The experiments were approved by the Medical Ethics Committee of Binzhou Medical University. Severn paired samples of lung adenocarcinoma and adjacent noncancerous lung were obtained from patients who had undergone surgery between October 1, 2018 and July 31, 2019 at Yantaishan Hospital, the Affiliated Hospital of Binzhou Medical University (Yantai, China). Informed consent was obtained from all patients. The tissues were fixed in 10% neutral formalin for immunohistochemistry analysis or extracted with total protein for Western blotting.

Immunohistochemistry analysis of paraffin-embedded tissue was performed as follows. The sections were dewaxed with xylene, treated with gradient ethanol for hydration, and repaired with EDTA solution (pH 8.0), followed by blocking with goat serum and incubation with normal IgG. The sections were incubated overnight at 4°C with anti-TRIB2 antibody (1:200 Bioss, Beijing, China), and then incubated in biotin-conjugated second antibody. Proteins were visualized using a pv-9000 two-step detection kit (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China).

A549, H1975, and HeLa cells (Shanghai Institute of Cell Biology, Shanghai, China) were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% CO₂ atmosphere.

Transfection of expression plasmids or short interfering RNAs (siRNAs) into HeLa, A549, and 293T cells was carried out using Lipofectamine™ 3000 (Thermo Fisher Scientific, Waltham, MA, USA; L3000015) according to the manufacturer’s recommendations. All siRNAs were transfected into cells at a final concentration of 50 nM. The sequences of the siRNAs used in this study are shown ( Supplementary Table S1 ).

Total cell lysates were prepared using RIPA buffer (Beyotime, Shanghai, China). All proteins were separated via SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Invitrogen, Carlsbad, CA, USA). The membranes were incubated overnight at 4°C with the following primary antibodies: p-IKB-α, IKB-α, RFWD2, ubiquitin (1:500, Bioworld Technology, Inc., Minneapolis, MN, USA), TRIB2 (1:1,000, Cell Signaling Technology, Danvers, MA, USA), and p-RFWD2 (1:500, Bioss Biotechnology). Next, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:6,000, Beijing Zhong Shan-Golden Bridge Technology Co., Ltd., Beijing, China), and the signals were detected with an Automatic Image Analysis System (Tanon 5200 Multi, Shanghai, China) following electrochemiluminescence immune reactions.

The DNA sequences of TRIB2-full and its truncated mutants were amplified using pcDNA-TRIB2 as a template. The primer sequences used are as shown in Supplementary Table S2 . These DNA fragments were inserted into the p3×flag-CMV-9-10 vector plasmid. To construct the GFP-TRIB2-expressing plasmid, the cDNA for TRIB2 was amplified from pcDNA-TRIB2 using the same primers with Flag-TRIB2-full via PCR and ligated into the pEGFP-C3 plasmid containing an N-terminal GFP tag.

Cells were seeded into 10-cm plates and transfected with Flag-tagged expression plasmid vectors using Lipofectamine 2000 (Invitrogen) for 48 h. The total lysate was extracted with NP-40 buffer (50 mM Tris-Cl at pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% SDS, protease inhibitor mixture) and incubated with 50 µl of Anti-Flag M2 Affinity Gel (Sigma-Aldrich, St. Louis, MO, USA) at 4°C for 2 h. Following centrifugation, the samples were washed with PBS and heated with 40 µl 1× loading buffer, and the supernatant was subjected to SDS-PAGE followed by immunoblotting.

To analyze the interactions among endogenous proteins, 500 µg of cell extracts were incubated with primary antibodies or control IgG overnight at 4°C, and then with protein G/A beads (Invitrogen) for 2 h at 4°C. The beads were washed with lysis buffer, mixed with protein loading buffer, and detected by SDS-PAGE.

Cells growing on glass coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.1% NP-40, and incubated with rabbit antihuman IKB-α/RFWD2 (1:100; Bioworld Technology) overnight at 4°C. The cells were then incubated with Alexa Fluor 488 donkey antirabbit IgG (H+L) or Alexa Fluor 594 donkey antimouse IgG (H+L) (Molecular Probes, Eugene, OR, USA) at 37°C for 1 h. Immunofluorescence was observed using a microscope (DM6000B, Leica, Wetzlar, Germany).

