Molecular Mechanism of TMEM126A Modulating PANoptosis and Proliferation via TRAF6/NF-κB Signalling Pathway in Non-Small Cell Lung Cancer

Molecular Mechanism of TMEM126A Modulating PANoptosis and Proliferation via TRAF6/NF-κB Signalling Pathway in Non-Small Cell Lung Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Molecular Mechanism of TMEM126A Modulating PANoptosis and Proliferation via TRAF6/NF-κB Signalling Pathway in Non-Small Cell Lung Cancer Xuyong Lin, Lai Wei, Ji Li, Quanxiu Jin, Huanyu Zhao, Yang Liu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7527532/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The mitochondrial transmembrane protein-126A (TMEM126A) is crucial for the accurate assembly of mitochondrial complex I and plays a significant role in preserving mitochondrial function. We aimed to investigate the expression of TMEM126A in non-small cell lung cancer (NSCLC), its biological impact on the malignant progression of NSCLC, and its underlying molecular mechanisms. Here we show TMEM126A was underexpressed in NSCLC and was closely correlated with clinicopathological factors and poor prognosis. In vitro and in vivo functional experiments validated the vital tumour-suppressing roles of TMEM126A in inhibiting cell proliferation and promoting PANoptosis and autophagy. Mechanistically, TMEM126A was identified to interact with and facilitate the autophagy-mediated degradation of TRAF6 via its own transmembrane domain, thereby suppressing the NF-κB signalling pathway and weakening the proliferation of NSCLC. In conclusion, TMEM126A plays a significant inhibitory role in NSCLC malignant progression, which provides experimental evidence to support the development of small-molecule inhibitors. Biological sciences/Cancer/Lung cancer Biological sciences/Cell biology/Cell death TMEM126A TRAF6 NF-κB signalling pathway autophagy PANoptosis NSCLC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Growth kinetics-related factors, such as the proliferation capacity and PANoptosis rate of tumour cells, determine the growth speed and malignant progression. PANoptosis is a newly proposed inflammatory programmed cell death pathway characterised by the key features of apoptosis, pyroptosis, and/or necroptosis. Briefly be described as follows: Caspase-8, released from the apoptotic complex, increases mitochondrial outer membrane permeability by activating Bid, which in turn releases cytochrome C and promotes the release of Caspase-3 and Caspase-7, ultimately inducing apoptosis. In contrast, the RIPK-1/3 complex facilitates the phosphorylation of MLKL, leading to necroptosis. Additionally, Gasdermin D (GSDMD) is cleaved into an N-terminal pore-forming domain (GSDMD-N) and a C-terminal self-inhibitory domain by Caspase-1. GSDMD-N binds to phospholipids in the cell membrane, creating pores that ultimately trigger inflammatory responses and induce cell death [ 1 – 5 ]. Currently, PANoptosis has garnered significant attention as a potential target for tumour therapeutic intervention [ 6 – 7 ]. Autophagy is a physiological process that transports damaged, denatured, and aging proteins and organelles from cells to lysosomes for degradation, thereby playing an important role in maintaining cellular homeostasis [ 8 – 9 ]. Research has indicated that autophagy disorders can lead to physiological abnormalities in cellular metabolism, inflammatory regulation, DNA damage, and other signalling pathways, and are closely related to PANoptosis [ 10 – 13 ]. However, the specific molecular mechanisms regulating autophagy and PANoptosis remain to be fully elucidated. The mitochondrial transmembrane protein TMEM126A (also known as OPA7), with a molecular weight of approximately 25 kDa, is crucial for the proper assembly and functionality of mitochondrial complex I and plays a significant role in maintaining mitochondrial function. TMEM126A comprises 195 amino acids, and its primary functional domains contain four transmembrane structures and four topological domains [ 14 – 16 ]. Current studies have mainly focused on the close association between TMEM126A mutations and autosomal recessive optic nerve atrophy [ 17 – 22 ]. Additionally, Kim EC and his colleagues discovered that in mouse bone marrow cells and macrophages, TMEM126A, in conjunction with its ligand CD137L and in synergy with TLR4 signalling, upregulates the expression of genes related to antigen presentation [ 23 – 24 ]. Therefore, TMEM126A is considered to play a regulatory role in the immune response. However, research on TMEM126A and its association with tumours remains limited. To date, only one study has revealed that silencing TMEM126A facilitates the generation of ROS and depolarisation of the mitochondrial membrane potential, subsequently promoting extracellular matrix remodelling, epithelial-mesenchymal transition, and metastasis in breast cancer cells [ 25 ]. The expression pattern and potential impact of TMEM126A on the malignant progression of lung cancer, as well as the underlying molecular mechanisms, remain unclear. In this study, we aimed to investigate the expression of TMEM126A in NSCLC and demonstrate its vital role of TMEM126A in PANoptosis, autophagy, and cancer cell proliferation in vivo and in vitro , thereby providing an experimental basis for developing small-molecule targeted therapeutic drugs for NSCLC. Methods Patients and samples This study was approved by the Ethics Committee of China Medical University (Approval Number: LS[2019]003) and conducted in accordance with the principles of the Helsinki Declaration. A total of 128 clinical lung cancer tissue samples and 12 pairs of fresh tumours and paired adjacent tissues involved in the study were obtained from the Department of Pathology, First Hospital of China Medical University (2019–2023). None of the patients had undergone radiation therapy or chemotherapy prior to surgery, and all signed informed consent forms. Based on the latest World Health Organization histological classification criteria for lung cancer (2021 edition) [ 26 ], 77 cases were defined as adenocarcinoma and 51 were squamous cell carcinoma. The average age of the patients with lung cancer was 60 years. According to the UICC/AJCC TNM staging criteria (2023 edition) [ 27 ], we categorised 52 cases into stages I and II, while 76 cases were classified as stage III. Immunohistochemical staining The experimental process is described in detail in the Supplementary Materials and Methods. Two pathologists scored the TMEM126A staining intensity and positivity rate based on the following criteria: staining intensity- 0 (uncoloured), 1 (light yellow), 2 (yellow), and 3 (dark yellow-brown); and positivity rate- 0 (1–25%), 1 (26–50%), 2 (51–75%), and 3 (76–100%). Finally, the total score for each slide was obtained by multiplying the scores for staining intensity and positivity rate. As the expression score of TMEM126A was typically > 6 in most adjacent cancer tissues, we defined a score of ≥ 6 as positive and < 6 as negative. For P65 and TRAF6, nuclear staining scores (≥ 4 points) and cytosolic staining scores (≥ 6) were considered as positive expression. Cell culture and immunofluorescence The HBE cell line was purchased from the American Type Culture Collection (#AC338600, ATCC, Manassas, VA, USA). The LK2 cell line was kindly donated by Dr. Hiroshi Kijima from the Department of Pathology and Biological Sciences, Hiroshima University Graduate School of Medicine, Japan. Other NSCLC cell lines, including H1299 (#SCSP-589), A549 (#TCHu150), H460 (#SCSP-584), SK-MES-1 (SCSP-5010), HCC827 (SCSP-538), and H1975 (SCSP-597) were obtained from the cell bank of the Shanghai Institute of Biological Sciences (Shanghai, China). All cell lines were identified through short tandem repeat (STR) DNA analysis and were cultured in RPMI 1640 medium containing 10% foetal bovine serum and 1% penicillin-streptomycin at 37°C with 5% CO 2 . The immunofluorescence assay process is described in detail in the Supplementary Materials and