The Cancer Genome Atlas-Liver HCC (TCGA-LIHC) (22), International Cancer Genome Consortium-Liver Cancer-Riken, Japan (ICGC-LIRI-JP) (23) and OEP00000321 (24) preprocessed datasets were downloaded from the HCC database (lifeome.net:809/#/download) (25). Based on the median expression of CCT2 calculated in HCC tissue (11.17 in TCGA-LIHC, 5.40 in ICGC-LIRI-JP and 11.41 in NODE-OEP00000321), patients were stratified into high and low CCT2 expression groups. The association between CCT2 expression and overall survival was assessed by Kaplan-Meier analysis using GraphPad Prism 10 software (Dotmatics).

The HCC cell lines Huh-7 (cat. no. STCC10102G), Hep3B (cat. no. STCC10103G), Li-7 (cat. no. STCC10107G) and HCCLM3 (cat. no. STCC10111G) were provided by Wuhan Servicebio Technology Co., Ltd. STR profiling was performed to ensure the authenticity of cells. Complete culture medium was prepared with DMEM (cat. no. C11995500BT) supplemented with 10% FBS (cat. no. A5256701; both Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin solution (cat. no. P4333; Sigma-Aldrich; Merck KGaA). The cells were incubated in complete culture medium at 37°C under a humidified environment with 5% CO₂. Recombinant human IL-6 (cat. no. HY-P7044; MedChemExpress) as used for STAT3 activation. In brief, Huh-7 and HCCLM3 cells were treated with IL-6 (50 ng/ml) or PBS at 37°C for 24 h and cells were collected for further experiments.

Huh-7 and HCCLM3 cells were seeded into 6-well plates at a density of 1×10⁵ cells/well and incubated at 37°C overnight. As previously described (26), shRNA plasmids and packaging plasmids were co-transfected into 293T cells at 37°C for 8 h. Mature lentiviral particles (1×10⁸ TU/ml) carrying CCT2-specific short hairpin (sh)RNA (sh-CCT2) or negative control shRNA (sh-NC) were provided by Shanghai GeneChem Co., Ltd. The sequence for human CCT2 shRNA was 5′-TTCATCCACAGACCATCATAG-3′. The sequence for human sh-NC was 5′-TTCTCCGAACGTGTCACGT-3′. The lentiviral transduction of HCC cells was performed at a multiplicity of infection of 10. Following 12 h transduction at 37°C, the viral medium was replaced with fresh complete medium. To select stably transduced cells, puromycin was applied at a concentration of 5 µg/ml for 48 h. The viable cells were maintained in culture medium containing 1.25 µg/ml puromycin for continuous passaging. Expression of CCT2 was detected by reverse transcription-quantitative PCR (RT-q)PCR and western blot analysis. Subsequent experiments were performed ≥72 h after lentiviral transduction.

Huh-7 and HCCLM3 cells were cultured in 6-well plates at an initial seeding density of 1×10⁵ cells/well. Following a 3 h light-protected incubation at 37°C with 0.1% EdU reagent (cat. no. C10310-1; Guangzhou RiboBio Co., Ltd.), the cells were fixed using 4% paraformaldehyde (PFA) for 30 min at room temperature. The incorporated EdU was detected by treating the cells with Apollo staining reagent (cat. no. C10371-1; Guangzhou RiboBio Co., Ltd.) for 30 min at room temperature. The cell nuclei were stained with Hoechst 33342 reagent for 30 min at room temperature. Fluorescence images were captured using the Operetta CLS high-content analysis system (PerkinElmer, Inc.). In total, five visual fields were chosen at random and the number of EdU positive cells was evaluated using ImageJ software (version 1.53 K; National Institutes of Health). The proportion of EdU-positive cells was calculated as follows: (EdU-positive cells/total cells) ×100%.

