The APC/C subunit APC7 promotes hepatocellular carcinoma progression by mediating ubiquitination and degradation of LATS1 to activate YAP/TAZ

Background

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase critically involved in cell cycle regulation. However, the pathological functions of its individual subunits, particularly in hepatocellular carcinoma (HCC), remain largely unexplored.

Aim

To systematically analyze the expression and prognosis of APC/C in pan-cancer, and also focus on studying the function and mechanism of APC7 in the progression of HCC.

Methods

Transcriptome data were downloaded from TCGA, ICGC, and GEO databases. The analysis of differential expression genes and clinical characteristics were performed to identify the key APC gene. Moreover, immune subtype analysis was conducted to elucidate potential functions, and immune cell infiltration was assessed using the CIBERSORT algorithm. Then, gain- and loss‐of‐function studies were employed to elucidate the role of APC7 in HCC. Finally, RNA sequencing and ubiquitination assays were employed to elucidate the underlying mechanisms of APC7 in HCC.

Results

This study systematically analyzed the expression and prognosis of various subunits of the APC/C complex in pan-cancer samples, and identified APC7 as a key subunit in HCC. APC7 exhibited the most significant upregulation in HCC and was closely associated with poor prognosis in patients. Moreover, the expression profiles of APC7 is closely related to immune checkpoint genes and tumor-infiltrating immune cells. The results of multiplex immunohistochemistry showed that APC7 overexpression markedly increased the infiltration of Foxp3⁺, CD25⁺, and CD4⁺ Treg cells. Functional studies demonstrated that the knockdown of APC7 inhibited HCC cell proliferation, migration, invasion, and G1/S phase transition, whereas overexpression of APC7 promoted these malignant phenotypes in vitro and in vivo. Mechanistically, RNA sequencing and molecular studies revealed that APC7 interacts with CDH1 to mediate ubiquitin-dependent degradation of LATS1 at K860, leading to YAP/TAZ activation. Rescue experiments confirmed that LATS1 ablation reversed the tumor-suppressive effects of APC7 knockdown.

Conclusions

Our findings identify APC7 as a key oncogenic driver in HCC, promoting tumor progression via the Hippo signaling pathway. APC7 may represent a prognostic biomarker and a potential therapeutic target in HCC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13062-026-00759-7.

Highlights

Pan-cancer analysis of APC/C identifies APC7 as the most significantly upregulated and is strongly associated with poor prognosis in patients with HCC.

APC7 expression is correlated with immune checkpoint genes and tumor-infiltrating immune cells, suggesting a role in the tumor immune microenvironment.

The overexpression of APC7 promotes HCC malignant progression by facilitating proliferation, migration, invasion, and cell cycle progression.

APC7 interacts with CDH1 to mediate ubiquitin-dependent degradation of LATS1 at K860, leading to YAP/TAZ activation and dysregulation of the Hippo pathway.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13062-026-00759-7.

Introduction

Hepatocellular carcinoma (HCC) is a major global health burden, exhibiting high mortality and particularly heavy disease burden in China [1, 2]. The development of HCC is a multistep process driven by factors like viral hepatitis and aflatoxin, involving genetic mutations and dysregulated signaling pathways [3]. Ubiquitination modification plays a crucial role by precisely regulating protein degradation processes or signal transduction pathways in the chronic progression of normal liver to hepatitis, cirrhosis, and ultimately HCC [4]. This ubiquitination cascade is executed by the sequential action of the E1 activating enzyme, E2 conjugating enzyme, and E3 ligase. Following ATP-dependent ubiquitin activation by E1 and transfer to E2, the E3 ligase directly catalyzes the formation of an isopeptide bond between ubiquitin and a lysine residue on the specific substrate, thereby defining the specificity of the entire pathway [5, 6].

The main types of E3 ubiquitin-ligase enzymes can be classified into three families based on their characteristic domains and the mechanism by which ubiquitin is transferred to substrate proteins: RING E3s, HECT E3s, and RBR E3s [7]. The APC/C, a multi-subunit RING-type E3 ubiquitin ligase, consists of the catalytic core (APC2, APC11, and APC10), the platform component (APC1, APC4, APC5, and APC15) and tetratricopeptide repeat (TPR) lobes (APC3, APC6, APC7, APC8, APC12, APC13, and APC16) [8]. While the function of the catalytic core is well characterized, the regulatory roles of TPR subunits such as APC7 remain poorly understood. APC/C activation begins with binding of coactivators CDC20 or CDH1, leading to conformational changes that facilitate the recruitment of E2 enzymes UBE2C and UBE2S. These catalyze initial ubiquitination and K11-linked polyubiquitin chain formation, respectively, marking substrates for proteasomal degradation [9–12]. Although APC/C is recognized for its mitotic regulatory functions [13–15], emerging evidence suggests its involvement in cell differentiation and maintenance of cell identity [16–20]. However, the pathological roles of specific APC/C subunits in in human cancers, including HCC, are largely unknown.

APC7, encoded by the gene ANAPC7, is a core subunit of the TPR lobe in the APC/C complex, and its depletion leads to impaired ubiquitination of APC/C substrates [21]. Emerging evidence has revealed the role of APC7 in the occurrence and development of tumors. For instance, it was highly expressed in esophageal squamous cell carcinoma, and high expression of APC7 was significantly correlated with adverse clinical features [22]. In gastric cancer, APC7 interacted with PD-L1, which promoted immune escape by enabling tumor cells to evade the killing effect of NK cells [23]. However, the expression patterns and functional significance of APC7 in HCC remain largely unknown.

A schematic diagram of the comprehensive research framework was provided in Fig. 1. In this study, we systematically investigated the expression and prognosis of APC/C subunits in pan-cancer, as well as the analysis of the correlation between APC7 and immune infiltration and immune checkpoints. In addition, we probed the expression of APC7 in HCC samples, and found that the upregulation of APC7 was associated with an unfavorable prognosis and the expression of APC7 protein was identified as an independent predictor. Loss- and gain-of-function experiments confirmed that overexpression of APC7 promoted malignant phenotypes in HCC cells. Moreover, we delineated the mechanism by which the APC7-CDH1 axis targets the Hippo kinases LATS1 for ubiquitin-mediated degradation, leading to YAP/TAZ activation and driving HCC progression.

