Silencing of TRAF5 enhances necroptosis in hepatocellular carcinoma by inhibiting LTBR-mediated NF-κB signaling

Cell culture

Human HCC cell lines (HepG2, HuH7, SMMC-LM3, and Hep3B), normal adult liver epithelial cells (THLE-2), and human embryonic kidney cells (HEK293T) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). All HCC cell lines and THLE-2 cells were cultured in high glucose-Dulbecco’s modified Eagle’s medium (H-DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C with 5% CO₂. HepG2, HuH7, and HEK293T cells were also cultivated in H-DMEM that contained 10% FBS and 1% penicillin-streptomycin at 37 °C with 5% CO₂ for subsequent functional assays.

Cell transfection and treatments

Two short hairpin RNAs targeting TRAF5 (sh-TRAF5-1 and sh-TRAF5-2) were constructed for the knockdown of TRAF5 expression in HepG2 and HuH7 cells. Besides, two short hairpin RNAs targeting LTBR (sh-LTBR-1 and sh-LTBR-2) were established to down-regulate LTBR expression in HepG2 and HuH7 cells. Overexpression of RNA targeting LTBR (oe-LTBR) was used to up-regulate LTBR expression in HepG2 cells. Scrambled shRNA (sh-NC) and empty vectors were used as controls. GeneChem (Shanghai, China) was used to synthesize all plasmids used in this study. Before transfection, HepG2 and HuH7 cells (2 × 10⁵ cells/ml) were seeded into six-well plates. Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) was used to transfect plasmids into HepG2 and HuH7 cells and stably transfected cell lines were established. After 48 h of incubation, the cells were collected and quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were conducted to evaluate transfection efficiency. The primers used in this study are listed in Table 1.

HepG2 and HuH7 cells were stably transfected with sh-NC, empty vectors, sh-TRAF5-1, sh-TRAF5-2, sh-LTBR-1, sh-LTBR-2 or oe-LTBR for 48 h. sh-NC- and empty vector-transfected cells served as control cells. To further investigate the mechanisms underlying HCC, HepG2 and HuH7 cells were pre-administered with 12 μM SHN (a necroptosis inducer) for 7 h. To determine the pathway mechanisms of HCC, HepG2 cells were administrated with 1 µM SC75741 (an NF-κB inhibitor) for 24 h.

qRT-PCR

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from cells according to the manufacturer’s instructions. Then, total RNA was reverse-transcribed into cDNA using an RT-PCR kit (Takara, Beijing, China). The Mx3000P Real-Time PCR System (Stratagene, San Diego, CA, USA) was used for all experiments. The performance was conducted using the following reaction program: 95 °C for 3 min, 40 cycles of 95 °C for 12 s, and 62 °C for 40 s. Gene expression was calculated using the 2^−ΔΔCt method. GAPDH served as an internal control. The primers used are listed in Table 1.

Western blotting

Total protein was isolated from HCC cells or tumor tissues after lysis with RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentration was quantified using a BCA kit (Beyotime, Shanghai, China). The obtained proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime, Shanghai, China), followed by transfer onto polyvinylidene difluoride membranes (PVDF; Beyotime, Shanghai, China). Subsequently, membranes were sealed with 5% nonfat milk (Beyotime, Shanghai, China) for 1 h. Afterward, the membranes were incubated with primary antibodies at 4 °C overnight, and subsequently with goat anti-rabbit IgG H&L (HRP) (1:10,000, #ab205718; Abcam, Cambridge, UK) secondary antibody for 1 h. Then, the membranes were visualized using enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA), and protein bands were captured using a Tanon-3500 Image Analyzer (Tanon, Shanghai, China). The detailed information of primary antibodies used was as follows: anti-TRAF5 (1:1,000, #ab137763; Cell Signaling Technology, Danvers, MA, USA), anti-RIP1 (1:1,000, #ARG40183; Arigo, Taiwan, China), anti-p-RIP1 (Ser166) (1:1,000, #ARG66476; Arigo, Taiwan, China), anti-MLKL (1:1,000, #ab184718; Abcam, Cambridge, UK), anti-p-MLKL (Ser345) (1:1,000, #ab196436; Abcam, Cambridge, UK), anti-LTBR (1:500, #ab70063; Abcam, Cambridge, UK), anti-NF-κB (1:1,000, #ab32536; Abcam, Cambridge, UK), anti-p-NF-κB (1:1,000, #ab76302; Abcam, Cambridge, UK), and anti-GAPDH (1:2,500, #ab9485; Abcam, Cambridge, UK). The protein expression was normalized using GAPDH as the internal control.