Huh-7 and HCCLM3 cells were cultured in 6-well plates at an initial seeding density of 1×10³ cells/well. The complete DMEM in the wells was refreshed at 3 day intervals. Following 14 day culture, 4% PFA and 0.1% crystal violet were added to the plates for fixation (30 min at room temperature) and staining (20 min at room temperature), respectively. The cell colonies consisting of ≥40 cells were observed using the ECLIPSE Ts2 inverted light microscope (Nikon Corporation) and counted using ImageJ software.

Early and late apoptotic cells were labeled using Annexin V-PE/7-AAD (cat. no. KGA1104; Nanjing KeyGen Biotech Co., Ltd.). Huh-7 and HCCLM3 cells were digested with EDTA-free trypsin. Then, 1×10⁵ cells were resuspended in 500 µl binding buffer working solution to prepare a single-cell suspension. For each 500 µl cell suspension, 1 µl Annexin V-PE and 5 µl 7-AAD were sequentially added. After mixing by vortexing, the tubes were kept in the dark for 10 min to ensure complete reactions. The FACSAria II flow cytometer (BD Biosciences) was used to measure the percentage of apoptotic cells. Cell apoptosis was analyzed by FACSDiva Software (v6.1.3, BD Biosciences).

Huh-7 or HCCLM3 cells were resuspended in DMEM at a density of 1×10⁵ cells/ml. Then, 100 µl Apo-ONE caspase-3/7 substrate working solution (cat. no. G7792; Promega Corporation) and 100 µl cell suspension were sequentially added to a black 96-well cell culture plate. The contents were mixed using a plate shaker. The cells were incubated at room temperature, protected from the light, for 8 h. The fluorescence of each well was measured using the FL 6500 spectrofluorometer (PerkinElmer, Inc.), as previously described (27).

Ibidi Culture-Insert 25 Well plates (cat. no. 80209; ibidi GmbH) were used for the gap closure assay. 3×10⁵ Huh-7 or HCCLM3 cells were cultured in the wells until they formed 100% confluent monolayers, at which point the inserts were removed. The cells were cultured in serum-free DMEM (Thermo Fisher Scientific, Inc.) at 37°C for an additional 48 h. In total, five independent repeats were performed for each group. Images were captured by the ECLIPSE Ts2 inverted light microscope at 0 and 48 h. The gap area was measured by ImageJ software and calculated as follows: Gap closure ratio (%)=[1-(area at 48 h/area at 0 h)] ×100%.

DMEM-Matrigel (10%; cat. no. 354234; Corning, Inc.) was used to precoat the upper surface of permeable Transwell insert membranes (cat. no. 3464; Corning, Inc.) for 2 h at 37°C. The inserts were placed in a 24-well plate. Next, 2×10⁴ Huh-7 or HCCLM3 cells were resuspended in 200 µl serum-free DMEM and added to the upper chamber of the Transwell insert, while the lower chamber was filled with 600 µl complete DMEM. Following a 24 h culture at 37°C, the inserts were incubated with 4% PFA and 0.1% crystal violet for fixation (30 min at room temperature) and staining (20 min at room temperature), respectively. The successfully invaded cells were counted under the ECLIPSE Ts2 inverted light microscope.

Suspended single Huh-7 or HCCLM3 cells (1×10³) were cultured with Tumor Stem Cell Pellet Culture Medium (serum-free; cat. no. P2401; Shanghai QiDa Biotechnology Co., Ltd) in ultra-low attachment plates (24-well; cat. no. 3473; Corning, Inc.). Following 14 days incubation at 37°C, cell spheres were counted under the ECLIPSE Ts2 inverted light microscope. The spheroid formation efficiency (SFE) was calculated as follows: SFE (%)=number of spheres/1,000×100%. Furthermore, tumor spheres were harvested and digested into a single-cell suspension for EdU incorporation assay and Transwell assay.