Fig. 1.

Study flowchart

Materials and methods

Tissue microarray

The tissue microarray of human HCC samples, containing 91 HCC and 91 adjacent tissues, was custom-designed from Outdo Biotech Co., Ltd. (Shanghai, China). The research was conducted with the approval of the Ethical Committee of Shanghai Outdo Biotech Company, with the ethical approval number HLivH180Su30 and HLivH180Su31.

Animal studies

Huh-7-NC, Huh-7-APC7, HCCLM3-shNC, and HCCLM3-sh-APC7 cells (3 × 10⁷ cells / mL) were subcutaneously injected into 6-week-old BALB/c male nude mice, with five mice per group. Starting on day 7 post-inoculation, tumor volume was measured every three days.

To assess the metastatic ability of APC7 in HCC in vivo, 1 × 10⁶ MHCC97H or HCCLM3 cells stably expressing firefly luciferase and with lentiviral-mediated APC7 overexpression or knockdown were injected into the tail vein of 6-week-old BALB/c nude mice (n = 5). Prior to sacrifice, mice received an intraperitoneal injection of D-luciferin potassium salt, and metastasis was evaluated by in vivo bioluminescence imaging using a Carestream Molecular Imaging System, following the manufacturer’s protocol.

To evaluate the effect of APC7 on the infiltration of Treg cells in vivo, 2 × 10⁶ Hepa1-6 cells stably overexpressing APC7 were orthotopically injected into the livers of 6-week-old male C57BL/6 mice. Tumor tissues were subsequently collected and subjected to multiplex immunohistochemistry (mIHC) staining.

All animal procedures were authorized by the Animal Ethics Committee of the National Translational Science Center for Molecular Medicine, Fourth Military Medical University.

Transcriptome profiles of HCC tissues

The HCC transcriptome data and corresponding clinical information were obtained from the TCGA database (50 adjacent and 374 HCC tissues) and the ICGC database (202 adjacent and 243 HCC tissues). Additionally, the datasets GSE22058 (97 adjacent and 100 HCC tissues), GSE76427 (52 adjacent and 115 HCC tissues), and GSE54236 (80 adjacent and 81 HCC tissues) were sourced from the GEO database.

Immunohistochemistry

The tissue sections were deparaffinized in xylene and rehydrated through a graded alcohol series. Antigen retrieval was performed using citrate buffer (pH 6.0). Subsequently, endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide, and nonspecific binding sites were blocked with goat serum. After blocking, the sections were incubated overnight at 4 °C in a humidified chamber with the APC7 primary antibody. On the following day, a chromogenic reaction was developed using a streptavidin-peroxidase detection kit (Zhongshan Jinqiao Co., Ltd., Beijing, China). Finally, staining was evaluated by scoring based on the percentage of positively stained cells and staining intensity.

Cell culture and transfection

The human HCC cell lines HCCLM3, HepG2, MHCC-97 H、Hep3B and the mouse cell line Hepa 1–6 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HLE and Huh-7 cells were procured from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan). All siRNAs used in this study were procured from Tsingke Biotechnology Co., Ltd., with their specific nucleotide sequences detailed in Table S3. The transfected reagent was used with Lipofectamine 2000 (Invitrogen, CA, USA) as per the manufacturer’s protocol. Lentiviruses carrying APC7 was custom-made from Genechem Co., Ltd. (Shanghai, China) for constructing stable cell lines.

Western blots

Cells were lysed using RIPA lysis buffer, and the extracted protein samples were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were then blocked with skim milk at room temperature for 1 h, followed by an overnight incubation with primary antibodies at 4 °C. The next day, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Protein bands were finally detected using the ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, USA). The primary antibodies used in this study included APC7 (PA5-114859, Invitrogen), PCNA (10205-2-AP, Proteintech), N-cadherin (22018-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), Cyclin D1 (60186-1-AP, Proteintech), pYAP (80694-2-RR, Proteintech), LATS2 (20276-1-AP, Proteintech), β-actin (66009-1-AP, Proteintech), LATS2 (sc-398560, Santa), YAP (F0366, Selleck), TAZ (F1356, Selleck).

Quantitative real-time PCR

Total RNA was extracted using the Steady Pure Rapid RNA Extraction Kit (AG21023, Aikori) and subsequently reverse-transcribed into cDNA with the PrimeScript™ RT Reagent Kit (TaKaRa, Japan). Quantitative PCR was then performed using the Taq™ Reagent (TaKaRa, Japan) on an Mx3005P Real-Time PCR System (Agilent, USA). The primers applied to qRT‒PCR was described in Table S3.

CCK-8 assay

HCC cells with overexpressed or knocked-down APC7 were plated at a density of 5 × 10³ cells per well in 96-well plates. At 0, 24, 48 and 72 h after cell culture, 10 µL CCK-8 solution was added to each well and incubated at 37 °C for 1 h. Finally, absorbance values were measured using a microplate reader at 450 nm wavelength for each well.

Cell migration and invasion

For the migration assay, harvested cells were resuspended in serum-free medium and seeded into the upper chamber of an uncoated Transwell insert. For the invasion assay, cells were plated into Matrigel-coated chambers. In both assays, the lower chamber was filled with complete medium containing 10% FBS as a chemoattractant. After 24 h of incubation, non-migrated/non-invaded cells on the upper membrane surface were removed. Cells that traversed the membrane were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified under an optical microscope.

Cell cycle assay

Cell cycle assay was performed using a cell cycle detection kit (Nanjing Keygen Biotechnology Co., Ltd., KGA9101). The cells were trypsinized and collected as a cell pellet. The cells were washed once with PBS, and the cell concentration was adjusted to 1 × 10⁶/mL. 500 µL of pre-cooled 70% anhydrous ethanol was added to the cell pellet, gently mixed, and fixed overnight in a -20 °C freezer. The ethanol was removed from the supernatant by centrifugation. 500 µL of PI/RNase A working solution was added. After mixing thoroughly, the cells were incubated for 30 min at room temperature in the dark. The cell cycle distribution was detected using a flow cytometer with an excitation wavelength of 488 nm. Data analysis was performed using ModFit L 3.3.11.