Cell counting kit-8 (CCK-8) assay

CCK-8 assay was performed to determine the viability of HepG2 and HuH7 cells. According to the supplier’s instructions, cells were seeded into 96-well plates (100 μL cell suspension/well) and subsequently treated with specific reagents for 24, 48, and 72 h at 37 °C in a 5% CO₂ atmosphere. Thereafter, 10 µL of CCK-8 solution was added to each well, and the cells were incubated for another 2 h. Optical density (OD) was measured at 450 nm using a microplate reader (DR-3518G; Hiwell Diatek, Wuxi, China).

Colony formation assay

HepG2 and HuH7 cells (200 cells/well) were seeded in six-well plates and cultured for 14 days at 37 °C with 5% CO₂. After rinsing twice with phosphate-buffered saline (PBS), the cells were fixed in methanol (1 mL) for 15 min and stained with 0.5% crystal violet (Beyotime, Shanghai, China) for 20 min. The plates were dried at 25 °C, and images were photographed under a microscope (CKX53; Olympus, Tokyo, Japan).

Cell wound healing assay

Cell wound healing assay was performed to detect the migratory ability of HepG2 and HuH7 cells. HepG2 and HuH7 cells (3 × 10⁵ cells/well) were plated in six-well plates and then cultivated at 37 °C overnight. Thereafter, the cell monolayer was scratched using a sterilized 200 pipette tip. Subsequently, the cells were incubated in serum-free DMEM for 24 h at 37 °C and 5% CO₂. Images of the wounds were captured under a microscope (CKX53; Olympus, Tokyo, Japan).

Cell invasion assay

Transwell chambers were used to evaluate the invasive ability of HepG2 and HuH7 cells. Cells were seeded in H-DMEM with 10% FBS at a density of 1 × 10⁵ cells/mL, and the upper chamber was pre-coated with 20% Matrigel. The lower chamber contained 600 μL H-DMEM supplemented with 20% FBS. Next, 200 μL of the cell suspension was added to the lower chamber. After 24 h of incubation, the cells were washed with PBS, fixed with methanol at 4 °C for 30 min, and stained with 0.1% crystal violet for 20 min. Images of 12 random fields were captured using a microscope (CKX53; Olympus, Tokyo, Japan). ImageJ software (v1.8.0; National Institutes of Health, Bethesda, MD, USA) was used to quantify cell invasion.

Flow cytometry

Flow cytometry was performed to assess cell survival, necrosis, and apoptosis. After washing twice with PBS, cells were reconstituted in 300 µL binding buffer and stained with 5 μL Annexin V-fluorescein isothiocyanate (FITC; Beyotime, Shanghai, China) for 15 min and 10 µL propidium iodide (PI; Beyotime, Shanghai, China) for another 10 min at 25 °C in the dark. A flow cytometer (CytoFLEX S; Beckman, Miami, FL, USA) was used to measure the survival, necrotic, and apoptotic ratios using Cell Quest software (BD Biosciences, Franklin Lakes, NJ, USA).

