RNA-sequencing (seq), microRNA (miR/miRNA)-seq, somatic mutation and clinical data from 274 patients with liver cancer in The Cancer Genome Atlas (TCGA; accession no. TCGA-LIHC; portal.gdc.cancer.gov/) cohort were obtained. Somatic mutation data were analyzed using the ‘maftools’ R package (version 2.18.0; bioconductor.org/packages/release/bioc/html/maftools.html) to calculate the frequency of variant classifications in liver cancer tissues and to determine the distribution of different variant gene types. Somatic variant profiles of liver cancer tissues were detected using the VarScan algorithm (30), and the top 10 most frequently mutated genes in liver cancer were visualized. A forest plot was generated to display the hazard ratio (HR) and P-value for each mutation, categorizing patients into wild-type and mutated groups.

RNA-seq profiles from TCGA liver cancer cohort were downloaded, and the matrix was normalized using the limma package (version 3.6.4; bioconductor.org/packages/release/bioc/html/limma.html). Probe names were converted to gene names prior to analysis. Based on the negative binomial distribution model, edgeR is able to model gene expression count data more accurately, thereby improving the accuracy of differential expression analysis (31). Differential gene expression analysis was carried out using edgeR (version 3.44.0; bioconductor.org/packages/release/bioc/html/edgeR.html) between liver cancer tissues with TTN mutations and wild-type controls. DEGs were defined as genes with a log₂ fold change ≥1 and P<0.05.

Functional annotation and visualization of the DEGs was carried out using the DAVID Bioinformatics Resources (david.ncifcrf.gov/), enabling the identification of enriched Kyoto Encyclopedia of Genes and Genomes (KEGG; kegg.jp/) pathways and Biological Process (BP) categories. It not only provides rapid access to a variety of heterogeneous annotation data in a centralized location, but also enriches the bio-informative level of individual genes. By using Benjamini-Hochberg multiplex testing, adjust P-values to control False Discovery Rate (FDR), terms with P<0.05 were considered to indicate a statistically significant difference and a bubble plot was used to visualize the top five terms.

In vivo drug responses to 198 drugs in patients with liver cancer were predicted using the OncoPredict R package (version 1.2; URL: cran.r-project.org/web//packages/oncoPredict/index.html) (32). This package calculates drug scores by fitting the gene expression matrix of each liver cancer sample into the gene expression matrix of cancer cell lines from the Broad Institute's Cancer Cell Line Encyclopedia (CCLE; portals.broadinstitute.org/cclelegacy/home). The corresponding half-maximal inhibitory concentration (IC₅₀) for each drug was determined. A higher drug score indicates greater drug resistance (32). Drug scores between patients with liver cancer exhibiting wild-type and mutant genes were compared using unpaired t-test, with P<0.05 considered to indicated a statistically significant difference.

Total RNA was extracted from 5×10⁶ liver cancer cells (SNU182, HuH7 and JHH7 from Procell Life Science & Technology Co., Ltd.; HepG2, Hep3B and Li7 cells were purchased from EIAab Group.) using the Total RNA Extraction Kit (Tiangen Biotech Co., Ltd.). According to the manufacturer's protocol, the Rever Tra Ace qPCR RT Master Mix (cat. no. RR037A; Takara Bio Inc.) was used to reverse transcribe total RNA into complementary DNA (cDNA). RT-qPCR was carried out using a real-time PCR instrument (cat. no. 12011319; CFX Opus 96 Real-Time PCR System), and relative expression was calculated using the 2^−ΔΔCq method (33). GAPDH was used as a loading control. The primers used were as follows: GAPDH, forward primer 5′-GTCTCCTCTGACTTCAACAGCG-3′, and reverse primer 5′-ACCACCCTGTTGCTGTAGCCAA-3′, TTN forward primer 5′-CCCCATCGCCCATAAGACAC-3′, and reverse primer 5′-CCACGTAGCCCTCTTGCTTC-3′. The thermocycling conditions were as follows: Initial denaturation at 95.0°C for 30 sec, followed by 40 cycles of 95.0°C for 5 sec, 62.0°C for 30 sec and 67.5°C for 5 sec.