Total RNA from Huh-7 and HCCLM3 cells was extracted using TRIzol™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RT-qPCR was performed using the SuperScript™ III Platinum™ One-Step qRT-PCR kit (cat. no. 11732020; Invitrogen; Thermo Fisher Scientific, Inc.). The one-step thermocycling conditions were as follows: 50°C for 15 min to synthesize cDNA; 95°C for 2 min to denaturalize the cDNA and activate the Taq DNA Polymerase; 40 cycles of 95°C for 15 sec (denaturation) and 60°C for 30 sec (annealing and extension). The 2^−∆∆Cq method was used to calculate the relative expression of the target genes (28,29). The primers were as follows: CCT2 forward, 5′-GCACTACCTCTGTTACCGTTTT-3′ and reverse, 5′-CTTCTCTCCAACCCGCTATGA-3′ and β-actin (reference gene) forward, 5′-CATGTACGTTGCTATCCAGGC-3′ and reverse, 5′-CTCCTTAATGTCACGCACGAT-3′.

RIPA buffer (cat. no. P0013B) and the BCA Protein Assay kit (cat. no. P0012; both Beyotime Institute of Biotechnology) were used to extract the total protein from Huh-7 and HCCLM3 cells and measure the protein concentration, respectively. The same mass of protein (40 µg/lane) was separated by 4–20% SDS-PAGE (cat. no. P0468S; Beyotime Institute of Biotechnology) and then transferred onto PVDF membranes (cat. no. IPVH00010; MilliporeSigma). The membranes were washed with TBS for 10 min at room temperature. The membranes were blocked with 5% fat-free milk for 1 h at room temperature followed by incubation with primary antibodies (1:1,000 using 5% fat-free milk) at 4°C overnight. The primary antibodies were as follows: CCT2 (cat. no. 24896-1-AP), β-actin (cat. no. 66009-1-Ig), MMP2 (cat. no. 10373-2-AP), myeloid cell leukemia sequence 1 (MCL1; cat. no. 16225-1-AP) and SRY-box transcription factor 2 (SOX2; cat. no. 11064-1-AP; all Proteintech Group, Inc.) and STAT3 (cat. no. 4904) and phosphorylated (p-)STAT3 (Tyr705; cat. no. 4113; both Cell Signaling Technology, Inc.) The membranes were washed three times in TBST (0.1% Tween-20) for 5 min each at room temperature. The proteins were labeled with HRP-conjugated secondary antibodies (1:5,000 in 5% fat-free milk; cat. nos. SA00001-1 and SA00001-2; Proteintech Group, Inc.) for 1 h at room temperature. The membranes were washed three times in TBST for 5 min each at room temperature. Chemiluminescent signals were detected by an enhanced chemiluminescence kit (cat. no. WBKLS0500; MilliporeSigma). The band densitometry was quantified by ImageJ software.

A total of 20 BALB/c (age, 4–6 weeks; weight, 16–18 g) male nude mice were acquired from SPF Biotechnology Co. Ltd. and raised in a specialized pathogen-free environment (25°C, 45% humidity, 12/12-h light/dark period and ad libitum access to diet and water). The institutional animal care and use committee of SPF Biotechnology Co., Ltd (Beijing, China). approved the animal experiment protocol (approval no. AWE2023102504). Nude mice were randomly assigned into four groups (sh-NC and sh-CCT2 for the xenograft and hematogenous lung metastasis model; n=5/group) using random number table method. For the xenograft model, a 150 µl single-cell suspension containing 2×10⁶ transduced HCCLM3 cells (with sh-NC or sh-CCT2) was administered via subcutaneous injection into the left forelimb. Once the subcutaneous tumors were palpable, they were monitored three times per week. Tumor diameters were measured using a vernier caliper. Tumor volumes were determined as follows: Volume (mm³)=a × b²x0.5, where a indicates the length and b indicates the width. The tumor volume did not exceed 2,000 mm³, in compliance with animal ethics requirements. For the lung metastasis model, a single cell suspension of 1×10⁵ transduced HCCLM3 cells (with sh-NC or sh-CCT2) in 100 µl sterile saline was administered into mice via tail vein injection. After 28 days, all animals were placed in a chamber ventilated with CO₂ at 30–70% of its volume/min. The mice were left in the chamber for ≥5 min after respiratory arrest and observed to ensure the cessation of breathing and heartbeat. Cervical dislocation (with the confirmation of a gap between the skull and spinal column) was used to ensure the death. For the xenograft model, subcutaneous tumors were dissected and weighed. For the lung metastasis model, both lungs were dissected. The tumor and lung were fixed with 4% PFA for 30 min at room temperature for further experiments.