Hoechst 33342/PI double-staining

A Hoechst 33342/PI double-staining kit (Solarbio, Beijing, China) was used to evaluate cell survival, necrosis, and apoptosis according to the manufacturer’s instructions. HepG2 and HuH7 cells were seeded into six-well plates (5 × 10⁵ cells/well) and cultivated at 37 °C with 5% CO₂ overnight. After washing twice with PBS, the cells were stained with Hoechst 33342 for 10 min and PI for another 10 min. After 30 min of incubation, the cells were washed with PBS, and images were captured under a microscope (CKX53; Olympus, Tokyo, Japan).

Co-immunoprecipitation (co-IP)

HEK293T cells were lysed using Pierce IP Lysis Buffer (Thermo Fisher, Rochester, NY, USA) according to the manufacturer’s instructions. Then, the cell lysates were incubated with anti-TRAF5 (1:1,000, #ab137763; Abcam, Cambridge, UK), anti-LTBR (1:500, #ab70063; Abcam, Cambridge, UK), anti-IgG (negative control, 1: 1:10,000, #ab109489; Abcam, Cambridge, UK), and Dynabeads^™ Protein G (Thermo Fisher, Rochester, NY, USA) at 25 °C overnight with shaking. Thereafter, the beads were washed and centrifuged (1,000 g for 1 min; 4 °C). After boiling denaturation with loading buffer, the protein precipitate was obtained and used for western blot analysis.

Immunofluorescence

HEK293T cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 1% Triton X-100 for 10 min, and blocked with 3% BSA (Beyotime, Shanghai, China) for 30 min. Cells were then incubated with anti-TRAF5 (1:100, #ab137763; Abcam, Cambridge, UK) and anti-LTBR (1:500, #ab70063; Abcam, Cambridge, UK) primary antibodies. After washing three times with PBS, the cells were incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 488) secondary antibody (1:10,000, #ab150077; Abcam, Cambridge, UK), and the nuclei were stained with DAPI. Fluorescence images were captured using a confocal imaging system (UltraVIEW VoX; Perkin Elmer, Waltham, MA, USA).

Animal model and treatments

Twelve pathogen-free BALB/c nude mice used in this study (age, 4–5 weeks; weight, 22 ± 2 g) were purchased from GemPharmatech Co. Ltd., Nanjing, China. All mice were housed under a 12-h light/dark cycle at 22 ± 3 °C with the humidity of 35–55% and allowed ad libitum access to food and water. All animal experiments were approved by the Animal Ethics Committee of Xiamen University (No. XMULAC20220034-4), and complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

BALB/c nude mice were randomly classified into the sh-NC and sh-TRAF5 groups (n = 6). The sh-NC- or sh-TRAF5-transfected HepG2 cell suspension (1 × 10⁶ cells/100 μL) was subcutaneously injected into the right dorsal flank of BALB/c nude mice in the corresponding group. Therefore, a xenograft model was established in this study. Tumor growth was monitored every day, and tumor volume (mm³, calculated as length × width²/2) was measured every three days. After four weeks of injection, all mice were euthanized by intraperitoneal injection of 150 mg/kg pentobarbital. Then, tumor weight was measured, and tumor tissues were collected for subsequent experiments.

Immunohistochemistry

Xenograft tumor tissues from mice were paraffin-embedded and sliced at 4 μm. Tumor sections were dewaxed with xylene for 20 min, followed by graded concentrations of ethanol (100%, 95%, 90%, 80%, and 70%) for 5 min at 25 °C. Antigens were obtained by boiling in sodium citrate solution (pH 6.0) for 2 min, and endogenous peroxidase was blocked by soaking in 3% H₂O₂ for 20 min at 25 °C. The sections were incubated with 50 μL of normal goat serum for 15 min at 25 °C without washing. Thereafter, 50 μL of anti-Ki67 (1:200, #ab15580; Abcam, Cambridge, UK) was added to the sections, which were then incubated at 4 °C overnight and washed three times with PBS. Subsequently, the sections were incubated with a goat anti-rabbit IgG H&L (Alexa Fluor® 488) secondary antibody (1:10,000, #ab150077; Abcam, Cambridge, UK) for 15 min at 25 °C. After development with 3, 3′-diaminobenzidine (DAB; Beyotime, Shanghai, China) at 25 °C, the sections were observed under a microscope (CKX53; Olympus, Tokyo, Japan). The sections were then counterstained with hematoxylin for 30 min, rinsed with running water for 10 min, and dehydrated with xylene and ethanol. Finally, images were captured using the microscope.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining

TUNEL kits (Beyotime, Shanghai, China) were used to measure cell apoptosis in tumor tissues in accordance with the manufacturer’s instructions. Prepared paraffin-embedded sections were dewaxed and dehydrated in graded concentrations of ethanol and then incubated with 50 μL of proteinase K solution at 37 °C for 30 min. Thereafter, the sections were incubated with the reaction solution for 1 h at 37 °C, washed thrice with PBS, and stained with DAPI for 10 min at 25 °C in the dark. Finally, the sections were photographed using a confocal imaging system (UltraVIEW VoX; Perkin Elmer, Waltham, MA, USA).

Statistical analysis

All data were presented as mean ± standard deviation. Every experiment was conducted thrice. GraphPad 7.0 software (La Jolla, CA, USA) was used for the statistical analysis. Student’s t-test or one-way ANOVA was performed to compare differences between groups. P < 0.05 was considered statistically significant.

TRAF5 knockdown restrains the malignant progression of HCC cells

TRAF5 contributes to the progression of HCC, as evidenced by previous studies (Ding et al., 2021b; Jiang et al., 2020). Thus, we initially examined TRAF5 expression in THLE-2 and HCC cell lines HepG2, HuH7, SMMC-LM3, and Hep3B. and confirmed the up-regulated expression of TRAF5 in these HCC cell lines when compared to THLE-2 cells (P < 0.05; Figs. 1A and 1B). Notably, TRAF5 expression levels in HepG2 and HuH7 cells were markedly higher than those in SMMC-LM3 and Hep3B cells (P < 0.05; Figs. 1A and 1B). Therefore, HepG2 and HuH7 cells were chosen for subsequent experiments.

HepG2 and HuH7 cells were stably transfected with sh-TRAF5-1 and sh-TRAF5-2 and their transfection efficiency was evaluated using qRT-PCR to determine the appropriate sh-TRAF5 for subsequent functional assays. Our results showed that TRAF5 mRNA expression in both HepG2 and HuH7 cells were significantly reduced following sh-TRAF5-1 and sh-TRAF5-2 transfection compared with empty vector transfection (P < 0.01; Fig. S1A). Meanwhile, sh-TRAF5-1 and sh-TRAF5-2 markedly inhibited the viability of HepG2 and HuH7 cells compared with empty vectors (P < 0.01; Fig. S1B). Besides, we did not find significant differences in TRAF5 mRNA expression and cell viability between sh-TRAF5-1- and sh-TRAF5-2- transfected HepG2 and HuH7 cells (P > 0.05; Figs. S1A and S1B), and we selected sh-TRAF5-1 for the following assays.

To further confirm the oncogenic role of TRAF5 in HCC, HepG2 and HuH7 cells were stably transfected with sh-NC (control) or sh-TRAF5. We observed that sh-TRAF5 notably down-regulated TRAF5 expression in HepG2 (Figs. 2A and 2B) and HuH7 cells (P < 0.01; Figs. S2A and S2B), indicating the successful inhibition of TRAF5 in HCC cells by shRNA. Functional assays showed that sh-TRAF5 markedly suppressed the viability (P < 0.05 at 72 h), colony formation (P < 0.01), migration (P < 0.01), and invasion (P < 0.01) of HepG2 cells (Figs. 2C–2F) and HuH7 cells (P < 0.01; Figs. S2C–S2F).