Methods. Plasmids, short-hairpin RNA (shRNA), reagents and transfection The information about plasmids and shRNA were listed in the Supplementary and Materials and Methods. Cells were transfected with Lipofectamine 8000 reagent (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. MG132 (#474790), cycloheximide (CHX; #C7698), and JSH23 (#481408-M) were purchased from Sigma-Aldrich (Shanghai, China). Protein extraction, western blotting, nuclear and cytoplasmic protein separation experiment, immunoprecipitation (IP) and mass spectrometry analysis Protein extraction, western blotting and nuclear and cytoplasmic protein separation experiment procedures, as well as the primary antibodies used in this study were described in detail in the Supplementary and Materials and Methods. For IP, cells were lysed using immunoprecipitation lysis buffer (P0013J, Beyotime Biotechnology) and centrifuged at 4°C and 12000 rpm for 20 min to collect the supernatant. Subsequently, 40 µl of protein A/G agarose magnetic beads (P2012, Beyotime Biotechnology) was added, and the setup was sealed at 4°C for 2–4 h, followed by centrifugation at 1000 rpm for 5 min, aspiration of the supernatant, and addition of the antibody or IgG control at 4°C overnight. The next day, 40 µl of magnetic beads was added to collect the immunocomplex. The magnetic beads were washed thrice with the prepared IP lysis buffer; the beads were retained, 40 µl of 2× loading buffer was added, boiled in 100°C boiling water for 10 min, and subjected to immunoblotting and mass spectrometry analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) qRT-PCR procedure was described in detail in the Supplementary and Materials and Methods. The primer sequences are presented in Table 1 . Table 1 Primers for real-time (RT)-qPCR Primer sequences (5′→3′) TMEM126A 5′- GCTTCCAGAAGCAGAAAGGAATC − 3′ 5′- CTGGCTGCTAGACCACCATT − 3′ TRAF6 5- CTGCAAAGCCTGCATCATAA − 3′ 5′- GGGGACAATCCATAAGAGCA − 3′ GAPDH 5′- GGACCTGACCTGCCGTCTAG − 3′ 5′- GTAGCCCAGGATGCCCTTGA − 3′ 5-Ethynyl-2′-deoxyuridine (EdU) staining and Oxazole yellow/ Propidium iodide (YO- PRO-1/PI) staining EdU staining and YO-PRO-1/PI staining assays were performed according to the manufacturer protocols. The procedures were described in detail in the Supplementary and Materials and Methods. Subcutaneous xenograft tumour experiment Animal experiments were conducted in accordance with the ethical standards for animal research at China Medical University (approval number: CMU20231358). The maximum tumour size permitted by the ethics committee was 2000 mm 3 , and none of the tumours in our study exceeded this size. The formula for calculating tumour volume was as follows: length × width 2 /2. Tumour tissue was partially fixed with 4% paraformaldehyde, embedded in paraffin, and serially sectioned for H&E and immunohistochemical staining. Statistical analysis The results of immunohistochemistry experiments were analysed using the IBM SPSS Statistics 27 (IBM, Armonk, NY, USA) system for chi-square tests and P -value statistics. The other experiments were quantitatively analysed using ImageJ software 18.0, and the remaining experimental results were subjected to t -test using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). The experiments were performed in triplicate, and statistical significance was set at P < 0.05. Results TMEM126A was under-expressed both in tissue specimens and NSCLC cell lines To explore the expression pattern of TMEM126A in NSCLC, we first performed immunofluorescence staining and immunohistochemistry (IHC) of NSCLC tissues (n = 128) and cell lines (n = 3; H1299, A549, H460). The results indicated that TMEM126A is mainly located in the cytoplasm and that its expression is strongly positive in the normal bronchial and alveolar epithelia (29/40, 72.5%) while being weakly positive or negative in adenocarcinoma and squamous cell carcinoma (59/128, 46.1%) (Fig. 1 A-C, P < 0.05). Statistical analysis showed that low expression of TMEM126A was significantly and