Apoptosis experiment

The apoptosis rate was quantified using the Annexin V-APC/7-AAD dual-stain kit (Nanjing Keygen Biotechnology Co., Ltd., KGA1106). Cells were collected by digestion with EDTA-free trypsin, and the cell pellet was washed twice with PBS. 500 µL of Binding Buffer was added to each sample and gently mixed to obtain a single-cell suspension. 5 µL of Annexin V-EGFP and 5 µL of PI solution were added to each sample, followed by gentle mixing. The samples were incubated at room temperature in the dark for 10 min. The results were detected and analyzed using a flow cytometer, with an excitation wavelength of 488 nm and an emission wavelength of 530 nm for Annexin V, and an excitation wavelength of 488 nm and an emission wavelength of 630 nm for PI. Data analysis was performed using FlowJo 10.6.2.

RNA sequencing analysis

This study conducted transcriptome sequencing on three independent RNA samples from Huh-7 cells stably overexpressing APC7 and their corresponding control cells. The sequencing and analysis were carried out by Gene Denovo Biotechnology Co. We identified statistically significant differentially expressed genes (DEGs) using a threshold of |log₂FC| >1 and FDR < 0.05.

Multiplex immunohistochemistry (mIHC)

Mouse tissue samples were embedded in paraffin, and the paraffin-embedded tissue sections were cut to a thickness of 4 μm, baked overnight at 37 °C, dehydrated in xylene, and rehydrated through a graded ethanol series. Antigen retrieval was performed in citrate buffer (pH = 6) for 15 min. Subsequently, sections were incubated with primary antibody for 1.5 h at 37 °C. Afterwards, the sections were incubated with a secondary antibody conjugated to streptavidin peroxidase for 10 min at room temperature to amplify the signal. Finally, TSA dye was applied for 10 min at room temperature. The remaining antibodies were stained using the aforementioned protocol, concluding with a final wash and sealing with a DAPI-containing mounting medium. Digital imaging was performed using an AKOYA Vectra Polaris system. The following primary antibodies were used: anti-CD4 (ab288724, Abcam), anti-Foxp3⁺ (ab75763, Abcam), anti-CD3 (ab16669, Abcam), and anti-APC7 (PA5-114859, Thermo Fisher Scientific).

Statistical analysis

Data analysis was performed using GraphPad Prism and SPSS 22. The association between APC7 expression and the clinical characteristics of HCC patients was assessed by the chi-square test. Overall survival was analyzed by the Kaplan–Meier method. Each experiment was repeated at least three times. Differences between two groups were analyzed using Student’s t-test, and comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). The prognostic value of APC7 was further evaluated by univariate and multivariate analyses, with statistical significance set at P < 0.05.

Results

Expression patterns, prognostic and immune analysis of APC7 in pan-cancer

For transcriptome data from TCGA in pan-cancer, we delved deeper into the relationships between mRNA expression levels of APC/C subunits. Our analysis revealed a positive correlation of APC7, APC5, APC4, CDC26, APC13 with other members of the family, specifically within the context of pan-cancer (Fig. 2A). Additionally, we conducted a differential analysis of the expression levels of the APC/C subunits in the pan-cancer dataset. The expression of the APC/C subunits were significantly higher in both CHOL and LIHC tissues compared to the adjacent tissues (Fig. 2B). Taking into account the fold change (|log₂FC| > 1) and P-values (P < 0.05), we found that the P-value of APC7 is the smallest in HCC, and the value is 4.632E-29 (Fig. 2B, Table S1). The expression of APC7 was significantly overexpressed in LIHC compared to the adjacent tissues (Fig. 2C). The univariate Cox regression analysis of APC/C subunits revealed that high expression of APC4, APC5, APC15, CDC27, CDC16, APC7, CDC26, APC13, APC1 and CDC23 was significantly linked to unfavorable prognoses in HCC, respectively (Fig. 2D, Table S2). However, there is no significant difference in the association between all APC/C subunits and the prognosis in CHOL (Fig. 2D, Table S2). In addition, we analyzed the changes of APC/C subunits in immune subtypes and found that the APC/C subunits exhibited varying degrees of change in six immune subtypes. A higher frequency of APC7 expression was noted in wound healing and IFN-gamma dominant samples in HCC (Fig. 2E). Based on the above findings, our subsequent study focused on the expression, function, and tumor-promoting mechanisms of APC7 in HCC. Given the importance of checkpoint-based immunotherapy, we explored the correlation between the expression profiles of APC7 and immune checkpoint genes in pan-cancer and found that several genes (CTLA4, HAVCR2, ITPRIPL1, LAG3, PDCD1, TIGIT) were significantly positively correlated with APC7 expression in HCC (Fig. 2F). To validate these findings, we examined the mRNA expression levels of CTLA4, HAVCR2, ITPRIPL1, LAG3, PDCD1, and TIGIT by qRT-PCR. The results showed that the expression of immune checkpoint-related genes were upregulated after overexpression of APC7 (Fig. 2G). To investigate the relationship between the APC7 expression profiles and tumor-infiltrating immune cells, we used the CIBERSORT algorithms to evaluate the differences in immune infiltration between the APC7 high-expression and the low-expression groups. The expression of APC7 was positively correlated with Treg and Macrophage M0 cells, while showing a negative correlation with Monocyte cells (Fig. 2H). To validate these findings in vivo, we established an orthotopic liver tumor model in C57BL/6 mice using Hepa1-6 cells with APC7 overexpression. We collected tissue samples from orthotopic liver tumors after 21 days and subjected them to multiplex immunofluorescence staining. The results revealed that APC7 overexpression markedly increased the infiltration of Foxp3⁺, CD25⁺, and CD4⁺ Treg cells (Fig. 2I). To examine the mRNA expression of APC7 across different cell types in the tumor microenvironment, we analyzed three HCC single-cell transcriptome datasets from the TISCH2 database. In GSE140228, APC7 expression was detected in Tprolif, Treg, endothelial, and malignant cells. This expression pattern was corroborated in GSE166635 (observed in Tprolif, Treg, malignant, and CD8⁺ T cells) and GSE98638 (observed in Tprolif, Treg and malignant cells) (Figure S1A, S1B). Using the TIMER database, a multivariable Cox proportional hazard model was constructed to assess the relationship between different immune cell types, APC7 mRNA expression levels, and the prognosis of HCC patients. The results showed that high infiltration levels of B cell, CD8⁺ T cell, dendritic cell, as well as high expression of APC7, were all significantly associated with an increased risk of death in patients (Fig. 2J). The above results indicate that APC7 may be an important unfavorable prognostic factor in HCC, with its expression closely linked to an immunosuppressive tumor microenvironment.