Cells transfected with siRNAs (GenePharma, Shanghai, China; siRNA sequences were shown in Supplementary Table S1 ) or pcDNA-TRIB2 plasmids were cultured in 96-well plates for 48 h as described previously (20). Next, 10 µl 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; 5 mg/ml, Sigma-Aldrich) was added to the medium in each well. The medium was removed 4 h later, and 100 µl dimethyl sulfoxide (Sigma-Aldrich) was added, and the OD value (570 nm) was detected with a microplate reader (Multiskan FC, Thermo Fisher Scientific). Each experiment was performed in triplicate and repeated at least three times.

Harvested cells were treated with Annexin V-FITC/PI (KeyGEN Biotech. Co., Ltd., Nanjing, China) according to the manufacturer’s instructions and counted by flow cytometry (Beckman Coulter, Brea, CA, USA). Each experiment was performed in triplicate and repeated at last three times.

Cells transfected with indicated siRNAs or plasmids were maintained in culture media for 14 days, followed by staining with crystal violet. Colonies containing more than 20 cells were counted. Each experiment was performed in triplicate and repeated at last three times.

Cells (10⁴ cells per well in 100 µl fetal bovine serum-free 1640 medium) were seeded into the upper chamber of a Corning Costar Transwell chamber (Sigma-Aldrich), and 600 µl of RPMI-1640 medium containing 20% calf serum was added to the lower chamber. After 16 h, cells in the upper chamber were removed using cotton swabs, and migrated cells in the lower chamber were fixed with 4% paraformaldehyde and incubated with 1% crystal violet (Sigma) for 15 min. After washing with ddH₂O, five fields per Transwell were examined under a microscope (DM6000B, Leica). The assay was repeated three times for each group.

Migration assays were also performed using an xCELLigence DP instrument following the manufacturer’s instructions (ACEA Biosciences Inc., San Diego, CA, USA); 1.5 × 10⁵ cells in 100 µl of medium were seeded into the upper compartment of a CIM plate in triplicate per group, after which 160 µl medium containing 20% fetal bovine serum was added to the lower compartment. The cell index, representing the number of migrated cells, was calculated via RECA (real-time cell analysis) software 1.2.1 (ACEA Biosciences).

Animals were grouped by simple randomization of using random number table. A549 cells stably expressing TRIB2 or treated with TRIB2-siRNA or RFWD2-siRNA were harvested, and 2 × 10⁶ cells were injected subcutaneously into the backs of female BALB/C-nude mice aged 6–8 weeks (HFK Bio-Technology, Beijing, China) as previously described (20). The second siRNA was injected into the xenografts on day 14 after the initial treatment. Tumor volumes were measured daily using calipers. The animals were sacrificed by intraperitoneal injection of a barbiturate at 30 days after injection. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Binzhou Medical University.

SPSS 22.0 software (IBM Corp., Armonk, NY, USA) was used to analyze statistical significance. Data were tested for normal distribution using a normality test. Data are expressed as mean ± SD. The Student’s t-test was used to compare two averages. ANOVA was used for mean comparison of multiple groups; depending on whether the assumption of homogeneity of variance was satisfied or not, the LSD test or the Games-Howell test was used to compare means of different samples. Abnormally distributed data were expressed as median (interquartile range); the Mann-Whitney U test and Kruskal-Wallis H test were used to compare two groups or multiple groups, respectively. Statistical significance was set at P < 0.05.

TRIB2 acts as an oncogene in various tumors. Our previous study demonstrated that the overexpression of TRIB2 is related to the poor survival of patients with lung cancer, and miR-206 and miR-140 induce lung cancer cell death and suppress cell proliferation by regulating oncogenic TRIB2 promoter activity through p-Smad3 (20). To further determine the mechanism of TRIB2 in the progression and development of lung adenocarcinoma (LUAD), we detected the expression of TRIB2 in lung cancer and adjacent normal lung tissues by immunohistochemistry analysis. TRIB2 was overexpressed in lung cancer tissue compared with the adjacent normal lung tissue ( Figure 1A ). Next, we collected seven lung cancer and adjacent normal lung-tissue samples for Western blotting to detect the expression of TRIB2. We observed that TRIB2 was overexpressed in LUAD ( Figures 1B, C ). In a previous study, we transfected A549 cells with three pairs of siRNA-targeting TRIB2 and confirmed their effectiveness for knocking down TRIB2 (20). Next, we transiently transfected pcDNA-3.1-TRIB2 or siRNA into A549 cells to determine the role of TRIB2 in cancer after its overexpression or downregualtion ( Figures 1D, E ). The results of an MTT assay, which was performed to clarify the role of TRIB2 in cancer progression, showed that TRIB2 knockdown inhibited the proliferation of A549 cells ( Figure 1F ), whereas TRIB2 overexpression promoted the proliferation of A549 cells ( Figure 1G ). Plate colony formation indicated that cell clones, which were smaller and less abundant following TRIB2 knockdown in A549 cells (p < 0.01; Figures 1H, I ), became larger and more abundant after TRIB2 overexpression (p < 0.01; Figures 1J, K ).