PFA-fixed subcutaneous tumor and lung tissue were first embedded in paraffin and then sliced into 4-µm-thick sections. The sections were dewaxed in xylene (three times for 10 min each) and rehydrated through graded ethanol (100, 95, 90, 80 and 70%, 5 min each). For immunohistochemistry, antigen retrieval was performed by microwaving at 95°C for 10 min with Tris-EDTA buffer (pH 9.0; cat. no. PR30002; Proteintech Group, Inc.). Endogenous peroxidase activity quenching and non-specific antigen blocking were performed using IHC Detect kit for Rabbit/Mouse Primary Antibody (cat. no. PK10006; Proteintech Group, Inc.). The primary antibodies, including p-STAT3 (cat. no. 4113, Cell Signaling Technology), MCL1 (cat. no. 16225-1-AP), MMP2 (cat. no. 10373-2-AP) and SOX2 (cat. no. 11064-1-AP; all Proteintech Group, Inc.), were diluted 1:100 in antibody diluent (cat. no. PR30016; Proteintech Group, Inc.) and applied at 4°C overnight. Afterward, the sections were washed with PBS to eliminate any unbound primary antibodies and subsequently incubated with 100 µl ready-to-use HRP-conjugated polymer secondary antibodies (cat. no. RGAU011, Proteintech Group, Inc.) for 30 min at room temperature. DAB was used to visualize proteins. Hematoxylin reagent was utilized to stain the nuclei for 1 min at room temperature.

For H&E staining, the sections were dipped in hematoxylin reagent for 5 min at room temperature followed by washing in flowing tap water. Then, the sections were immersed in acid alcohol (1% hydrochloric acid in 70% alcohol) for 5 sec and washed in flowing tap water until they turned blue. Eosin solution (1%) was used to stain the sections for 20 sec at room temperature.

All sections were imaged using the ECLIPSE Ts2 inverted light microscope. The mean optical density was calculated by ImageJ software to assess the expression of the target protein in the subcutaneous tumor tissues. The number of lung nodules was counted to evaluate the tumor metastatic capacity.

Quantitative data are reported as the mean ± standard deviation of ≥3 independent experimental repeats. The unpaired Student's t-test was performed to evaluate differences between two groups when data passed Shapiro-Wilk normality and equal variance test; otherwise, the Mann-Whitney test was used. The differences between >2 groups were compared by one-way ANOVA, with Tukey's post hoc test. GraphPad Prism 10 software (Dotmatics) was used for data analysis. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the clinical significance of CCT2 in HCC, the association between CCT2 expression and OS was analyzed using multiple public HCC datasets. The results across all datasets demonstrated CCT2 expression was upregulated in HCC tissues compared with tumor-adjacent tissue (Fig. 1A-C). Kaplan-Meier curve analyses demonstrated that patients with HCC with high levels of CCT2 had a poorer OS prognosis. The hazard ratios (HRs) for OS were as follows: HR=1.51 [95% confidence interval (CI), 1.06–2.16; P=0.024; Fig. 1D] in TCGA-LIHC cohort, HR=3.12 (95% CI, 1.59–6.11; P<0.001; Fig. 1E) in the ICGC-LIRI-JP cohort and HR=2.59 (95% CI, 1.51–4.43; P<0.001; Fig. 1F) in the NODE-OEP00000321 cohort. Taken together, high expression of CCT2 may be considered a risk factor for patients with HCC.

