly regulate PEG10 expression in vivo.

Fig. 6.

The protein expression of SIAH1/2 is correlated with PEG10 in vivo. A The images of xenograft tumors from nude mice inoculated subcutaneously with Huh7-Vector/OE-SIAH1 cells (n = 5 per group). B The statistical results for tumor weight (g). C Representative IHC images of SIAH1 and PEG10 in tumor tissues. Scale bar, 100 μm. D The images of xenograft tumors from nude mice inoculated subcutaneously with MHCC97H-shNC/shSIAH2#3 cells (n = 5 per group). E The statistical results for tumor weight (g). F Representative IHC images of SIAH2 and PEG10 in tumor tissues. Scale bar, 100 μm. G Protein levels of SIAH1/2 and PEG10 in normal liver tissues (n = 7) and tumor tissues (n = 7). H The correlation of the SIAH1 and PEG10 protein levels in normal liver tissues and tumor tissues. r = -0.7773, P = 0.0011. I The correlation of the SIAH2 and PEG10 protein levels in normal liver tissues and tumor tissues. r = 0.7761, P = 0.0011. J Representative IHC images of SIAH1/2 and PEG10 in human normal liver tissues and HCC tissues. Scale bar, 50 μm. K Schematic illustration of this study. **P < 0.01, ***P < 0.001

Furthermore, a total of seven normal human liver tissue samples and seven human HCC tissue samples were randomly selected. Total protein was extracted, and the expression levels of SIAH1/2 and PEG10 proteins were subsequently analyzed. The analysis revealed that SIAH1 expression was significantly downregulated in HCC tissues, whereas SIAH2 and PEG10 exhibited marked upregulation (Fig. 6G). Furthermore, statistical analysis demonstrated a significant negative correlation between SIAH1 and PEG10 protein levels (Fig. 6H), while a significant positive correlation was identified between SIAH2 and PEG10 protein levels (Fig. 6I). Subsequently, we conducted further analysis of the expression levels of SIAH1/2 and PEG10 in clinical tissue samples using immunohistochemical techniques. The obtained results were consistent with our previously reported findings. In HCC tissues, SIAH1 was observed to be expressed at relatively low levels, while SIAH2 and PEG10 demonstrated significantly elevated expression (Fig. 6J).

Discussion

Ubiquitination is a highly dynamic and multifaceted post-translational modification that regulates a wide range of cellular processes. The process is primarily mediated through a sequential three-enzyme cascade comprising the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. This critical modification underpins numerous essential biological functions, such as autophagy [22, 23], DNA damage response signaling [24], and the EMT [25, 26] during tumor progression.

Ubiquitin ligases serve as critical regulators throughout the ubiquitination pathway, exerting pivotal functions in substrate recognition and ubiquitin transfer. SIAH represents a highly conserved cyclic E3 ubiquitin ligase family that consists of three structurally related members: SIAH1, SIAH2, and SIAH3. Notably, SIAH3 lacks the canonical RING domain and exhibits restricted expression profiles, which may account for its relatively limited functional characterization. Consequently, SIAH1 and SIAH2 remain the predominant subjects of investigation within this protein family [1]. Our previous investigations, in conjunction with findings from other research teams, have established the involvement of SIAH1 and SIAH2 in the initiation and progression of HCC [2–4, 15–17, 27–30]. To further clarify the functional roles of these proteins in regulating the proliferation and metastatic capacity of HCC cells, we performed a comprehensive validation in the present study. Experimental data revealed that SIAH1 significantly inhibited both cell proliferation and metastasis, while SIAH2 demonstrated a promoting effect on these processes.

