Knockdown of TRIM35 suppresses cell proliferation and metastasis by modulating the PPAR signalling pathway in papillary thyroid cancer cells

Introduction

Thyroid cancer has undergone a pronounced rise in incidence in recent years, ranking seventh among all cancers by 2022 (1). Papillary thyroid cancer (PTC) accounts for approximately 85% of cases. Despite a high survival rate exceeding 90% following surgery (total or partial thyroidectomy) and radioiodine (I-131) treatment, 10–20% of patients with PTC experience distant metastasis or local recurrence (2). Two-thirds of these cases may progress to radioiodine-refractory PTC (3). Investigating the molecular mechanisms underlying PTC development could provide valuable insights for its diagnosis, treatment, and prognosis.

The tripartite motif-containing (TRIM) family represents a key class of regulators involved in multiple biological processes, such as cell death, innate glucose metabolism, and immune responses (4,5). Earlier studies have emphasized their roles in cancer development, influencing cell cycle regulation, apoptosis, differentiation, metabolism, and immune modulation (6,7). TRIM35, a member of the TRIM family, implicated in tumour suppression in breast cancer and hepatocellular carcinoma (8-10). In non-small cell lung cancer, it enhances anti-tumour immunity via K63-linked ubiquitination-mediated inhibition of lysine-specific histone demethylase 1A (11). At present, the functional role and underlying regulatory mechanisms of TRIM35 in PTC are not clear.

Otto Warburg observed that cancer cells exhibited increased glucose consumption and lactate production, even when oxygen is available, a phenomenon referred to as aerobic glycolysis or the Warburg effect (12,13). Glycolysis can provide essential precursors, such as amino acids and nucleotides, for tumour growth (14,15). Excess lactate production creates an acidic microenvironment that protects malignant cells and promotes tumour proliferation and metastasis (14,16). Key proteins associated with aerobic glycolysis, including pyruvate kinase M2, hexokinase (HK), and lactate dehydrogenase (LDH), have been linked to PTC growth and metastasis (17-19). This underscores the importance of further investigation into the regulatory mechanisms and therapeutic targets associated with aerobic glycolysis in PTC.

This study aimed to examine TRIM35 expression in PTC tissues, to evaluate its functional influence on cell proliferation, metastatic potential, and glycolysis in PTC cell lines, and to explore the associated mechanisms through transcriptomic analysis of TRIM35-knockdown cells. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2769/rc).

Methods

Specimen collection

A total of 20 paraffin-embedded tissue samples, comprising PTC lesions and paired adjacent non-neoplastic tissues, were procured from the archives of the Pathology Department at our institution. These specimens were collected from patients diagnosed with PTC during the period from February to December 2024. The cohort included 13 females and 7 males, aged 25–75 years old. TNM staging distribution was as follows: 3 specimens in stage I, 9 in stage II, 6 in stage III, and 2 in stage IV. Inclusion criteria were: (I) pathologically confirmed PTC; (II) TNM stages I–IV; and (III) voluntary participation with informed consent. Exclusion criteria included other malignancies, organ dysfunction, thyroid disorders, or a history of recent medication use that could affect thyroid function. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Medical Ethics Committee of Longhua District People’s Hospital (No. 2024-098) and informed consent was taken from all the patients.

Immunohistochemistry

Paraffin sections were first dewaxed, and antigen retrieval was carried out using citric acid solution. Following, slides underwent endogenous peroxidase inactivation with 3% H₂O₂ and were blocked with serum. Subsequently, they were incubated overnight at 4 °C with a primary antibody against TRIM35 (#40249, Signalway Antibody) at a 1:50 dilution in 10% goat serum. After overnight incubation, slides were washed and then incubated with a goat anti-rabbit IgG secondary antibody at 37 °C for 45 minutes. After a further rinse, 3,3’-diaminobenzidine (DAB) staining was performed using 50 µL of freshly prepared DAB solution, and the reaction was stopped by rinsing with water. Nuclei were then counterstained with haematoxylin, differentiated with hydrochloric acid alcohol, and washed thoroughly. Finally, slides were briefly incubated with a bluing solution, washed, and mounted for microscopic analysis. Random images were captured after scanning. For each experiment, a known positive tissue section was included as a positive control, and the primary antibody was omitted for the negative control. A semi-quantitative assessment of TRIM35 protein expression was conducted as previously defined (20). The positivity rate of the cells and staining intensity were assessed, and the total TRIM35 expression score was calculated by multiplying these values. PTC patients were stratified into TRIM35 high- and low-expression groups based on immunohistochemistry scores using a cutoff score of 4 (20).