To explore the necroptosis mechanism underlying the oncogenic role of TRAF5 in HCC, we assessed the protein expression levels of p-RIP1 (S166), RIP1, p-MLKL (S345), and MLKL in HCC cells. In this study, we found that sh-TRAF5 significantly up-regulated the protein expression ratios of p-RIP1 (S166)/RIP1 (P < 0.05) and p-MLKL (S345)/MLKL (P < 0.01) in HepG2 cells (Fig. 2G), indicating the pro-necroptosis effect of sh-TRAF5 on HepG2 cells. Besides, sh-TRAF5 notably decreased the survival ratio of HepG2 cells, but increased the necrosis and apoptotic ratios (P < 0.01; Fig. 2H). Results of Hoechst 33342/PI double-staining showed that sh-TRAF5 enhanced the number of necrotic and apoptotic HepG2 cells (Fig. 2I). Additionally, the detection results in HuH7 cells were comparable with those in HepG2 cells (Figs. S2G–S2I).

TRAF5 knockdown promotes the necroptosis of HCC cells

To further determine the necroptosis-promotive role of TRAF5 silencing on HCC cells, HepG2 and HuH7 cells were treated with sh-TRAF5 or/and the necroptosis inducer SHN. qRT-PCR and western blotting showed that SHN did not affect TRAF5 expression in HepG2 cells (P > 0.05; Figs. 3A and 3B). However, SHN exerted a notable inhibitory effect on HepG2 cell viability and a promotive effect on cell necrosis (P < 0.01; Figs. 3C and 3D). Moreover, SHN strengthened the ability of sh-TRAF5 to decrease the viability, inhibit the survival, and promote necrosis of HepG2 cells (P < 0.01; Figs. 3C and 3D), which were also confirmed by Hoechst 33342/PI double-staining assay (Fig. 3E). Furthermore, SHN enhanced the up-regulatory effect of sh-TRAF5I on the ratios of p-RIP1 (S166)/RIP1 (P < 0.01) and p-MLKL (S345)/MLKL (P < 0.05) in HepG2 cells (Fig. 3F). Similar results were observed in HuH7 cells (Figs. S3).

TRAF5 knockdown suppresses LTBR expression and NF- $\text{κ}$B pathway

Research has suggested that the occurrence of HCC is related to LTBR signaling (Zhu et al., 2017). In the present study, down-regulation of LTBR expression (P < 0.01) induced by sh-TRAF5 was found in HepG2 cells with decreased TRAF5 expression (P < 0.01) (Fig. 4A). Besides, the interaction between TRAF5 and LTBR in HCC cells was determined using the co-IP assay (Fig. 4B). Immunofluorescence demonstrated that TRAF5 and LTBR co-localized in the cytoplasm of HEK293T cells (Fig. 4C).

Subsequently, HepG2 and HuH7 cells were stably transfected with sh-LTBR-1 and sh-LTBR-2 to preliminarily investigate the involvement of LTBR in HCC development. Our data showed that sh-LTBR-1 and sh-LTBR-2 markedly down-regulated LTBR mRNA expression in both HepG2 and HuH7 cells relative to empty vector transfection (P < 0.01; Fig. S1C), indicating the successful knockdown of LTBR in HCC cells. Furthermore, the viability of HepG2 and HuH7 cells was significantly suppressed by sh-LTBR-1 and sh-LTBR-2 compared with empty vector transfection (P < 0.01; Fig. S1D). This suggested the beneficial role of sh-LTBR in preventing HCC cell malignant progression. To further validate the interaction between TRAF5 and LTBR in HCC, HepG2 cells were stably transfected with sh-TRAF5 or/and oe-LTBR. qRT-PCR and western blotting showed that oe-LTBR-transected HepG2 cells had markedly higher LTBR expression than control cells (P < 0.01; Figs. 5A and 5B), suggesting the successful transfection of HepG2 cells. Notably, oe-LTBR markedly abolished the effect of sh-TRAF5 on reducing LTBR expression in HepG2 cells (P < 0.01), whereas it exerted no significant impact on TRAF5 expression (P > 0.05; Figs. 5A and 5B). Meanwhile, oe-LTBR notably enhanced HepG2 cell viability (P < 0.01) and decreased the apoptotic ratio (P < 0.05) of HepG2 cells, which weakened the viability-suppressive effect (P < 0.05) and apoptosis-promotive (P < 0.01) effects of sh-TRAF5 (Figs. 5C and 5D). Moreover, oe-LTBR notably decreased the protein ratios of p-RIP1 (S166)/RIP1 (P < 0.01) and p-MLKL (S345)/MLKL (P < 0.01) in HepG2 cells (Fig. 5E). Similarly, oe-LTBR significantly eliminated the up-regulatory effect of sh-TRAF5 in the protein ratios of p-RIP1 (S166)/RIP1 (P < 0.01) and p-MLKL (S345)/MLKL (P < 0.05) in HepG2 cells (Fig. 5E).