Fig. 2.

Pan-cancer analysis of APC/C subunits identified APC7 as a key factor in HCC. (A) Correlation analysis of APC/C subunits expression in pan-cancer transcriptome data from TCGA. (B) Differential expression analysis of APC/C subunits in tumor and adjacent tissues in pan-cancer. (C) APC7 expression in pan-cancer transcriptome data from TCGA. (D) Cox regression analysis evaluating the prognostic significance of APC/C subunits in pan-cancer. (E) The correlation between APC7 expression and the immune subtype. (F) The correlation between APC7 expression and the immune checkpoint genes. (G) RT-PCR analysis of CTLA4, HAVCR2, ITPRIPL1, LAG3, PDCD1, and TIGIT mRNA expression in APC7-overexpressing Huh-7 cells. (H) The correlation between APC7 expression and the immune cell infiltration. (I) mIHC analyzed the percentage of Foxp3⁺, CD25⁺, and CD4⁺ positive cells in tumor tissues. (J) Multivariable risk model analysis of the impact of immune cell infiltration and APC7 expression on overall survival. *P < 0.05, **P < 0.01, ***P < 0.001

APC7 is upregulated in HCC and associated with poor prognosis

To investigate the expression of APC7 in HCC, we analyzed the mRNA expression of APC7 based on TCGA_LIHC, ICGC_LIRI and GEO datasets (GSE22058, GSE76427, and GSE54236). Consistently across these datasets, the mRNA expression of APC7 was significantly higher in HCC tissues compared to adjacent tissues (Fig. 3A-3E, Figure S2A). Furthermore, the proteomic data in the CPTAC database also indicated that APC7 in HCC tissues was significantly higher than that in adjacent tissues (Fig. 3F). Kaplan‒Meier analysis revealed that higher expression of APC7 was associated with poorer overall survival in two different HCC datasets, TCGA_LIHC and ICGC_LIRI (Fig. 3G and H). We further analyzed the correlation between the expression of APC7 and clinical characteristics in HCC patients. The results revealed the expression of APC7 was positively correlated with tumor grade and stage, but not related to age or gender (Figure S2B-S2E). Furthermore, ROC analysis demonstrated that APC7 had a certain predictive ability for patient risk, with AUC values of 0.737, 0.655, and 0.581 for 1-year, 3-year, and 5-year survival, respectively (Figure S2F). The univariate and multivariate analyses revealed significant correlations between tumor stage, APC7 mRNA expression and overall survival (Figure S2G, S2H).

Fig. 3.

APC7 was upregulated in HCC and associated with poor prognosis. (A) APC7 mRNA expression in TCGA_LIHC (Adjacent: N = 50, HCC: N = 374). (B) APC7 mRNA expression in TCGA_LIHC paired samples (Adjacent and HCC: N = 50). (C) APC7 mRNA expression in ICGC_LIRI (Adjacent: N = 202, HCC: N = 243). APC7 mRNA expression in GSE22058 (Adjacent: N = 97, HCC: N = 100) (D) and GSE76427 (Adjacent: N = 52, HCC: N = 115) (E). (F) APC7 protein expression in HCC and adjacent tissues (N = 165) from the CTPAC samples. Kaplan-Meier analysis of HCC patients in TCGA (G) and ICGC (H). (I) Representative IHC images of APC7 in HCC and paired adjacent tissues from tissue microarray. (J) IHC scores associated with APC7 expression in HCC and paired adjacent tissues (N = 91). (K) Kaplan–Meier analysis of HCC patients with high or low APC7 expression. Univariate (L)and multivariate (M) Cox regression analyses of HCC patients. **P < 0.01, ***P < 0.001

In the Human Protein Atlas database, the expression of APC7 in HCC was higher than that in adjacent tissues (Figure S2I). Furthermore, we performed immunohistochemical staining to detect the expression of APC7 in an HCC tissue microarray comprised of 91 matched pairs of HCC tumors and their adjacent tissues. The results showed that the protein expression of APC7 was markedly higher in HCC tissues as opposed to paired adjacent tissues (Fig. 3I and J). The correlation between APC7 protein expression and clinicopathological parameters revealed that high APC7 expression was significantly associated with tumor size larger than 5 cm (P = 0.005), grade (P = 0.045) and TNM stage (P = 0.039) (Table 1). Kaplan-Meier analysis revealed higher APC7 protein expression correlated with significantly poorer prognosis (P = 0.0296) (Fig. 3K). Univariate analysis indicated that age (P < 0.001), tumor grade (P = 0.004), TNM stage (P = 0.02) and APC7 protein expression (P = 0.025) were notably related to overall survival (Fig. 3L). We further performed a multivariate Cox regression analysis and confirmed that APC7 expression serves as an independent predictor for overall survival (P = 0.006) (Fig. 3M). Collectively, these findings demonstrate that elevated APC7 expression contributes to HCC malignancy, positioning it as a promising prognostic biomarker.

Table 1.

Correlation of APC7 expression and clinicopathologic features in patients with HCC

Clinicopathological variables	APC7 expression		P value
	High (n = 46)	Low (n = 45)
Gender
Male	41	38	0.508
Female	5	7
Age
< 50 years	10	17	0.094
≥ 50 years	36	28
HBsAg
Positive	30	27	0.607
Negative	16	18
Serum AFP
< 20 (ng/ml)	18	23	0.251
≥ 20 (ng/ml)	28	22
Tumor size
< 5 cm	15	31	0.005
≥ 5 cm	31	14
Grade
1–2	20	29	0.045
3	26	16
TNM stage
1	11	20	0.039
2–3	35	25

HbsAg hepatitis B virus surface antigen, AFP alpha-fetoprotein, TNM tumor-node metastasis. Values in bold indicate statistically significant differences