positively correlated with tumour size ( P = 0.021), poor differentiation ( P < 0.001), positive lymph node metastasis ( P = 0.035), and advanced TNM stage ( P < 0.001), though not related to the patient's sex ( P = 0.155), age ( P = 0.725), or histological classification ( P = 0.539) (Table 2 ). We selected a normal bronchial epithelial cell line (HBE) and a panel of NSCLC cell lines (n = 7) to detect the expression of TMEM126A. Immunoblotting results indicated that the expression of TMEM126A in multiple NSCLC cell lines (6/7) was significantly lower than that in HBE (Fig. 1 D, E). In addition, we measured TMEM126A protein expression in 12 pairs of fresh NSCLC tissue samples and adjacent normal specimens. TMEM126A expression in the NSCLC tissues was significantly lower than in adjacent tissues (11/12, P = 0.003, Fig. 1 F, G). Therefore, our preliminary results demonstrated a low expression pattern of TMEM126A in NSCLC. Table 2 Association between TMEM126A expression and clinicopathological characteristics in NSCLC. Clinicopathological characteristics N TMEM126A negative TMEM126A positive χ2 P (two-side) Gender male 58 27(46.55%) 31(53.45%) 2.039 female 70 42(60.00%) 28(40.00%) 0.155 Age (years) ≤ 60 67 35(52.24%) 32(47.76%) 0.157 > 60 61 34(55.74%) 27(44.26%) 0.725 Tumor diameter ≤ 3 cm 67 43(64.18%) 24(35.82%) 5.971 > 3 cm 61 26(42.62%) 35(57.38%) 0.021 Histological type AC* 77 40(51.95%) 37(48.05%) 0.298 SCC # 51 29(56.86%) 22(43.14%) 0.593 Differentiation Well-moderate 58 44(75.86%) 14(24.14%) 20.576 poor 70 25(35.71%) 45(64.29%) <0.001 Lymph node metastasis Negative 63 40(63.49%) 23(36.51%) 4.588 Positive 65 29(44.62%) 36(55.38%) 0.035 TNM classification Ⅰ- Ⅱ 52 13(25.00%) 39(75.00%) 29.451 Ⅲ 76 56(73.68%) 20(26.32%) <0.001 *: Adenocarcinoma, #: Squamous cell carcinoma Table 3 Potential binding sites predicted by the molecular docking model for the interaction between TMEM126A and TRAF6. TMEM126AResidues TRAF6 Residues Distance(A) Specific Interactions A:Lys 158 B:Gln 263 1.6 1x hb to B:Gln 263 A:Ile 149 B:Arg 314 1.7 1x clash to B:Arg 314 A:Trp 148 B:Glu 321 1.7 1x hb to B:Glu 321 A:Arg 64 B:Cys 218 2 1x hb to B:Cys 218 A:Ser 52 B:Asn 256 1.6 1x hb to B:Asn 256 A:Leu 48 B:Arg 255 1.7 1x clash to B:Arg 255 Ectopic expression of TMEM126A inhibited the proliferation ability and promoted PANoptosis in NSCLC cells An imbalance between the survival and death of tumour cells is crucial for determining tumour growth and malignant progression. Therefore, we investigated whether TMEM126A plays a regulatory role in the survival and PANoptosis of lung cancer cells. We selected H460 and H1299 cells, which exhibit low and moderate expression of TMEM126A, respectively, for TMEM126A plasmid gradient transfection. Western blotting results demonstrated the transfection efficiency and effectiveness of the subsequent functional experiments (Fig. 2 A, B). EdU staining results demonstrated that TMEM126A overexpression significantly inhibited the proliferation of NSCLC cells (Fig. 2 C-F). Immunoblotting was performed to assess the expression of cell proliferation-related proteins after transfecting a TMEM126A-overexpressing plasmid. The results indicated that, compared to those in the control group, ectopic TMEM126A expression led to a decrease in the expression levels of the cell cycle-dependent kinases CDK4 and CDK6, as well as the cell cycle protein CyclinD1 (Fig. 2 G). In contrast, YO-PRO-1/PI staining indicated that, compared to that in the control group, the number of YO-PRO-1-positive cells significantly increased in TMEM126A-overexpressed group, suggesting that TMEM126A promoted the PANoptosis of tumour cells (Fig. 2 H-K). Accordingly, immunoblotting analysis revealed that overexpression of TMEM126A elevated the expression of apoptosis-related proteins, such as cleaved-caspase-3 and Bax, while suppressing the expression of the anti-apoptotic protein Bcl-2. Additionally, it led to an increase in the phosphorylation levels of necrotic apoptosis-related proteins, including