Liver cancer cell lines SNU182, HuH7 and JHH7 were obtained from Procell (Procell Life Science & Technology Co., Ltd.), while HepG2, Hep3B and Li7 cells were purchased from EIAab Group. According to mutation data from the CCLE database, JHH7, HepG2 and Li7 exhibit TTN mutations, whereas HuH7, Hep3B and SNU182 have wild-type forms of TTN. The authenticity of all cell lines used in the present study has been confirmed by STR analysis. All cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc.) at 37°C and 5% CO₂.

TTN overexpression was induced via transfection with pcDNA3.1(+) plasmids (Shanghai GeneChem Co., Ltd.) using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). Empty vector was used as a negative control. Transfection was performed at 37°C, 5% CO₂ incubator for 4–6 h and switch to fresh complete medium. After 48 h, the expression level of TTN protein was detected by Western blot to verify the overexpression effect.

Stable TTN knockdown was achieved through transduction with TTN-targeting lentivirus (iGene Biotechnology Co., Ltd.). Using a second-generation lentiviral generation system, lentiviral vector plasmids (10 µg), packaging plasmids, and envelope plasmids were co-transfected into HEK293T cells (Procell Life Science & Technology Co., Ltd.) at 50–70% confluence (4:3:1), incubated at 37°C for 6–8 h, and then replaced with fresh complete medium. Viral supernatants were collected 48 h after transfection and viral particles were collected by ultracentrifugation (4°C, 50,000 g, 2 h). JHH7 and HepG2 cells (50–60% confluence, lentiviral particles (MOI: 20) were added to the cell culture medium along with polybrene (5 µg/ml) and after 24 h of incubation at 37°C, 5% CO₂ incubator, they were replaced with fresh complete medium. 48 h after transfection, puromycin (1 µg/ml; cat. no. A1113803; Thermo Fisher Scientific, Inc.) was added for 1 weeks of screening, followed by a maintenance culture using puromycin at a concentration of 0.5 µg/ml. Western blot was used to measure the expression level of TTN protein to verify the knockdown effect. All shRNAs (Shanghai Genechem Co., Ltd.) used are as follows for the sense (SS) and antisense strands (AS): sh-NC SS sequence 5′-TTCTCCGAACGTGTCACGT-3′, and AS sequence 5′-ACGTGACACGTTCGGAGAA-3′; sh1-TTN SS sequence GGTTGTTTATGTTATGTTA, and AS sequence TAACATAACATAAACAACC; sh2-TTN SS sequence GCTTGTTACTAATATAATA, and AS sequence TATTATATTAGTAACAAGC; sh3-TTN-SS sequence CACAGAAGTTAGAGTTCAA, and AS sequence TTGAACTCTAACTTCTGTG.

Total protein was extracted from liver cancer cells using phenylmethanesulfonyl fluoride (1:100) in high-strength RIPA lysis buffer (Sangon Biotech Co., Ltd.). The protein concentration was determined using a BCA protein assay. SDS-PAGE electrophoresis was carried out using 12% gels (20 µg/lane), first at 80 V for 30 min and then at 120 V for 90 min. Proteins were transferred to a 0.45 µm polyvinylidene fluoride membrane for 90 min at 300 mA. The membrane was blocked with 5% skim milk powder for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibodies against GAPDH (1:5,000; cat. no. AB-2937024; Abmart Pharmaceutical Technology Co., Ltd.) and TTN (1:2,000; cat. no. H00007273-M09; Thermo Fisher Scientific, Inc.). After washing with TBS with 0.1% Tween-20, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (H+L) (1:10,000; cat. no. SA00001-2; Proteintech Group, Inc.) or HRP-conjugated Goat Anti-Mouse IgG (H+L) (1:10,000; cat. no. SA00001-1; Proteintech Group, Inc.) for 2 h at room temperature. The membrane was then treated with enhanced chemiluminescence (ECL; Omni-ECL^™, Shanghai Epizyme Biotech Co., Ltd.) solution for ~1 min, followed by imaging using Tanon 5200 automatic chemiluminescence image analysis system. All experiments were carried out in triplicate as previously described (34). Optical density analysis was performed using ImageJ version 1.53t software (National Institutes of Health).

Liver cancer cells were cultured to 50–70% confluence, the old medium was removed and a fresh medium containing cycloheximide (CHX; cat. no. S7418; Selleck Chemicals) with a final concentration of 1.0 µM was added at 37°C and 5% CO₂, cells were collected at 0, 1, 2, 3 and 4 h and protein was extracted, and the expression of TTN was detected by western blotting.