Co-expression analysis of APC7 in HCC

Based on the median expression level of APC7, we stratified the HCC patients in the TCGA_LIHC cohort by the high and low expression of APC7, resulting in a total of 4431 DEGs. Among them, 4288 were up-regulated DEGs and 143 were down-regulated DEGs, with the screening threshold set at |log₂FC| > 1 and FDR < 0.05. The heatmap showed representative DEGs (Fig. 4A). GO analysis revealed that the aforementioned DEGs were significantly enriched in biological processes such as nuclear division and the meiotic cell cycle, cellular components including condensed chromosomes, and molecular functions like gated channel activity (Fig. 4B). KEGG pathway analysis highlighted enrichments in critical pathways such as the cell cycle, DNA replication, cAMP signaling pathway and cell adhesion molecules (Fig. 4C). Furthermore, GSEA results further supported the above findings, showing that the gene expression profile in TCGA_LIHC with high APC7 expression was significantly enriched for gene sets related to the cell cycle, ABC transporters, DNA replication, ubiquitin mediated proteolysis, and RNA degradation (Fig. 4D). In order to have a more comprehensive understanding of the role of APC7 in the gene regulatory network, we conducted an in-depth analysis of the co-expressed genes of APC7 using the co-expression network. The results showed that APC7 was positively correlated with MCRS1, DENR, SMARCD1, SRRT, KMT5A and NUP37, and negatively correlated with SLC27A5, TAT, LDHD, ADH1B and TTC36 (Fig. 4E). Using the GeneMANIA dataset, we identified APC7 subunit associated molecules, such as CDC16, PIK3CA, CDC20, etc (Fig. 4F). These findings not only deepen our understanding of the oncogenic functions of APC7 but also provide valuable clues and directions for subsequent research.

Fig. 4.

Co-expression analysis of APC7 in HCC. (A) Heatmaps showing the representative DEGs in the high and low APC7 expression groups. GO (B), KEGG (C) and GSAE (D) pathway analysis showing the most enriched pathways of DEGs. (E) The co-expression network of APC7 and its related genes. (F) Interaction gene networks of APC7

Knockdown of APC7 inhibits the malignant phenotype of HCC cells

We investigated the correlation between the expression of APC7 and the functional pathways, and the results showed that there was a significant positive correlation between the expression of APC7 and tumor proliferation signature, DNA replication and epithelial-mesenchymal transition (EMT) markers (Fig. 5A-5C). In addition, we analyzed the CRISPR whole-genome knockout screening dataset from DepMap, and the results revealed that gene effect score for APC7 exhibited negative value, indicating that knockout of APC7 expression suppressed growth or induced death in liver cancer cells (Fig. 5D). The bioinformatic findings strongly suggest a potential role of APC7 in promoting the development of HCC. To experimentally validate these results, we first evaluated mRNA and protein expression of APC7 in various liver cancer cell lines using qRT-PCR and Western blot assays. The results showed that the mRNA and protein expression of APC7 was highly expressed in HCCLM3 and HepG2 and lowly expressed in Huh-7 and MHCC97H (Fig. 5E and F).

Fig. 5.

APC7 knockdown inhibited the malignant phenotype of HCC cells. Correlation analysis of APC7 expression with tumor proliferation (A), DNA replication (B), and EMT markers (C). (D) Gene effect score for APC7 in HCC cell lines from CRISPR screens. (E) qRT-PCR analysis of APC7 mRNA expression in six HCC cell lines. (F) Western blot analysis of APC7 protein expression in six HCC cell lines. (G-H) qRT-PCR and Western blot analysis to validate the knockdown of APC7 in HCCLM3 cells. The effect of siRNA knockdown of APC7 on the proliferation (I), migration and invasion (J), cell cycle (K) of HCC cells in vitro. *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean ± SD collected from three independent experiments

Furthermore, to elucidate the role of APC7 in HCC, APC7 knockdown was performed in the HCCLM3 cell line. We verified the interference efficiency of siRNA targeting the expression of APC7 at the mRNA and protein levels (Fig. 5G and H and Figure S3A, S3B). CCK-8 assay unequivocally demonstrated that APC7 knockdown significantly inhibited the proliferation of HCC cells (Fig. 5I). The results of transwell assay indicated that the migration and invasion abilities of HCC cells were significantly reduced after APC7 gene knockdown (Fig. 5J). The cell cycle assay revealed the oncogenic properties of APC7 in HCC cells. Knockdown of APC7 significantly inhibited the transition from the G1 to S phase, thereby impeding cell cycle progression (Fig. 5K). In addition, the apoptosis assay demonstrated that APC7 did not exert a discernible impact on the level of apoptosis (Figure S3C), highlighting the promoting effect of APC7 on the proliferation and motility of HCC cells rather than cell death.

Overexpression of APC7 promotes the malignant phenotype of HCC cells

We established stable overexpression of APC7 in Huh-7 and MHCC97H cell lines and validated the overexpression efficiency by qRT-PCR and western blot (Fig. 6A and B and Figure S4A). Subsequently, we employed the CCK-8 assay to investigate the proliferative capacity of HCC cells following APC7 overexpression. The results unequivocally showed that APC7 overexpression significantly promoted cell proliferation in both HCC cell lines (Fig. 6C). Transwell assays revealed a striking enhancement in migration and invasion abilities upon APC7 overexpression in Huh-7 and MHCC97H cells (Fig. 6D). Cell cycle analysis demonstrated that APC7 overexpression led to a reduction in the proportion of cells in the G1 phase and an increase in the proportion of cells in the S phase, thereby facilitating the G1/S phase transition in both cell lines (Fig. 6E). Consistent with the results of the APC7 knockdown, the apoptosis assay did not show any significant changes in the proportion of apoptotic cells between the APC7 overexpression and control groups (Figure S4B). Furthermore, we analyzed the expression of key regulatory proteins involved in cellular behavior by Western blot. In the HCC cells with overexpression of APC7, the level of the proliferation marker PCNA was increased. Concurrently, changes in EMT markers were observed, characterized by downregulation of E-cadherin and upregulation of N-cadherin. Critically, and in full support of the cell cycle data, the expression of Cyclin D1, a key promoter of the G1/S transition, was upregulated. Meanwhile, the opposite was observed in APC7-knockdown HCCLM3 cells (Figure S4C, S4D). Overall, our integrated approach, combining bioinformatic analysis and experimental validation, provides compelling evidence for the pivotal role of APC7 in driving the malignant progression of HCC cells.