phosphorylated RIPK3 and phosphorylated MLKL, and a significant upregulation of the activated form of the pyroptosis-related protein GSDMD-N (Fig. 2 L). TMEM126A knockdown promoted proliferation and weakened PANoptosis of NSCLC cells We selected A549 and H1299 cells (with relatively high and moderate TMEM126A expression, respectively) for the knockdown experiments to demonstrate the biological role of TMEM126A in NSCLC (Fig. 3 A, B). The EdU staining assay showed that downregulation of TMEM126A expression promoted the proliferation of cancer cells (Fig. 3 C-F), and correspondingly upregulated the expression levels of CDK4 and CDK6 proteins, as well as CyclinD1 (Fig. 3 G). YO-PRO-1/PI staining results showed that TMEM126A silencing decreased the number of YO-PRO-1-positive cells, indicating the inhibition of PANoptosis in NSCLC (Fig. 3 H-K). Immunoblotting analysis revealed that compared to those in the control group, TMEM126A knockdown resulted in the downregulation of apoptosis-related protein (cleaved-caspase-3, Bax) expression, necrotic apoptosis-related protein (phosphorylated RIPK3 and phosphorylated MLKL) expression, and cell death-related protein (GSDMD-N) expression, and the upregulation of anti-apoptotic protein (Bcl-2) expression (Fig. 3 F). Thus, our results suggested that TMEM126A is an anti-oncogenic protein that inhibits proliferation and promotes the PANoptosis of NSCLC cells. TMEM126A could be a negative regulator of the NF-κB signalling pathway For exploring the potential molecular mechanisms by which TMEM126A exerts its anti-cancer effect on the malignant progression of NSCLC, we first transfected small interfering RNA sequences into A549 cells to knock down the expression of TMEM126A; RNA-sequencing, Gene Oncology enrichment (GO), and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses indicated that TMEM126A is closely related to the NF-κB signalling pathway (Fig. 4 A, B). NF-κB signalling pathway plays a crucial role in numerous physiological and pathological processes of eukaryotic organisms, including inflammation, cell proliferation, transformation, tumorigenesis, and apoptosis inhibition. The primary mechanism of NF-κB activation involves the phosphorylation of the IKK complex, consisting of two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ). This phosphorylation leads to the degradation of IκBα, thereby releasing NF-κB (P65), which is then transported into the nucleus and binds to specific DNA sequences to initiate transcription [ 28 – 33 ]. In addition, TNF receptor-associated factor-6 (TRAF6) interacts with the activated form of mitogen-activated protein kinase (phosphorylated TAK1) and activates the NF-κB pathway [ 34 ]. Hence, we initially performed immunoblotting to detect the impact of TMEM126A on the changes in the expression levels of key proteins in the NF-κB signalling pathway, both in H460 and H1299 cells. Results indicated that the phosphorylation levels of TAK1, IKK, and IκB-α decreased upon TMEM126A gradient overexpression (Fig. 4 C). Therefore, we conducted immunofluorescence and nuclear-cytoplasmic protein separation assays to detect the effect of TMEM126A on the nuclear-cytoplasmic distribution of P65, and found ectopic TMEM126A expression to significantly reduce the level of P65 nuclear translocation in H460 and H1299 cells (Fig. 4 D, E). Notably, we utilised the NF-κB signalling pathway inhibitor JSH23 (a P65 nuclear entry inhibitor) and discovered that it abrogates the promoting effect of TMEM126A knockdown on P65 nuclear import (Fig. 4 F, G), indicating that TMEM126A plays a crucial role in inhibiting NF-κB signalling pathway. We further explored whether TMEM126A exerts its biological behaviour in inhibiting the malignant progression of lung cancer by suppressing the activity of the NF-κB signalling pathway. We silenced TMEM126A in H1299 and A549 cells treated with JSH23 and DMSO as the experimental and the control groups, respectively. EdU staining experiments indicated that JSH23 could