PEG10 is believed to have originated from the Ty3/Gypsy family of retrotransposons. It comprises two overlapping open reading frames, designated RF1 and RF2. The gene is expressed in both adult and embryonic tissues, with particularly high expression observed in the placenta, and has been implicated in fundamental cellular processes such as proliferation, differentiation, and apoptosis [31, 32]. Accumulating evidence suggests that its overexpression is associated with a range of malignancies, including HCC [7, 10, 11]. The research findings demonstrate that SIAH1/2 can directly interact with PEG10 [15]. Moreover, SIAH1 has been experimentally confirmed to decrease the expression level of the PEG10 protein [16]. Nevertheless, the exact molecular mechanism underlying SIAH1/2-mediated regulation of PEG10 remains to be fully clarified. Furthermore, it is yet to be determined whether this regulatory process plays a role in the overexpression of PEG10 observed in HCC. In this study, our findings demonstrated that the effects of SIAH1 and SIAH2 on the proliferation and metastasis of HCC cells were mediated through their interaction with PEG10. SIAH1 predominantly promoted PEG10 degradation via K48-linked ubiquitination at Lys 36, whereas SIAH2 mainly stabilized PEG10 through K63-linked ubiquitination at Lys 19 and Lys 155.In addition, in vivo analysis revealed a significant negative correlation between SIAH1 and PEG10 expression levels, while SIAH2 expression was positively correlated with PEG10 expression. Collectively, these results provide strong evidence for a regulatory relationship between SIAH1/SIAH2 and PEG10.

Conclusions

In conclusion, our findings provide compelling evidence that dysregulation of SIAH1 and SIAH2 expression plays a pivotal role in HCC progression. Specifically, downregulation of SIAH1 reduces ubiquitination-mediated degradation of PEG10, leading to its accumulation at the protein level. Meanwhile, upregulation of SIAH2 enhances polyubiquitination of PEG10, thereby increasing its protein stability. Together, these complementary mechanisms synergistically drive elevated PEG10 protein expression, which significantly promotes the proliferation and metastatic potential of HCC cells (Fig. 6K). However, several limitations within this study remain to be addressed. One critical issue concerns the relative dominance of SIAH1 versus SIAH2 in regulating PEG10 expression. To investigate this, we attempted to simultaneously modulate the expression levels of both SIAH1 and SIAH2 and monitor the resulting changes in PEG10 protein levels. However, due to the distinct expression patterns of SIAH1 and SIAH2 in HCC, combined with variability in transfection efficiency across experimental conditions, a precise quantitative comparison could not be achieved. These findings highlight the need for future studies to adopt more refined strategies—such as the development of isoform-specific small molecule inhibitors or advanced bioengineering tools—to accurately determine the individual contributions of SIAH1 and SIAH2 in HCC pathogenesis.

Abbreviations

HCC

hepatocellular carcinoma

SIAH1

Seven in absentia homologs 1

SIAH2

Seven in absentia homologs 2

SIAH1/2

SIAH1 and SIAH2

PEG10

Paternally expressed gene 10

Authors’ contributions

Z.L., K.C., H.S., and B.Z. designed and supervised the study. Z.L., Q.L., and Q.H. conducted experiments, analyzed the data, prepared figures, and wrote the main manuscript. K.W., and J.S. conducted the animal experiments. N.X. and J.X. collected the clinical samples. Z.L., Q.H., K.C., H.S., and B.Z. provided study materials and technical support. All the authors read and approved the manuscript.

Funding

The work was supported by the Frontier Technology Research and Development Program of Jiangsu (Healthcare Field) (BF2025632), New Round of Xuzhou“Pengcheng Talent Program”——High-level Healthcare Talent Recruitment and Development Project (2025TD03), Advanced Program of The Affiliated Hospital of Xuzhou Medical University (PYJH2025305), Construction Project of High Level Hospital of Jiangsu Province (GSPJS202401, GSPJS202502), Natural Science Project of The Affiliated Hospital of Xuzhou Medical University (2024ZL11), Xuzhou Municipal Health Commission Science and Technology Project (XWKYHT20250088), and Jiangsu Postgraduate Research Innovation Program (KYCX25_3317).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Clinical human tissues used in this study was approved by the Ethics Review Committee of the Affiliated Hospital of Xuzhou Medical University (approval no.: XYFY2019-KL129-01). Informed consent was obtained from all patients before treatment. The study was conducted in accordance with the Declaration of Helsinki. The patients signed an internal regulatory document stating that the remaining samples could be used for academic studies without other informed consent. The animal studies have been conducted in accordance with the Institutional Animal Care and Use Committee of Xuzhou Medical University (approval no.: 202303T028).

Consent for publication

Written informed consent for publication was obtained from all participants.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.