Fig. 6.

APC7 overexpression promoted the malignant phenotype of HCC cells. qRT-PCR (A) and Western blot analysis (B) to validate the overexpression of APC7 in Huh-7 and MHCC97H cells. The effect of overexpression APC7 on the proliferation (C), migration and invasion (D), cell cycle (E). *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean ± SD collected from three independent experiments

APC7 promotes HCC tumor growth in vivo

To further investigate the function of APC7 in vivo, we established a xenograft tumor model by subcutaneously inoculating Huh-7-APC7 and HCCLM3-shAPC7 cells into nude mice. The results showed that overexpression of APC7 significantly promoted the increase in tumor volume and weight in the xenograft mice (Fig. 7A-7C). Immunohistochemical analysis further confirmed that Ki67 expression was elevated after APC7 overexpression (Fig. 7D). Conversely, knockdown of APC7 markedly suppressed tumor volume and weight in mice (Fig. 7E-7G), accompanied by a reduction in Ki67 expression (Fig. 7H). Taken together, these data indicate that APC7 also significantly promotes the development of HCC in vivo. We established a tumor metastasis model, MHCC97H or HCCLM3 cells, which stably express firefly luciferase and feature either APC7 overexpression or silencing, were injected into the tail veins of nude mice, and discovered that luciferase intensity in the lung was significantly increased by APC7 overexpression while depressed by APC7 knockdown (Fig. 7I). These findings further support the role of APC7 in promoting HCC metastasis in vivo.

Fig. 7.

APC7 promotes HCC tumor growth in vivo. (A) Representative tumor tissue image showing APC7 overexpression. (B) Tumor volume was observed in the APC7 overexpression group. (C) Tumor weights were measured on 19 days in the APC7 overexpression group. (D) Hematoxilin-eosin (H&E) staining and IHC analysis detection of Ki-67 and APC7 expression in tumor tissues of xenograft nude mouse models with APC7 overexpression. (E) Representative tumor tissue image showing APC7 knockdown. (F) Tumor volume was observed in the APC7 knockdown group. (G) Tumor weights were measured on days 19 in the APC7 knockdown group. (H) Hematoxilin-eosin (H&E) staining and IHC analysis detection of Ki-67 and APC7 expression in tumor tissues of xenograft nude mouse models with APC7 knockdown. (I) Effects of APC7 knockdown or overexpression on lung metastasis in NCG mice (N = 5). *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean ± SD collected from three independent experiments

APC7 suppresses the Hippo pathway through ubiquitin-dependent degradation of LATS1

To further investigate the mechanism by which APC7 promotes HCC progression, we performed RNA sequencing to identify its downstream regulatory genes. The screening threshold for identifying differentially expressed genes is set at FDR < 0.05 and |log₂FC| > 1, and the results showed that overexpression of APC7 led to the downregulation of 326 genes and the upregulation of 951 genes, respectively (Fig. 8A). DEGs in representative pathways are shown as heatmaps (Fig. 8B). GO analysis of differentially expressed genes revealed that cell periphery (P = 3.798e-09), regulation of biological quality (P = 6.598e-09) and regulation of cell differentiation (P = 3.403e-08) were significantly enriched (Figure S5). KEGG enrichment analysis based on the RNA-seq data indicated significant enrichment of the Hippo signaling pathway (P = 0.011), PPAR signaling pathway (P = 3.11e-04), ABC transporters (P = 0.016) and TGF-beta signaling pathway (P = 0.040) (Fig. 8C). GSEA enrichment indicated that APC7 expression was positively associated with Hippo signaling pathway (P = 0.008), IL-17 signaling pathway (P = 0.002), and NF-kappa B signaling pathway (P = 0.022) (Fig. 8D). APC/C^Cdh1 plays a critical role in the intrinsic regulation of the Hippo signaling pathway during cell cycle progression [24] and we mainly focused on the regulation of the Hippo pathway by APC7. To validate these findings, we examined changes in key molecules of the Hippo pathway in APC7-knockdown and APC7-overexpression cell lines. The results demonstrated that APC7 knockdown significantly increased YAP phosphorylation levels while markedly decreasing total YAP and TAZ protein levels, whereas APC7 overexpression exhibited the opposite trend (Fig. 8E and Figure S6A, S6B). Further immunofluorescence experiments confirmed that in APC7-knockdown cells, YAP remained localized in the cytoplasm (Fig. 8F). Conversely, APC7 overexpression promoted the translocation of YAP into the nucleus (Fig. 8F). These results demonstrate that APC7 exerts its functions by modulating the activity of the Hippo signaling pathway.

Fig. 8.

APC7 suppressed the Hippo pathway through ubiquitin-dependent degradation of LATS1. (A) Volcano plots showing the DEGs regulated by the overexpression of APC7 analyzed by RNA-seq (up: 951, down: 326). (B) Heatmaps showing the representative DEGs regulated by the overexpression of APC7 analyzed by RNA-seq. KEGG (C) and GSAE (D) pathway analysis showing the most enriched pathways of DEGs identified by RNA-seq. (E) Western blot analysis of APC7 overexpression and knockdown effects on the Hippo signaling pathway. (F) Immunofluorescence detection of YAP localization in APC7 knockdown and overexpression cell lines. Scale bars: 40 μm. (G) Co-immunoprecipitation analysis of CDH1 or CDC20 interacting with APC7. (H) Immunoprecipitation combines with western blot to analyze the effects of APC7 knockdown on LATS1 ubiquitination in the presence of MG132 (10 µM). (I) Immunoprecipitation combines with western blot to analyze the effects of APC7, CDH1 or CDC20 knockdown on LATS1 ubiquitination in the presence of MG132 (10 µM). (J) Western blot analysis of the interaction between CDH1 and LATS1 after APC7 knockout. (K) Immunoprecipitation combined with western blot was used to analyze the effect of APC7 knockout on CDH1-mediated LATS1 ubiquitination. (L) Western blot analysis of APC7, CDH1 or CDC20 knockdown effects on the Hippo signaling pathway. (M) Immunoprecipitation combines with western blot to analyze APC7-mediated LATS1 ubiquitination sites. (N) Western blot analysis of pathway recovery following LATS1/2 knockdown. (O) Effect of APC7 overexpression and YAP interference on hepatocyte proliferation assessed by CCK8 assay. (P) Western blot analysis of APC7 and Hippo pathway molecules expression in HCC and adjacent tissues