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Shanghai Yeasen Biotechnology Co., Ltd.). Cells were seeded at a density of 4×10³ cells/well in a 96-well plate and incubated at 37°C with 5% CO₂. Once cells adhered, Nilotinib (cat. no. HY-10159; 0.00, 0.62, 1.25, 2.50, 5.00, 10.00 and 20.00 µM), GSK1904529A (cat. no. HY-10524; 0.000, 0.031, 0.062, 0.125, 0.250, 0.500 and 1.000 µM), 5-Fluorouracil (5-FU; cat. no. HY-90006; 0.00, 0.62, 1.25, 2.50, 5.00, 10.00 and 20.00 µM) and Sapitinib (cat no. HY-13050; 0.0, 0.5, 1.0,2.0, 4.0, 8.0 and 16.0 nM) were added to the wells for 48 h. All drugs were purchased from MedChemExpress. After treatment, the culture medium containing the drug was discarded, and 100 µl of DMEM containing 1/10 of the CCK-8 reagent (Shanghai Yeasen Biotechnology Co., Ltd.) was added to each well and incubated for 2 h. Absorbance was measured at 450 nm to assess cell viability.

Liver cancer cells were suspended in 200 µl of DMEM (Gibco; Thermo Fisher Scientific, Inc.) at a density of 1,000 cells per well, cultured in 96-well clear ultra-low attachment U-plates (Invitrogen; Thermo Fisher Scientific, Inc.) was incubated at 37°C for 2 days. Culture medium was refreshed with a complete medium containing 5-FU (Huh7: 4.5 µM; Hep3B: 6.5 µM; JHH7: 2.0 µM; HepG2: 2.5 µM) or with hMAO-B-IN-9 (1.58 µM) (cat. no. HY-163879, MedChemExpress). The culture medium was replaced every 3 days, and the cells were continuously cultured in a cell incubator at 37°C and 5% CO₂ for 13 days. The size of the spheroids derived from liver cancer cells were captured using an inverted microscope.

To assess cell proliferation, the BeyoClick^™ EdU-555 kit (cat. no. C0075S, Beyotime Institute of Biotechnology) was used to evaluate DNA synthesis in liver cancer cells. The experiment was carried out according to the manufacturer's instructions. All experiments were carried out in triplicate, as described previously (34).

Liver cancer cells were seeded in culture dishes and collected when the cell density reached 80%. Cells were lysed using Western and IP cell lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology), and then the supernatant was collected by centrifugation at 12,000 g at 4°C for 10 min for subsequent assays. MDA content in the cells was measured using the Lipid Peroxidation (MDA) Assay Kit (cat. no. HY-K0319; MedChemExpress) according to the manufacturer's instructions. Absorbance was measured at 532 nm using a microplate reader, and the resulting values were compared with a standard curve to determine the concentration of MDA. GSH and GPX4) activity were also tested using Reduced Glutathione (GSH) Colorimetric Assay (cat. no. E-BC-K030-M) and Glutathione Peroxidase 4 (GPX4) Activity Assay Kit (E-BC-K883-M; both Elabscience Biotechnology Co., Ltd.) following the manufacturer's instructions.

Control Huh7 and Hep3B cells, and TTN-overexpressing Huh7 and Hep3B cells were seeded in 6-well plates at 500 cells per well and cultured for 2 weeks with replacement of the culture medium containing 5-FU (Huh7: 4.5 µM; Hep3B: 6.5 µM) every 3 days. After 2 weeks of incubation, cells were gently washed with PBS three times (3 min each), fixed with 4% paraformaldehyde for 30 min at room temperature, and washed three times with PBS for 3 min each. Cells were then stained with 1% crystal violet for 30 min at room temperature. After washing with PBS, the dishes were dried, and colonies with diameter >100 µm were counted. Clone count and size measurements were performed using ImageJ version 1.53t software (National Institutes of Health).