To elucidate the biological functions of CCT2 in HCC malignancy, the expression of CCT2 in HCC cell lines was detected by western blotting (Fig. 2A). Huh-7 and HCCLM3 cell lines were selected for subsequent experiments due to their relatively high levels of CCT2 expression. CCT2 expression was knocked down in Huh-7 and HCCLM3 cells by lentivirus-mediated sh-CCT2 transduction. RT-qPCR and western blotting confirmed that, compared with the sh-NC group, the mRNA (Fig. 2B and D) and protein (Fig. 2C and E) levels of CCT2 were significantly downregulated in the sh-CCT2 group.

Functional experiments were performed to determine whether CCT2 knockdown affected HCC cell viability. CCT2 knockdown significantly decreased the percentage of EdU-positive cells (Fig. 3A and B) and the number of colonies formed (Fig. 3C and D). Additionally, compared with the sh-NC group, the proportion of apoptotic cells (Fig. 3E and F) and the activity of caspase-3/7 (Fig. 3G and H) increased in the CCT2 knockdown group. Collectively, the findings indicated that CCT2 may be involved in the regulation of cell proliferation and apoptosis in HCC.

The effects of CCT2 knockdown on cancer cell motility were investigated. CCT2 knockdown significantly suppressed the migration and invasion of Huh-7 and HCCLM3 cells, as shown by the gap closure (Fig. 4A and B) and Transwell invasion (Fig. 4C and D) assays. These data demonstrated that CCT2 may be involved in the migration and invasive characteristics of HCC.

The effect of CCT2 on cancer cell self-renewal was detected by the tumor-sphere formation assay. The number of spheres was lower in the sh-CCT2 compared with the sh-NC group (Fig. 5A and B). Compared with the parental cells in sh-NC group, the spheroid cells in sh-NC group exhibited enhanced proliferative and invasive capacities (Fig. 5C-F). Furthermore, knockdown of CCT2 inhibited these malignant behaviors in both parental and spheroid cells (Fig. 5C-F). Taken together, these findings demonstrated that CCT2 may regulate the stemness traits of HCC cells.

Subcutaneous xenograft and hematogenous lung metastasis models were used to verify the anticancer effects of CCT2 knockdown in vivo. Knockdown of CCT2 in HCCLM3 cells significantly decreased the tumor proliferation rate and final tumor weight in xenograft mice (Fig. 6A and B). Moreover, compared with the sh-NC group, the number of lung metastatic nodules decreased in the sh-CCT2 group (Fig. 6C). In brief, the in vivo results confirmed the roles of CCT2 in HCC progression.

Phosphorylation of STAT3, particularly at residue Tyr705, serves as a hallmark of its canonical activation (15). Western blotting showed that knockdown of CCT2 attenuated the phosphorylation levels of STAT3 rather than changing its total protein levels (Fig. 7A and B). In addition, the protein levels of MCL1, MMP2 and SOX2 were downregulated following CCT2 knockdown (Fig. 7A and B). Immunohistochemical staining of subcutaneous tumor tissue also demonstrated that the p-STAT3, MCL1, MMP2 and SOX2 levels were decreased in the sh-CCT2 group (Fig. 7C-F). These results suggested that the STAT3 signaling pathway may be a key downstream pathway regulated by CCT2 in HCC cells.

To assess whether the oncogenic effects of CCT2 were dependent on the activation of STAT3, recombinant human IL-6 (50 ng/ml) was used to activate STAT3 signaling in HCC cells. The western blotting results confirmed that IL-6 treatment increased the levels of p-STAT3 in Huh-7 and HCCLM3 cells (Fig. 8A and B). Furthermore, compared with the PBS treatment group, the inhibitory effects of CCT2 knockdown on the proliferation (Fig. 8C and D) and invasion (Fig. 8E and F) of Huh-7 and HCCLM3 cells were significantly abrogated by IL-6 treatment. These results indicated that CCT2 exerts its tumor-promoting effects via STAT3 activation.