APC/C exerts ubiquitination functions through interactions with two activating factors, CDH1 and CDC20, which guide APC/C to target specific substrates at different stages of the cell cycle [12, 25]. Therefore, whether APC7, as the TPR subunit of the APC/C, regulates downstream pathways by binding to CDH1 or CDC20 to mediate ubiquitination remains unclear and requires further investigation. We further confirmed the interaction of APC7 with CDH1 and CDC20 via CO-IP experiments (Fig. 8G). While the LATS1/2 kinase cascade suppresses YAP/TAZ by promoting their degradation, this suppression is reversed in tumors due to the ubiquitin-mediated degradation of LATS1/2 itself, thereby unleashing the YAP/TAZ oncogenic pathway [26]. Therefore, we conducted a ubiquitination assay to investigate the effect of APC7 on the ubiquitination of LATS1/2. The result showed that APC7 knockdown reduced the ubiquitination levels of LATS1 and LATS2 (Fig. 8H, Figure S6C). To verify whether the ubiquitin function of APC7 depends on CDH1 or CDC20, we conducted ubiquitination assay and the results showed that knockdown of CDH1 had the same effect as knockdown of APC7, both reducing the ubiquitination level of LATS1, whereas knockdown of CDC20 had no effect on LATS1 ubiquitination (Fig. 8I). Subsequently, we examined the interaction between CDH1 and LATS1 with APC7 knockdown in HCCLM3 cells. The results showed that after APC7 knockdown, the interaction between CDH1 and LATS1 was significantly weakened (Fig. 8J). Ubiquitination assays revealed that in APC7 knockdown cells, overexpression of CDH1 failed to restore LATS1 ubiquitination levels (Fig. 8K). Similarly, knockdown of CDH1 or APC7 significantly upregulates the expression of LATS1/2, leading to increased phosphorylation of YAP. This is accompanied by a decrease in the total protein levels of both TAZ and YAP, thereby modulating the activation state of the Hippo signaling pathway. However, knockdown of CDC20 did not affect this pathway (Fig. 8L and Figure S6D). These results indicate that APC7 interacts with CDH1 to recruit the substrate LATS1 and mediate its ubiquitination, thereby regulating the Hippo signaling pathway.

More importantly, APC7-mediated ubiquitination sites by LATS1 were explored in further experiments. First, through the PhosphoSitePlus database, the ubiquitination sites of LATS1 on the Pkinase domain were identified as K728, K734, K860, K976, and K1005 (Figure S6E). The IUUCD 2.0 database identified the ubiquitination sites of LATS1 as K688, K860, and K1005 (Figure S6F). By constructing site-directed mutagenesis plasmids for the shared ubiquitination sites, namely K860A and K1005A. We found that the ubiquitination level of LATS1 was reduced in the K860A group, while there was no significant difference in the K1005A group, indicating that the K860 is the ubiquitination site where APC7 mediates the ubiquitination of LATS1 (Fig. 8M). Furthermore, rescue experiments confirmed that simultaneous knockdown of LATS1 and LATS2 reversed the regulatory effects of APC7 knockdown on this pathway (Fig. 8N, Figure S6G). The CCK-8 assay confirmed that YAP knockdown and the YAP inhibitor verteporfin could inhibit the proliferation of Huh-7 cells induced by APC7 overexpression (Fig. 8O, Figure S6H, S6I). Finally, we validated these findings in human HCC tissue samples, which revealed that APC7 was highly expressed in tumor tissues compared to adjacent normal tissues. The expression of LATS1 and pYAP(S127) in tumor tissues was significantly lower than in adjacent normal tissues, further corroborating the results from the cell experiments (Fig. 8P, Figure S6J).

Based on the aforementioned research, our findings revealed that APC7 promotes HCC progression by interacting with CDH1 to mediate the ubiquitination and degradation of LATS1. This event attenuates the LATS1 mediated inhibition of YAP/TAZ, leading to the activation of the Hippo signaling pathway and ultimately driving malignant progression of HCC.

Discussion

Accumulating evidence has highlighted the critical roles of various APC/C subunits in tumorigenesis. For instance, in urothelial bladder cancer, APC11 exerts its oncogenic function by mediating the ubiquitination-dependent degradation of FOXO3, leading to downregulation of p21 and GULP1 [27]. In non-small cell lung cancer, APC10 interacts with GAC to regulate glutamine metabolism and induce autophagy [28]. CDC23 promotes cancer progression in lung cancer cell lines by modulating the expression of epithelial-mesenchymal transition (EMT) markers [29], whereas CDC27 facilitates colorectal cancer cell proliferation through the ID1-p21 signaling axis [30]. Although the APC/C complex have been demonstrated to be involved in the malignant behaviors of tumors [31–34], the pathological functions of the TPR lobe subunit, APC7, in the APC/C complex remain largely unexplored. In this study, we performed differential expression analysis of APC/C subunits in a pan-cancer dataset and found that, considering factors such as fold change and statistical P-value, APC7 was the most significantly up-regulated subunit in patients with HCC compared to adjacent normal tissues. Moreover, our HCC tissue microarray analysis consistently demonstrated that APC7 was up-regulated in HCC and associated with the tumor size. In addition, elevated APC7 was related to poorer prognosis. The univariate and multivariate analyses confirmed that APC7 protein expression serves as an independent predictor for overall survival. These results suggest that the potential of APC7 as a novel prognostic biomarker for HCC patients and indicate the tumor-promoting function of APC7 in HCC progression.

APC7 is a component of the APC/C, which is a cell cycle-regulated E3 ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle [35]. Although APC7 has been implicated in various cancers [22, 23, 36], such as esophageal squamous cell carcinoma, gastric cancer, and colorectal cancer, the mechanisms underlying its pro-oncogenic effects remain unclear. Moreover, the role of APC7 in HCC has not been investigated. We found that the expression of APC7 was significantly correlated with tumor proliferation signature, DNA replication and EMT markers based on TCGA_LIHC database. The CRISPR whole-genome knockout screening database indicated that the deletion of APC7 could inhibit the growth of HCC cells. In this study, loss-of‐function and gain‐of‐function assays indicated that enhanced expression of APC7 promoted HCC cell proliferation, migration, invasion, cell cycle progression and the ability to form tumors subcutaneously in nude mice.