eliminate the tumour cell proliferation promoted by TMEM126A knockdown (Supplementary Fig. S1 A, B) as well as the upregulation of protein expression of CDK4, CDK6, and CyclinD1 (Supplementary Fig. S1 C). In contrast, YO-PRO-1/PI staining demonstrated that JSH23 reversed the biological effect of TMEM126A knockdown on PANoptosis inhibition (Supplementary Fig. S1 D, E) as well as its regulatory effects on the expression levels of cleaved caspase-3, Bax, Bcl-2, p-RIPK3, p-MLKL, and GSDMD-N proteins (Supplementary Fig. S1 F). In summary, we concluded that TMEM126A exerts its biological function by inhibiting cell proliferation and promoting PANoptosis, thereby suppressing the NF-κB signalling pathway. Interaction of TRAF6 in the NF-κB pathway with TMEM126A We further explored the specific molecular mechanisms by which TMEM126A regulates the NF-κB signalling pathway. Mass spectrometry and co-immunoprecipitation were used to identify the tumour necrosis factor receptor-associated factor 6 (TRAF6), which interacts with TMEM126A (Fig. 5 A-E). Accumulating research has shown that TRAF6 plays a pivotal role in activating the NF-κB pathway, and regulating apoptosis and autophagy [ 35 – 36 ]. The molecular docking model between the proteins was validated ( https://hdock.phys.hust.edu.cn ), and the results revealed that TMEM126A and TRAF6 share six binding sites, as listed in Table-3 and Fig. 5 F-G, suggesting a strong binding affinity between them. Immunofluorescence analysis revealed that they co-localised in the cytoplasm of H1299 and H460 cells (Fig. 5 H). The structural basis of this interaction was explored next. Based on the six binding sites identified by molecular docking, three mutants were designed; the first involved mutations at positions 48, 52, and 64 within the transmembrane domain, the second involved mutations at positions 148, 149, and 158 in the topological domain, and the third involved mutations at all six aforementioned positions (Fig. 5 F). Immunoprecipitation results indicated that when mutations occurred at positions 48, 52, and 64, TMEM126A was unable to bind TRAF6, suggesting that TMEM126A interacts with TRAF6 primarily through its transmembrane domain (Fig. 5 I, J). Since TRAF6 is recognised as an upstream factor in the NF-κB signalling pathway, we further investigated whether TMEM126A inhibits the NF-κB signalling pathway via TRAF6. We transfected wild-type and mutant TMEM126A plasmids, respectively, into H460 and H1299 cells, and explored the regulatory effects of TMEM126A on proteins related to the NF-κB signalling pathway and on the malignant phenotype. Immunoblotting revealed that wild-type TMEM126A could gradually downregulate the phosphorylation levels of TAK, IKK, and IκB α in a dose-dependent manner, whereas mutant TMEM126A (i.e., unable to bind to TRAF6) could not (Fig. 5 K). EdU and YO-PRO-1/PI staining assays confirmed that the TMEM126A with a transmembrane-domain mutation was unable to exert its anti-cancer effects by inhibiting cell proliferation (Supplementary Fig. S2 A-D) and downregulating the proliferation-related proteins (CDK4, CDK6, CyclinD1, Supplementary Fig. S2 E); promotion of PANoptosis (Supplementary Fig. S2 F-I) and upregulation of PANoptosis-related proteins (cleaved caspase-3, Bax, Bcl2, p-RIPK3, p-MLKL, and GSDMD-N, Supplementary Fig. S2 J) were also abrogated. Finally, a rescue assay indicated that TRAF6 silencing could abrogate the regulatory effect mediated by TMEM126A on the expression levels of p-TAK1, p-IKΚ, and p-IκB α proteins (Fig. 5 L). In summary, we concluded that TMEM126A exerts a key anti-cancer effect by binding to TRAF6 through its transmembrane domain, thereby inhibiting the activity of the NF-κB signalling pathway and modulating the proliferative ability and PANoptosis process of NSCLC. TMEM126A promoted the metabolic degradation of TRAF6 via autophagy We found that the overexpression of TMEM126A significantly downregulated the protein expression of TRAF6 (Fig. 6 A, B). Subsequently, we conducted