All animal experiments were conducted following the ‘3R’ principles (replacement, reduction and refinement) and were approved by the Animal Care Committee of Guizhou Medical University under approval no. 2400641. A total of 10 female and 10 male (age, 4–6 weeks of 16–18 g BALB/c nude mice were purchased from Beijing Huafukang Biotechnology Co., Ltd., and after receiving the mice, they were acclimatized for 2 weeks in a specific pathogen-free environment at 25°C and humidity of 30%. The mice were exposed to a 12-h light/dark cycle, and were given free access to mouse feed sterilized and water sterilized by autoclaving. For tumor growth experiments, BALB/c mice were randomly assigned to shNC and shTTN groups, 10 mice per group). HepG2/shNC or HepG2/shTTN cells (6×10⁵ cells/100 µl) were resuspended in PBS containing 1% Matrigel and subcutaneously injected into the right abdomen of BALB/c mice. After 1 week, when tumors reached ~10 mm³, the mice were randomly divided into two groups: One treated with Di-methyl sulfoxide (DMSO) control, and the other with 5-FU (25 mg/kg; 100 µl; intraperitoneal injections, once daily for 3 weeks). Tumor size was measured every 3 days, and tumor volume was calculated using the formula: Volume=1/2 (length × width²). The mice were monitored for signs of poor health, including weight loss, postural abnormality, changes in activity levels, breathlessness, and signs of distress. Humane endpoints included maximum tumor volume >2,000 mm³, ulceration, necrosis or infection on the surface of the tumor, weight loss >20%, abnormal posture, tumor affecting motor function, dyspnea, inability to eat and drink normally, the experiment was terminated, all mice were anesthetized with sodium pentobarbital at 5.0 mg/100 g body weight and sacrificed by cervical dislocation, and death was confirmed without breathing and heartbeat for more than 5 min. Tumors were harvested for weight measurement and histological analysis.

The tumor tissue was fixed in 4% paraformaldehyde at room temperature for 24 h, dehydrated in graded ethanol and embedded in paraffin, embedded in paraffin, and sectioned into 4 µm slices. After paraffin was removed with xylene at room temperature, and the tissue sections were gradually hydrated using ethanol. Antigen retrieval was performed using tris-EDTA antigen retrieval solution (pH 9.0; tris-EDTA antigen retrieval solution; Beyotime Institute of Biotechnology) at 95–100°C for 15 min for antigen retrieval. To reduce non-specific binding, tissues were blocked with a solution containing 3% hydrogen peroxide for 10 min at room temperature, and 5% bovine serum albumin (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature. Tissues were incubated overnight at 4°C with primary antibodies: Anti-Ki67 (1:400; cat. no. 2807-1-AP) and anti-PCNA (1:400; cat. no. 60097-1-IG) (both from Proteintech Group, Inc.). After washing with PBS, tissues were incubated with the secondary antibody (goat anti-rabbit/mouse HRP-labeled polymer; cat. no. PR30009 Proteintech Group, Inc.) for 2 h at 37°C. The tissues were then stained with 3,3′-Diaminobenzidine tetrahydrochloride (Shanghai Yeasen Biotechnology Co., Ltd.) and counterstained with hematoxylin for 3 min at room temperature to visualize the cell nuclei. The antigen-antibody complex binding was detected using a light microscope.

Statistical analysis was carried out using Prism software (version 6.0; Dotmatics). Data are presented as the mean ± standard deviation. All experiments were conducted in triplicate. Statistical significance was determined using an unpaired Student's t-test or one-way ANOVA with Tukey's post hoc test. The Benjamini-Hochberg method was used to control the false discovery rates. P<0.05 was considered to indicate a statistically significant difference.