Evidence has demonstrated that the activation of NF-κB signaling can promote the malignant progression of HCC (Ding et al., 2020). Thus, we also assessed the levels of NF-κB pathway-related proteins (p-NF-κB and NF-κB). Here, we observed that sh-TRAF5 significantly down-regulated the p-NF-κB/NF-κB protein ratio in HepG2 cells (P < 0.01), while oe-LTBR markedly up-regulated this ratio (P < 0.01; Fig. 5E). Notably, oe-LTBR abolished the effect of sh-TRAF5 on decreasing the p-NF-κB/NF-κB protein ratio in HepG2 cells (P < 0.01; Fig. 5E).

TRAF5 knockdown elevates the necroptosis of HCC cells by blocking the NF- $\text{κ}$B pathway

To further ascertain whether TRAF5 knockdown could inhibit the NF-κB signaling in HCC cells, HepG2 cells were stably transfected with sh-NC or sh-TRAF5 and the NF-κB pathway was further analyzed. Western blotting showed that sh-TRAF5 notably down-regulated the p-NF-κB/NF-κB protein level in HepG2 cells (P < 0.01; Fig. 6A). Also, to further verify whether TRAF5 silencing could enhance HCC cell necroptosis by blocking NF-κB signaling, the NF-κB inhibitor SC75741 was administered to sh-TRAF5-transfected HepG2 cells. Herein, we found that SC75741 significantly down-regulated both NF-κB mRNA level (P < 0.01) and p-NF-κB/NF-κB protein ratio (P < 0.01) in HepG2 cells compared with those in control cells, without affecting TRAF5 expression (P > 0.05; Figs. 6B and 6C). Moreover, SC75741 enhanced the ability of sh-TRAF5 to suppress the NF-κB pathway (P < 0.01) and elevate the protein ratios of p-RIP1 (S166)/RIP1 (P < 0.01) and p-MLKL (S345)/MLKL (P < 0.01) in HepG2 cells (Figs. 6B and 6C).

TRAF5 knockdown inhibits the malignant progression of HCC in vivo

To further figure out whether TRAF5 silencing could suppress HCC tumor progression in vivo, a xenograft model was constructed by administering sh-TRAF5-transfected HepG2 cells to mice. We found that sh-TRAF5 markedly reduced the volume (P < 0.01) and weight (P < 0.01) of xenograft tumors (Fig. 7A). Immunohistochemistry demonstrated the decreased expression of Ki67 (a cell proliferation marker) in xenograft tumor tissues by sh-TRAF5 (Fig. 7B). Besides, sh-TRAF5 promoted cell apoptosis in xenograft tumor tissues (Fig. 7C). Meanwhile, the protein ratios of p-RIP1 (S166)/RIP1 (P < 0.01) and p-MLKL (S345)/MLKL (P < 0.01) in xenograft tumor tissues were markedly increased by sh-TRAF5 (Fig. 7D).