Flow cytometry detection was conducted to investigate whether TRIB2-regulated cell proliferation is related to apoptosis. The results obtained demonstrated that the apoptotic rate of A549 cells significantly increased following TRIB2 knockdown but decreased following TRIB2 overexpression ( Supplementary Figures S1A, B ). Tumor cells often exhibit strong migration capabilities. Cell migration experiments indicated that TRIB2 knockdown significantly inhibited cell migration compared with that in the control group ( Supplementary Figures S1C, D ; p < 0.05). Following TRIB2 overexpression, the number of migrated cells increased significantly ( Supplementary Figures S1E, F ; p < 0.05), suggesting that TRIB2 promoted the migration of lung cancer cells. This finding was also supported by cell migration experiments using HeLa cells ( Supplementary Figure S2 ).

To investigate the mechanism of TRIB2 in promoting cancer cell proliferation, the interactions between TRIB2 and other proteins were analyzed with inBio Discover™ online (https://inbio-discover.com/#login). With inBio Discover™, we can explore the interactions between proteins, and gain valuable biological functions. The inBio Discover™ results demonstrated that TRIB2 can perform its functions by interacting with RFWD2, C/EBPα, WEE2, etc. ( Figure 2A ). TRIB2 is also specifically regulated by Wnt signaling in liver cancer cells, which is associated-Ub E3 ligase-TrCP, RFWD2, and Smurf1 reducing TCF4/β-catenin expression (21). To further verify whether the roles of TRIB2 are related to RFWD2 or other Ub E3 ligases, we extracted the total protein from 293T cells transiently expressing Flag-TRIB2 and performed coimmunoprecipitation experiments using an M2 affinity chromatography column binding anti-Flag antibodies. Our results confirmed the interaction between TRIB2 and RFWD2 but did not support the interaction between TRIB2 and polyubiquitin-C ( Figure 2B ). Coprecipitation experiments with the total lysates of 293T cells showed that anti-RFWD2 antibodies precipitated RFWD2, together with TRIB2 ( Figure 2C ). These results indicate that TRIB2 can interact with RFWD2, which may be related to TRIB2-regulating cancer cell proliferation.

RFWD2 is an E3 Ub ligase that plays a vital role in the regulation of cell proliferation and apoptosis (12). The abovementioned results showed that TRIB2 can interact with RFWD2, which indicates that the function of TRIB2 may affect the proteasome-mediated degradation of proteins through the interaction between TRIB2 and RFWD2. To investigate this hypothesis, we first detected the effect of TRIB2 on RFWD2 levels after the overexpression or knockdown of TRIB2 by Western blot. The levels of RFWD2 were significantly reduced following TRIB2 knockdown for 48 h in A549 cells but increased following TRIB2 overexpression ( Figure 2D ). These results indicate that the role of TRIB2 is related to its interaction with and effects on RFWD2. The roles of RFWD2 in cancer is related to targeting its substrates for ubiquitination and degradation, such as p-IκB-α (22), p53, Jun, STAT3, β-catenin, p27, and C/EBPα (12). Next, we detected the levels of RFWD2-related ubiquitination substrate p-IκB-α and p53 after the overexpression or knockdown of TRIB2 by Western blot. Our results showed that TRIB2 overexpression inhibited the p-IκB-α and p53 levels, whereas TRIB2 knockdown resulted in their increase ( Figures 2E, F ). The abovementioned results indicate that TRIB2 can affect p-IκB-α and p53 levels by regulating RFWD2.

To further investigate the above hypothesis, we selected IκB-α as a research object for coprecipitation experiments with the total lysates of 293T cells. The results showed that anti-RFWD2 antibodies precipitated RFWD2, also together with IκB-α. Further coimmunoprecipitation experiments indicated that IκB-α also interacted with RFWD2 ( Figures 2C , 3A ). To determine whether TRIB2 interacts directly with IκB-α, we conducted coimmunoprecipitation with cells overexpressing Flag-tagged TRIB2. The results demonstrated that anti-Flag M2 affinity gel precipitated TRIB2 with p-IκB-α ( Figure 3B ). We next confirmed the endogenous interaction between TRIB2 and p-IκB-α in 293T cells ( Figure 3C ). We further used eukaryotic expressed and purified TRIB2 and IκB-α proteins in coimmunoprecipitation experiments and observed that IκB-α antibodies may precipitate IκB-α and TRIB2 ( Figure 3D ). These results indicate that TRIB2, IκB-α, and RFWD2 form a ternary complex with RFWD2 though direct physical interactions, which may be related to the effect of TRIB2 on the proteasome-mediated degradation of proteins with RFWD2.