Genome-wide mutation profiling in liver cancer was conducted by analyzing somatic mutation data, revealing that missense mutations were the most common variant types, followed by nonsense and frameshift deletion mutations, with single nucleotide polymorphisms (SNPs) accounting for the majority of variant types. The C>T transition emerged as the most prevalent single nucleotide variant (SNV; Fig. 1A). The number of mutant bases per patient was quantified, with a median value of 1. TTN was identified as the gene with the highest mutation frequency in liver cancer. The top 10 most frequently mutated genes-TP53, TTN, CTNNB1, MUC16, ALB, PCLO, MUC4, RYR2, ABCA13 and APOB-demonstrated high mutation frequencies and distinct somatic mutation patterns (Fig. 1B). Analysis of tissue mutation characteristics in TCGA database revealed a positive correlation between TTN mutations and poor prognosis in liver cancer (HR=136.69; 95% CI=1.36–13,766.33; Fig. 1C). To elucidate the molecular mechanisms underlying TTN mutations in liver cancer, differential expression analysis was conducted on TTN-mutant and TTN-wild-type liver cancer tissues from TCGA database. The analysis revealed 140 downregulated genes and 30 upregulated genes (Fig. 1D and E). in TTN-mutant and -wild-type liver cancer (Fig. 1E). BP term enrichment analysis of the upregulated DEGs identified significant enrichment in terms such as ‘Complement activation’, ‘positive regulation of cell activation’, ‘stress response to metal ions’, ‘glutathione metabolism’ and ‘cellular response to metal ions’ (Fig. 1F). KEGG pathway analysis further indicated enrichment in pathways including ‘mineral absorption’, ‘neuroactive ligand-receptor interaction’, ‘ferroptosis’, ‘fat digestion and absorption’ and ‘pancreatic secretion’ (Fig. 1G). Notably, the BP terms impacted by TTN-mutant DEGs were associated with stress responses to metal ions and GSH metabolism. Additionally, KEGG pathway enrichment highlighted the association of these differential genes with ferroptosis. Given that numerous studies have established a connection between GSH metabolism and ferroptosis (35,36), these observations suggest that TTN mutations may influence liver cancer progression through the modulation of ferroptosis-related metabolic processes.

To assess the impact of TTN mutations on drug sensitivity in liver cancer, the OncoPredict algorithm was used. A total of 198 drugs were analyzed, revealing significant changes in the drug scores of four drugs in liver cancer tissues with TTN mutations (Fig. 2A and B). Specifically, the drug scores for GSK1904529A and nilotinib were decreased in TTN-mutant liver cancer tissues, while 5-FU and sapitinib scores were increased (Fig. 2C and D). This suggests that TTN mutations may increase sensitivity to GSK1904529A and nilotinib while conferring resistance to 5-FU and sapitinib. To validate these observations, liver cancer cell lines with and without TTN mutations were analyzed using the CCLE database. Three TTN-mutant cell lines (Huh7, Hep3B and SNU182) and three wild-type cell lines (JHH7, HepG2 and LI7) were selected for further experiments. CCK-8 assays were carried out to determine IC₅₀ values. For GSK1904529A, the IC₅₀ values in 48 h were as follows: JHH7 (0.4156 µM), HepG2 (0.3501 µM), LI7 (0.2572 µM), Huh7 (0.0720 µM), Hep3B (0.0836 µM) and SNU182 (0.1107 µM). For nilotinib, IC₅₀ values were: JHH7 (10.470 µM), HepG2 (6.200 µM), LI7 (5.049 µM), Huh7 (3.342 µM), Hep3B (3.675 µM) and SNU182 (2.010 µM). The IC₅₀ values of 5-FU were as follows: JHH7 (2.251 µM), HepG2 (2.831 µM), LI7 (2.347 µM), Huh7 (4.618 µM), Hep3B (6.624 µM) and SNU182 (4.950 µM). Finally, for sapitinib, IC₅₀ values were: JHH7 (2.140 nM), HepG2 (1.946 nM), LI7 (1.737 nM), Huh7 (7.749 nM), Hep3B (5.203 nM) and SNU182 (4.375 nM; Fig. 2E). Statistical analysis using the unpaired student's t-test indicated that the IC₅₀ values for 5-FU and sapitinib were increased in TTN-mutant liver cancer cells, while the IC₅₀s for GSK1904529A and nilotinib were decreased when compared with their wild-type counterparts (Fig. 2F). These results suggest that TTN mutations may enhance sensitivity to GSK1904529A and nilotinib while contributing to resistance against 5-FU and sapitinib.

To validate the effect of TTN mutations on gene expression, the transcription levels of TTN were analyzed in liver cancer tissues from TCGA cohort. No significant difference was observed in the transcriptional levels of TTN between mutant and wild-type liver cancer tissues (Fig. 3A). Similarly, RT-qPCR experiments revealed no marked difference in TTN mRNA expression between TTN-mutant and wild-type liver cancer cell lines (Fig. 3B). However, despite similar mRNA levels, TTN protein expression was notably higher in TTN-mutant liver cancer cells (JHH7, HepG2 and Li7) (Fig. 3C). To explore the underlying mechanisms, protein synthesis was inhibited using cycloheximide (CHX; 1 µM), revealing that TTN protein stability was increased in TTN-mutant cells, with slower degradation when compared with wild-type cells (Fig. 3D). These results indicate that single nucleotide missense mutations in TTN enhance the stability of the encoded protein.