Cell transfection

TPC-1 (catalog number: MZ-2159) and K1 (catalog number: MZ-0824) cells were purchased from Ningbo Mingzhou Biotechnology Co., LTD (Ningbo, China). Two specific small interfering RNAs (siRNAs) targeting TRIM35 mRNA, namely siRNA-TRIM35-1 and siRNA-TRIM35-2, along with an siRNA used as a negative control (siRNA-NC) (Suzhou GenePharma, Suzhou, China), were transfected into TPC-1 and K1 cells using Hieff Trans^®in vitro siRNA/miRNA Transfection Reagent (YEASEN, Shanghai, China).

Detection of TRIM35 mRNA expression levels

TRIM35 mRNA expression analysis was conducted as described previously (20), including steps for total RNA extraction, complementary DNA (cDNA) synthesis, and quantitative polymerase chain reaction (PCR). The sequences of primers used for quantitative PCR analysis are listed below: TRIM35 (forward: 5'-GCTCATCATGTCCCAGGAAG-3'; reverse: 5'-CCCCTTCACACACTGACATC-3') and 18S ribosomal RNA (forward: 5'-CCTGGATACCGCAGCTAGGA-3'; reverse: 5'-GCGGCGCAATACGAATGCCCC-3'). Relative TRIM35 expression was calculated via the 2^−ΔΔCt method with normalization to 18S ribosomal RNA. Three independent experiments were performed with three technical replicates.

Western blot

Total protein from 1×10⁶ cells was isolated using 1 mL of radioimmunoprecipitation assay buffer lysis buffer, which contained 10 µL of PMSF (100 mM) and 10 µL of a cocktail of protease inhibitors. After quantifying the protein concentration using a BCA Protein Content Assay Kit (KeyGEN Biotech, Nanjing, China), sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to separate the proteins. Following electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane. The membrane was rinsed with tris-buffered saline with tween 20 (TBST) for 5 min, followed by blocking with 5% non-fat milk solution at room temperature for 2 h. The membrane was washed once with TBST for 8 min. It was then incubated overnight at 4 °C with primary antibody. The primary antibodies used were: anti-TRIM35 (#40249, Signalway Antibody), anti-PPARγ (#ab209350, Abcam, Cambridge, UK), and anti-CD36 (#ab252923, Abcam), all at a 1:1,000 dilution. The next day, the membrane was washed with TBST three times for 8 min each and subsequently incubated with goat anti-rabbit IgG-horseradish peroxidase (HRP) at 37 °C for 50–60 min. After washing the membrane with TBST three times for 8 min each, Immobilon Western Chemilum HRP substrate (Millipore, Billerica, MA, USA) was applied to the membrane, and the chemiluminescence signal was scanned using the ChemiDoc MP Universal Imaging System (Bio-Rad, Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal reference and was detected using an HRP-conjugated antibody (Shanghai Kangcheng Biosciences, Cat# KC-5G5). Three independent experiments were performed with one technical replicates. The integrated optical density of each band was quantified using Image Pro-Plus 6.0 software (Media Cybernetics, Rockville, MD, USA), and the relative expression level of each protein was calculated as the ratio to the GAPDH.