Prior studies also supported that TRIB2 regulates the degradation of certain proteins via the Ub-proteasome pathway (23, 24). To further test whether TRIB2 can regulate the degradation of IκB via the Ub–proteasome pathway, we treated A549 cells with various concentrations of MG132, an inhibitor of the proteasome degradation pathway. Western blotting indicated that p-IκB-α expression increased in an MG132 concentration-dependent manner ( Figure 3E ). The findings confirmed that IκB-α was phosphorylated and entered the ubiquitination degradation pathway. However, IκB-α degradation was blocked when A549 cells were treated with 20 μM MG132 following TRIB2 knockdown with siRNA, suggesting that TRIB2 affected IκB-α degradation by regulating its phosphorylation ( Figure 3F ).

To test whether TRIB2 affects the proteasome-mediated degradation of protein IκB-α through a ternary complex TRIB2-RFWD2-IκB-α, we performed immunofluorescence staining to evaluate the interactions and colocalization among TRIB2, RFWD2, and IκB-α. Laser confocal microscopy revealed partial colocalization of exogenously expressed GFP-TRIB2 with FRWD2 in A549 cells ( Figure 4A ) and colocalization of endogenously expressed IκB-α with RFWD2 ( Figure 4B ). Following the transfection of A549 cells with Flag-TRIB2, immunofluorescence analysis revealed the colocalization of Flag-TRIB2 with IκB-α ( Figure 4C ). Altogether, these results support that TRIB2, IκB-α, and RFWD2 form a ternary complex. We also used a network tool (PRODIGY, https://nestor.science.uu.nl/prodigy/) to model the three-dimensional structures of TRIB2 and IκB-α and analyzed their potential for interaction with RFWD2. The PRODIGY results indicated that these three proteins directly interact ( Figure 4D ; Supplementary Table S3 ).

TRIB2 contains an N-terminal PEST region, pseudokinase domain, and C-terminal region (25). To identify the specific domain associated with the interaction of TRIB2 with RFWD2 and IκB-α, we constructed Flag-tagged TRIB2-domain deletion mutants ( Figure 5A ) and transiently transfected them into 293T cells. After 48 h, coimmunoprecipitation analysis of the total protein extract showed that RFWD2 precipitated together with Flag-TRIB2-Full, Flag-TRIB2-C, and Flag-TRIB2-E, indicating that TRIB2 interacted with RFWD2 mainly via its carboxyl terminus ( Figures 5B, C ). Flag-TRIB2-Full, Flag-TRIB2-B, and Flag-TRIB2-D coprecipitated with IκB-α, indicating that the pseudokinase domain is mainly required for the interaction between TRIB2 and IκB-α. Because of the affection of the binding of RFWD2 and the structure of mutant Flag-TRIB2-E, a relatively weak band was found in Flag-TRIB2-E. The abovementioned results indicate that TRIB2 may bind to RFWD2 via its C-terminus, whereas it binds to IκB via its pseudokinase domain. Therefore, the combination of TRIB2 with RFWD2 causes no effect on its interaction with IκB-α.

These above results indicate that TRIB2 can regulate the proteasome-mediated degradation of RFWD2. To further understand the biological role of RFWD2 in TRIB2 affecting the proteasome-mediated degradation of RFWD2-related protein, we screened siRNAs that effectively knock down RFWD2 ( Figure 5D ). siRNAs specific for RFWD2 were cotransfected with the control vector or TRIB2 expression plasmids into A549 cells, and the expression of related proteins was detected. The results showed that the levels of RFWD2-related protein p-IκB-α increased following the RFWD2 knockdown compared with the control treatment ( Figure 5E ). Our results indicate that the function of TRIB2 in regulating proteasome-mediated degradation requires the participation of RFWD2.