As crucial regulators of the immune system, immune checkpoints are primarily categorized into two groups: stimulatory and inhibitory immune checkpoint molecules [37]. Among these, inhibitory checkpoint molecules typically function by suppressing T cell activation and proliferation [38]. Tumor cells can exploit this mechanism of inhibitory checkpoints to evade immune surveillance and attack. Our results demonstrated that the expression of immune checkpoint‑related molecules (CTLA4, HAVCR2, ITPRIPL1, LAG3, PDCD1, and TIGIT) was upregulated upon APC7 overexpression. Concurrently, APC7 overexpression markedly increased the infiltration of Foxp3⁺, CD25⁺, and CD4⁺ Treg cells. Furthermore, our RNA-seq data indicated that high expression of APC7 activated the Hippo signaling pathway. Increasing evidence suggests that the Hippo/YAP pathway also plays a key role in regulating the tumor immune microenvironment. For example, activation of YAP/TAZ has been reported to directly upregulate the expression of immune checkpoint molecules such as programmed death-ligand 1 (PD-L1), thereby helping tumor cells evade immune surveillance [39]. Additionally, YAP signaling can regulate the expression of various chemokines and cytokines (e.g., CCL2, CXCL8), which can recruit immunosuppressive cell subsets such as regulatory T cells (Tregs) and tumor-associated macrophages (TAMs) to the tumor site [40]. However, whether APC7 modulates the tumor immune microenvironment through the Hippo pathway remains to be substantiated by further evidence.

Previous studies have shown that activation of the APC/C complex primarily relies on two adaptor proteins—CDC20 or CDH1. Both adaptor proteins facilitate the recruitment of substrates to APC/C, thereby activating its E3 ubiquitin ligase activity. Specifically, CDC20 activates APC/C from metaphase to anaphase, mediating the degradation of substrates such as Cyclin B and securin. In contrast, CDH1 functions from late anaphase through G1 phase, promoting APC/C-mediated substrate recognition and degradation. These observations indicate that CDC20 and CDH1 exhibit distinct substrate regulatory patterns [41]. Our findings indicate that APC7 binds to both CDC20 and CDH1. Importantly, APC7-mediated ubiquitination and degradation of LATS1, a key regulator of the Hippo pathway, is critically dependent on CDH1 but not CDC20. Future studies are needed to determine whether APC7 utilizes CDC20 to target other substrates.

The mammalian Hippo pathway comprises core components including the Ste20-like kinases MST1 and MST2, the scaffold protein Salvador, the large tumor suppressor kinases LATS1/2 (homologs of Warts), and the transcriptional coactivators YAP and TAZ (homologs of Yorkie, with TAZ also designated as WWTR1). Within this signaling cascade, MST1/MST2 kinases activate LATS1/2, which subsequently phosphorylate YAP/TAZ [42]. This phosphorylation leads to cytoplasmic retention and proteasomal degradation of YAP/TAZ, thereby executing their growth-suppressive function. In this study, RNA-seq analysis identified the Hippo signaling pathway as a potential downstream target of APC7. We experimentally demonstrated that APC7 interacts with the coactivator CDH1 to promote the K860 ubiquitination-mediated degradation of LATS1. Mutation of the K860 residue significantly reduced, but did not abolish, the ubiquitination level of LATS1, suggesting that additional lysine residues on LATS1 may also serve as ubiquitination sites. This degradation relieves the inhibitory phosphorylation of YAP, resulting in their transcriptional activation and consequent acceleration of HCC progression. In rescue experiments, concurrent knockdown of LATS1/2 reversed the tumor-suppressive effects elicited by APC7 depletion, providing compelling evidence for the functional hierarchy within this regulatory axis.

Although our findings suggest that APC7 may serve as a potential therapeutic target for HCC, as a subunit of the APC/C complex involved in cell cycle regulation, targeting APC7 could interfere with the normal activity of the complex, thereby affecting the proliferation and division of normal cells and leading to potential off-target effects. Therefore, its selectivity, specificity, and potential toxicity risks require comprehensive evaluation. Future research should focus on developing more specific targeting strategies, such as targeting the interaction interface between APC7 and its substrates, to minimize adverse effects on normal cells. Additionally, we will conduct studies with larger, multicenter sample cohorts to validate the reliability of APC7 as a prognostic marker for HCC.

Conclusions

In summary, our work uncovers a previously unelucidated oncogenic function of APC7 in HCC and delineates a complete signaling axis from ubiquitin-mediated degradation to transcriptional activation. These findings not only extend our understanding of APC/C biology beyond its canonical roles but also establish APC7 as a promising diagnostic and therapeutic target in hepatocellular carcinoma.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Author contributions

YS and ZKL drafted the manuscript. YS, RYZ, YLG, DW (Dong Wu), HLW, ZCT, MMH collected and analyzed the data. YS, DW (Dong Wu) and CL performed the cell and animal experiments involved in this study. RYZ, YLG, YTW, ZYF, CZ and WLL drew the figures and tables. YS, DW (Ding Wei), HJB and ZKL revised the manuscript. YS, HJB and ZKL participated in the design and coordination. All authors read and approved the final manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant numbers: 82130084 and 82203336), the Project Program of the State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers (Fourth Military Medical University) (2025GTEP008), the China Postdoctoral Science Foundation (2022TQ0088 and 2024M754270).

Data availability

RNA-sequencing data have been uploaded to GEO (No. GSE319107). The original contributions presented in the study are included in the article/Supplementary material. Further inquiries can be directed to the corresponding authors.

Declarations

Ethics approval and consent to participate

In accordance with the Declaration of Helsinki, this study was approved by the Ethical Committee of Shanghai Outdo Biotech Company (HLivH180Su30, HLivH180Su31). All animal procedures conformed to the ethics and welfare of Institutional Animal Care and Use Committee (IACUC) of National Translational Science Center for Molecular Medicine and were authorized by the Animal Ethics Committee of the National Translational Science Center for Molecular Medicine, Fourth Military Medical University (2025-NTSCMM-ID003).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.