Transwell cell migration assay

Following a 24-hour transfection period, 1×10⁵ cells suspended in 100 µL serum-free medium were introduced into the upper chamber. The lower chamber was then filled with 600 µL of cell-free complete medium. The setup was placed in a 5% CO₂ and 37 °C environment for 24 h. After incubation, cells that did not migrate through the membrane were carefully removed. Then, cells adhered to the membrane were fixed with 4% paraformaldehyde for 15 minutes. After rinsing, cells were stained with crystal violet for 10 minutes. Following a final wash, migrated cells were quantified and imaged for statistical analysis. Three independent experiments were performed with one technical replicates.

Matrigel-based Transwell cell invasion assay

This experiment was conducted using a protocol analogous to the Transwell cell migration assay, with the additional step of pre-coating the upper chamber with Matrigel (Corning, New York, USA). Three independent experiments were performed with one technical replicates.

Wound healing assay

Guide lines were marked on the bottom of the culture plates to aid in scratch alignment. Cells were harvested, resuspended in complete medium, and plated to reach appropriate density overnight. A sterile pipette tip was used to create uniform scratches along the guide lines, and the plates were gently rinsed to remove detached cells. To restrict cell proliferation, cells were cultured in serum-free medium in a 5% CO₂ atmosphere at 37 °C. Images of the scratch area was photographed at 24 h. The scratch area at 0 h and 24 h was measured using Image Pro-Plus 6.0 software and the wound healing rate was determined using the following equation: wound healing rate = (scratch area at 0 h – scratch area at 24 h)/scratch area at 0 h. Three independent experiments were performed with one technical replicates.

Cell proliferation analysis

This assay was conducted was using the Cell Counting Kit-8 (KeyGEN Biotech). Optical density (OD) at 450 nm (OD 450 nm) was recorded at 0, 24, 48, and 72 h after transfection. The OD 450 nm values were used to calculate proliferation rates. Three independent experiments were performed with three technical replicates.

Enzyme-linked immunosorbent assay (ELISA)

HK and lactate dehydrogenase A (LDHA) levels in cell lysate supernatants were quantified using the human HK ELISA Kit (KL-E11082, Shanghai Kang Lang Biological Technology Co., Ltd., Shanghai, China) and the human LDHA ELISA Kit (E-EL-H0556, Elabscience, Wuhan, China). Three independent experiments were performed with three technical replicates.

Measurement of lactate levels

The CheKine Micro Lactate Assay Kit (KTB1100, Abbkine, Wuhan, China) was used to quantify lactate levels in collected culture medium supernatants. Three independent experiments were performed with three technical replicates.

Transcriptome sequencing

K1 cells transfected with siRNA-NC or siRNA-TRIM35-1 (n=3) were harvested for transcriptome sequencing performed by Suzhou Genewiz Biotechnology Co., Ltd. (Suzhou, China). Following RNA extraction, mRNA was enriched and processed into cDNA. After library construction, the products were amplified and sequenced on an Illumina NovaSeq 6000 platform (2×150 PE). Raw reads were processed with Cutadapt, aligned to the reference genome using HISAT2, and quantified with HTSeq. Differentially expressed genes (DEGs) were identified using DESeq2 with thresholds of |log2FC| ≥1 and FDR <0.05. Subsequently, functional enrichment analysis of the DEGs was performed, interpreting their biological roles by mapping to Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

Statistical analysis

The bar graph data are presented as means ± standard deviation. The statistical significance of differences between two or more groups was evaluated using a t-test or one-way analysis of variance. Associations between TRIM35 expression and key clinicopathological features were analysed using Fisher’s exact test. A P value of less than 0.05 was defined as statistically significant. GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA) was used in statistical analysis.

Results

TRIM35 expression features in PTC tissue

All tissue samples exhibited positive TRIM35 expression. Representative immunohistochemical staining results for TRIM35 are shown in Figure 1A. Statistical analysis revealed a significantly increase in TRIM35 levels in cancerous tissues (Figure 1B). As shown in Table 1, the results of Fisher’s exact test revealed that high TRIM35 expression was significantly associated with larger tumour size (P=0.003) and the presence of lymphovascular invasion (P=0.03). No significant correlations were observed between TRIM35 expression and age, gender, TNM stage, or lymphocytic thyroiditis (all P>0.05).