The abovementioned results showed that TRIB2 promoted cell proliferation and migration, and that the function of TRIB2 in regulating gene expression requires the participation of RFWD2. We next evaluated whether blocking the RFWD2 activity suppresses the oncogenic role of TRIB2. Using siRNA to knockdown RFWD2, we investigated the effect of RFWD2 on the proliferation and migration of A549 cells. MTT assay showed that proliferation was significantly inhibited in siRNA-treated cells compared with that in the control oligo treatment group (p < 0.01; Figure 6A ). Fluorescence-activated cell sorting assay indicated that the apoptosis rate was elevated in siRNA-treated cells ( Figure 6B ). Real-time cell analysis of migration further showed that RFWD2 knockdown inhibited cell migration ( Figures 6C, D ; p < 0.01). The results in H1975 cells also supported that siRNA-RFWD2 inhibited cell proliferation and migration, and increased lung cancer cell apoptosis ( Supplementary Figure S3 ). These results indicate that RFWD2 knockdown suppresses A549 cell proliferation.

Next, we analyzed the effect of RFWD2 on TRIB2-induced cell proliferation and migration. The MTT assay suggested that either TRIB2-siRNA or RFWD2-siRNA suppressed the proliferation of A549 cells; this suppressive effect was evident when both proteins were downregulated simultaneously ( Figure 6E ). Although TRIB2 overexpression promoted cell proliferation, knockdown of RFWD2 partially weakened the oncogenic role of TRIB2 in A549 cells ( Figure 6F ). The apoptosis assay demonstrated that although the knockdown of either RFWD2 or TRIB2 individually increased the apoptosis rate, knocking them both down significantly increased the apoptotic rate of A549 cells ( Figure 6G ). Moreover, RFWD2 downregulation partially attenuated the role of TRIB2 ( Figure 6H ). The RTCA migration assay demonstrated that the simultaneous knockdown of TRIB2 and RFWD2 suppressed cell migration more substantially than the knockdown of TRIB2 alone ( Figures 6I, J ), and that knockdown of RFWD2 reduced the role of TRIB2 in cell migration ( Figure 6K, L ). The results in H1975 cells further supported that siRNA-RFWD2 can reverse the role of TRIB2 in cell proliferation, migration, and apoptosis ( Supplementary Figure S3 ).

A plate colony-formation assay showed that RFWD2 knockdown inhibited the colony-forming ability of A549 cells ( Supplementary Figures S4A, B ); TRIB2 had the same effect. However, when both TRIB2 and RFWD2 were knocked down simultaneously, the suppressive effect on colony formation became much more remarkable ( Supplementary Figures S4C, D ). Moreover, RFWD2 knockdown reversed the role of TRIB2 in promoting colony formation ( Supplementary Figures S4E, F ). These results indicate that the oncogenic role of TRIB2 is inseparable from RFWD2.

The abovementioned results demonstrated that RFWD2 knockdown suppressed cancer cell proliferation. To further analyze its roles in tumorigenesis, we investigated the RFWD2 expression in cancer based on datasets from The Cancer Genome Atlas online (http://ualcan.path.uab.edu/). The analyzed results demonstrated that RFWD2 is remarkably upregulated in the tissues of numerous kinds of tumors, including bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma (CESC), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), stomach adenocarcinoma (STAD), etc., compared with control tissues ( Figure 7A ). RFWD2 is also overexpressed in LUAD and lung squamous cell carcinoma (LUSC) tissues compared with normal tissues ( Figure 7B ). Kaplan-Meier plot analysis further (http://kmplot.com/analysis/) demonstrated that high RFWD2 levels from patients with lung cancer were related to extremely poor clinical outcomes ( Figure 7C ). These results indicate that RFWD2 may play an important role in promoting lung cancer cell proliferation.

Furthermore, siRNA- and control-treated cells were injected subcutaneously into the backs of BALB/C-nude mice to produce xenografts to investigate the effects of TRIB2 and RFWD2 on the tumorigenesis of lung cancer cells in vivo. On day 14, similar doses of siRNA and control were injected into the xenografts. After 4 weeks, the volume of transplanted tumors in the siRNA-TRIB2 and siRNA-RFWD2 groups was considerably smaller than that in the control group ( Figures 7D, E ). The tumor growth curve further demonstrated that the downregulation of TRIB2 and RFWD2 inhibited tumor formation ( Figure 7F ).

To further verify whether the oncogenic role of TRIB2 is inseparable from RFWD2 in vivo, we knocked down RFWD2 in A549 cell xenografts stably overexpressing TRIB2. The weight and growth curve of the xenografts showed that TRIB2 overexpression promoted tumor formation, whereas RFWD2 knockdown inhibited tumor formation and reduced tumorigenesis in xenografts stably expressing TRIB2 ( Figures 7G–I ). These results suggest that RFWD2 is important for the oncogenic roles of TRIB2 in vitro and in vivo.