5-FU, an uracil analog, is an effective anti-cancer drug. Previous studies have demonstrated its potent antitumor activity, both as a monotherapy and in combination with other chemotherapeutic agents, across a variety of different types of cancer, including lung cancer, hepatocellular carcinoma, colorectal cancer and gastric cancer (37–40). The present study further investigated the role of TTN protein in mediating 5-FU resistance in liver cancer cells. To enhance expression of TTN, plasmids were introduced into Huh7 and Hep3B cells (Fig. 4A). Studies have highlighted the role of metal ions in various physiological processes, including cellular homeostasis, metabolic regulation, biosynthesis, signal transduction and energy conversion (41,42), As studies examining the relationship between metal ions and cancer treatment progress, several metal ions have been found to induce apoptosis and enhance sensitivity to chemotherapeutic drugs (43,44). BP and KEGG pathway analyses revealed associations between TTN mutations and both metal ion metabolism and GSH metabolism (Fig. 1F), with ferroptosis pathway enrichment also observed (Fig. 1F). Building on these findings, the effects of TTN overexpression on the levels of ferrous and other metal ions in liver cancer cells were explored. The results revealed that TTN overexpression or 5-FU treatment had minimal impact on the levels of zinc, calcium and copper ions in the cells (Fig. 4B). Notably, TTN overexpression significantly inhibited the increase in ferrous ion levels in 5-FU-treated Huh7 and Hep3B cells compared with 5-FU-treated negative control (Fig. 4C). Further analysis of ferroptosis-related markers revealed that TTN overexpression reduced MDA content in 5-FU-treated cells and increased levels of GSH and GPX4 compared with the 5-FU-treated negative control group, effectively reversing 5-FU-induced ferroptosis (Fig. 4D). Additionally, while TTN overexpression did not significantly impact colony formation, tumor spheroid formation or DNA synthesis rates, it notably mitigated the inhibitory effects of 5-FU on these processes (Fig. 4E-G). These results suggest that increased TTN expression levels may inhibit ferroptosis in tumor cells, thereby enhancing liver cancer resistance to 5-FU.

TTN protein function in liver cancer cells harboring TTN mutations was further investigated. Three specific shRNAs were employed to knockdown TTN expression in JHH7 and HepG2 cells (Fig. 5A and B). TTN knockdown did not significantly affect the cellular levels of zinc, calcium, copper or ferrous ions. However, following 5-FU treatment, ferrous ion levels in the sh2 and sh3 groups increased significantly compared with shNC group (Fig. 5C and D). Additionally, MDA levels were significantly higher in the sh2 and sh3 groups following 5-FU treatment compared with shNC, while GPX4 and GSH levels were significantly decreased (Fig. 5E).

TTN knockdown in JHH7 and HepG2 cells also enhanced the inhibitory effect of 5-FU on DNA synthesis rates (Fig. 6A), and tumor spheroid proliferation (Fig. B). Treatment with the ferrous ion chelator hMAO-B-IN-9 slightly reversed these inhibitory effects (Fig. 6A and B).

In vivo, the synergistic effect of TTN deletion with 5-FU was explored by transplanting HepG2/shNC or HepG2/shTTN cells into the subcutaneous tissue of immunocompromised BABL/c mice. Tumor-bearing mice were treated with DMSO or 5-FU (25 mg/kg; 100 µl) via intraperitoneal injection once daily for 21 days (Fig. 7A). TTN knockdown did not significantly impact tumor growth in HepG2/shNC cells (Fig. 7B-D). However, stable TTN knockdown increased the sensitivity of tumors to 5-FU, significantly inhibiting tumor growth in mice transplanted with HepG2/shTTN cells (Fig. 7B-D). IHC staining of tumor tissue revealed that, similar to the in vitro results, the levels of Ki67 and PCNA were significantly lower in TTN-knockdown tumors compared with the control following 5-FU treatment (Fig. 7E-G). These results underscore the potential role of TTN mutations in modulating 5-FU sensitivity in liver cancer.