Figure 1 Expression levels of TRIM35 in PTC cancerous tissues compared to adjacent normal tissues detected by immunohistochemistry. (A) Representative images illustrating high and low TRIM35 expression levels. (B) Statistical analysis of immunohistochemistry score of TRIM35 in cancerous versus normal tissues. PTC, papillary thyroid cancer; TRIM35, tripartite motif-containing 35.

TRIM35 knockdown inhibits PTC cell proliferation

The role of TRIM35 in PTC was explored by silencing its expression in K1 and TPC-1 cell lines using two distinct siRNAs to minimise off-target effects. Both siRNAs effectively reduced TRIM35 expression, as shown in Figure 2A,2B. Transfection with siRNA-TRIM35-1 or siRNA-TRIM35-2 significantly reduced cell proliferation rate, indicating that TRIM35 knockdown effectively suppresses cellular growth (Figure 2C).

Figure 2 TRIM35 knockdown suppresses the proliferation of K1 and TPC-1 cells. (A,B) Transfection with siRNA-TRIM35-1 and siRNA-TRIM35-2 reduces TRIM35 mRNA and protein expression levels. For panel B, the right bar graph is the statistical results of the relative expression levels of TRIM35. (C) Both siRNAs significantly decrease the cell proliferation rate. The symbol ‘&’ indicates a P value of less than 0.05 compared to siRNA-NC. siRNA-NC, negative control siRNA; TRIM35, tripartite motif-containing 35.

TRIM35 knockdown suppresses PTC cell migratory and invasive properties

The impact of TRIM35 knockdown on the migratory and invasive properties of K1 and TPC-1 cells was assessed through wound healing, Transwell migration, and Matrigel-based Transwell invasion assays. Results showed that transfection with siRNA-TRIM35-1 or siRNA-TRIM35-2 significantly reduced migrated cell number (Figure 3A), decreased the wound healing rate (Figure 3B), and diminished invaded cell number (Figure 3C). These results suggest that TRIM35 knockdown effectively inhibits both migration and invasion of PTC cells.

Figure 3 TRIM35 knockdown suppresses the migratory and invasive properties of K1 and TPC-1 cells. (A,B) Results from the Transwell cell migration assay and wound healing assay show that both siRNAs significantly impaired cell migratory ability. (C) The Matrigel-based Transwell cell invasion assay demonstrated that both siRNAs markedly reduced cell invasive capacity. The symbol ‘&’ indicates a P value of less than 0.05 compared to siRNA-NC. siRNA-NC, negative control siRNA; TRIM35, tripartite motif-containing 35.

TRIM35 knockdown suppresses glycolysis

To assess the role of TRIM35 in glycolysis, lactate levels and expression levels of glycolytic enzymes HK and LDHA were measured following siRNA-mediated TRIM35 knockdown. Both siRNAs significantly reduced lactate levels in the culture medium and decreased the levels of HK and LDHA in cell lysate supernatants (Figure 4). These findings indicate that TRIM35 silencing suppresses glycolysis in PTC cells.

Figure 4 TRIM35 knockdown decreases lactate levels and the expression levels of HK and LDHA. (A) Lactate levels in culture medium supernatant. (B,C) Levels of HK and LDHA in cell lysate supernatant. The symbol ‘&’ indicates a P value of less than 0.05 compared to siRNA-NC. HK, hexokinase; LDHA, lactate dehydrogenase A; siRNA-NC, negative control siRNA; TRIM35, tripartite motif-containing 35.

Identification of DEGs in TRIM35-knockdown PTC cells

To further explore the molecular regulatory mechanism by which TRIM35-knockdown inhibits malignant behaviour and glycolysis in PTC cells, we identified differentially expressed mRNAs in TRIM35-knockdown K1 cells using transcriptome sequencing and the raw data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE308716. A heatmap showing the expression patterns of the DEGs is presented in Figure 5A. Among the DEGs, 75 were downregulated and 103 were upregulated (Figure 5B). The fold changes of the top 20 identified known genes were shown in Table 2. Subsequently, we performed GO and KEGG pathway analyses on these DEGs. GO analysis indicated that the biological processes involving these genes were associated with cell migration (Figure 5C). KEGG pathway analysis revealed that the DEGs were related to the synthesis and metabolism of various substances, especially lipid (Figure 5D). Additionally, these genes were enriched in the PPAR signalling pathway (Figure 5D). As members of the nuclear receptor superfamily, PPARs participate in the regulation of lipid metabolism and serve as an important link between lipid metabolism and cancer progression (21). Based on the above analysis, we hypothesize that TRIM35 may influence the occurrence and development of PTC by regulating PPAR-mediated lipid metabolism.

Figure 5 Identification of DEGs in TRIM35-knockdown K1 cells. (A) Heatmap showing the expression profiles of DEGs. (B) Volcano plot displaying the distribution of DEGs (log₂ fold change versus −log₁₀ P value). (C) Bar chart of the top 20 enriched GO terms. (D) Bar chart of the top 20 enriched KEGG pathways. DEGs, differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; TRIM35, tripartite motif-containing 35.

TRIM35 knockdown inhibits PPAR signal pathway

To further analyze the relationship between TRIM35 and the PPAR signalling pathway, we detected the expression of PPARγ and CD36 in TRIM35-knockdown K1 and TPC-1 cells. PPARγ is an important member of the PPAR family, and CD36 is a classical target gene of PPARγ, as well as a key fatty acid transporter. The results showed that TRIM35 knockdown significantly suppressed the expression of both PPARγ and CD36 (Figure 6).

Figure 6 Western blot analysis of PPARγ and CD36 protein expression in TRIM35 knockdown cells. The lower bar graph is the statistical results of the relative expression levels of PPARγ and CD36. The symbol ‘&’ indicates a P value of less than 0.05 compared to siRNA-NC. CD36, cluster of differentiation 36; PPARγ, peroxisome proliferator-activated receptor gamma; siRNA-NC, negative control siRNA; TRIM35, tripartite motif-containing 35.

Discussion

Patients diagnosed with PTC generally have a favourable prognosis; however, they remain at risk of postoperative recurrence, which can significantly affect their quality of life (2,22). Identifying biomarkers involved in PTC initiation and progression is essential for improving patient outcomes. This study aimed to evaluate TRIM35 expression feature in tumour tissues collected from PTC patients and investigate the effects and mechanism of TRIM35 knockdown on PTC cell lines. Currently, research on TRIM35 is limited, and there are no definitive studies have established a correlation between TRIM35 expression and PTC development. This study fills this gap by offering critical insights into the role of TRIM35 in PTC, potentially contributing to improved diagnostic and therapeutic strategies.

TRIM35 expression was elevated in PTC tissues, and significant associations were found between high TRIM35 expression and two aggressive parameters: larger tumour size and the presence of lymphovascular invasion. Our present findings strongly suggest that TRIM35 functions as an oncogene in PTC progression. To further validate this, siRNA technology was used to knockdown TRIM35 expression in PTC cell lines. TRIM35 knockdown markedly reduced the proliferation, migration, and invasion of PTC cells, supporting its role as an oncogene. Interestingly, this finding contrasts with TRIM35’s roles in other cancers, such as hepatocellular carcinoma and breast cancer, where it is associated with tumour suppression (8-10).

Tumour cells primarily depend on glycolysis as their main energy production pathway (23). Therefore, identifying the key genes regulating this glycolytic process is critical for effectively controlling tumour growth and metastasis. In our study, we observed that K1 and TPC-1 cells with reduced TRIM35 expression secreted significantly lower levels of lactate. Additionally, there was a marked decrease in the levels of key regulatory proteins associated with glycolysis, specifically HK and LDHA. These findings strongly suggest that the glycolytic pathway is inhibited in TRIM35-knockdown PTC cells. This underscores the possibility that TRIM35 functions as an oncogene in PTC progression by modulating energy metabolism.

Through transcriptomic sequencing, we identified an association between TRIM35 and the PPAR signaling pathway. PPARs are transcription factors that can be activated by endogenous or exogenous ligands and belong to the nuclear hormone receptor superfamily. PPARs play a crucial role in regulating lipid metabolism, glucose homeostasis, and energy balance, and are also involved in other biological processes such as cell proliferation, differentiation, apoptosis, and angiogenesis (21,24). PPARs have been widely implicated in various human diseases, including atherosclerosis, inflammation, immune regulation, cancer, and metabolic disorders such as obesity and diabetes (21,24,25). In mammals, three subtypes have been identified: PPARα, PPARβ/δ, and PPARγ. Our experimental validation confirmed that TRIM35 knockdown significantly suppressed the expression of PPARγ and the known PPARγ target gene CD36. Although PPARγ is classically recognized as a master regulator of lipid metabolism, emerging evidence has revealed its broader role in glucose metabolism, including the direct regulation of glycolysis (26-28). Recent studies have demonstrated that PPARγ can influence glycolytic flux through key glycolytic regulatory proteins GLUT1, PKM2, and PDK1 (29-31). This provides a mechanistic link between the PPAR pathway and glycolytic regulation. Therefore, the concurrent inhibition of glycolysis and the PPAR pathway observed upon TRIM35 knockdown may represent a coordinated metabolic regulatory mechanism, suggesting that TRIM35 modulates glycolysis at least partially through the PPAR signaling pathway. Furthermore, GO and KEGG enrichment analyses revealed that additional biological processes and signaling pathways were also enriched, suggesting that the PPAR signaling pathway may not be the only downstream route affected by TRIM35. It is possible that TRIM35 participates in the development and progression of PTC through a more complex regulatory network.

Several limitations exist in the present study. First, while our in vitro findings demonstrate an oncogenic role for TRIM35 in PTC, these results have not been validated in vivo. Future studies utilizing animal models, such as xenograft or genetically engineered mouse models, are necessary to confirm the physiological relevance of our observations. Second, although we have shown that TRIM35 knockout affects the PPAR signaling pathway and alters PTC cell behavior, the precise molecular mechanism by which TRIM35 regulates this pathway remains to be fully elucidated. TRIM35 is an E3 ubiquitin ligase, and its canonical function involves regulating substrate protein stability or activity through ubiquitination (32). Therefore, a key question for subsequent research is whether TRIM35 modulates the PPAR signaling pathway via direct ubiquitination of its components (e.g., PPARs themselves or associated cofactors) or through non-canonical, ubiquitination-independent mechanisms. In addition, further investigations, including protein interaction assays and promoter activity analyses, are warranted to clarify the specific relationship between TRIM35 and the PPAR signaling pathway in PTC. Third, our investigation primarily focused on PPARγ. Whether TRIM35 also regulates other members of the PPAR family, such as PPARα and PPARβ/δ, and how this might contribute to PTC progression, warrants further exploration. Addressing these questions in future work will provide a more comprehensive understanding of the TRIM35-PPAR signaling axis in PTC.

Conclusions

TRIM35 levels were elevated in PTC tissues and associated with tumour size and the presence of lymphovascular invasion. TRIM35 knockdown markedly inhibited the proliferation, migration, invasion, and glycolysis of PTC cells, likely by modulating the PPAR signalling pathway. These results identify TRIM35 as a potential biomarker for PTC progression and yield novel insights into the molecular basis of the disease.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2769/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2769/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2769/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2769/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. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Medical Ethics Committee of Longhua District People’s Hospital (No. 2024-098) and informed consent was taken from all the patients.

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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