RT-qPCR to determine that TMEM126A had no significant regulatory effect on TRAF6 mRNA levels (Fig. 6 C, D). Spearman's analysis of western blotting results of HBE and a panel of lung cancer cell lines (n = 7) revealed a negative correlation between TMEM126A and TRAF6 (Fig. 6 E, F, R = − 0.786, P = 0.028). The results indicated that TMEM126A downregulated TRAF6 expression at the post-transcriptional level. Notably, we overexpressed TMEM126A in H460 and H1299 cells and introduced cycloheximide (CHX) at various time points. The findings indicated that ectopic TMEM126A expression accelerated the degradation of TRAF6 and shortened its half-life (Fig. 6 G-J). Protein degradation pathways are known to depend on two systems, namely the ubiquitin-proteasome degradation system and the autophagy-lysosome pathway degradation system. Therefore, we opted to utilise the proteasome inhibitor MG132 and the autophagosome inhibitor 3-methyladenine (3-MA), respectively, to investigate the specific reasons behind the degradation of TRAF6 by TMEM126A at both exogenous and endogenous levels. Results indicated that the inclusion of 3-MA eradicated the downregulation of TRAF6 expression facilitated by TMEM126A overexpression, whereas MG132 did not (Fig. 6 K, L). This suggested that the degradation of TRAF6 by TMEM126A occurred primarily through the autophagic lysosomal pathway. We conducted further experiments on the correlation between TMEM126A expression and autophagy. Western blotting results indicated that TMEM126A overexpression in H460 and H1299 cells promoted the transformation of the autophagy-related protein LC3 (I to II, Fig. 6 M), and downregulated P62 expression, whereas TMEM126A silencing exerted the opposite effect (Fig. 6 N). Transmission electron microscopy revealed that TMEM126A overexpression increased the number of autophagosomes in lung cancer cells (Supplementary Fig. S3 A, B). Immunofluorescence analysis revealed that TMEM126A treatment increased the number of autophagic spots (Supplementary Fig. S3 C-F), demonstrating that TMEM126A plays a crucial role in activating autophagic activity in NSCLC. To further confirm the possibility of TMEM126A degrading TRAF6 through the promotion of autophagy, we reported that TMEM126A-mediated TRAF6 degradation disappeared upon the addition of 3-MA (Fig. 6 O), thereby suggesting that TMEM126A facilitates the metabolic degradation of TRAF6 by activating the autophagy pathway. In summary, TMEM126A promotes the autophagic degradation of TRAF6, thereby inhibiting the downstream NF-κB signalling pathways. This in turn could suppress cell proliferation, promote PANoptosis, and lead to the suppression of malignant progression in NSCLC. TMEM126A suppressed the growth of transplanted tumours in nude mice in vivo The above-mentioned experimental results were next validated at the cellular level. To ensure the rigor and effectiveness of our findings, we conducted experiments in vivo to validate our results (Fig. 7 A). A nude mouse xenograft experiment demonstrated that overexpression of wild-type TMEM126A in H460 cells significantly suppressed both the volume and weight of the transplanted tumours compared to that in the control group. Conversely, transfection with mutant TMEM126A, which has a transmembrane domain mutation preventing it from binding TRAF6, had no such effect (Fig. 7 B-D). On the other hand, knocking down the expression of TMEM126A in A549 significantly promoted the growth of transplanted tumours, whereas the addition of NF-κB signalling pathway inhibitor (JSH23, 3 mg/kg) or TRAF6 knockdown blocked the effect (Fig. 7 E-G). Subsequently, we embedded the transplanted tumours in paraffin, prepared sections, and conducted haematoxylin-eosin and immunohistochemical staining. The experimental results revealed that TRAF6 expression was downregulated in H460 cells transfected with wild-type TMEM126A and that the nuclear import level of P65 decreased. However, this phenomenon was not observed in the group transfected with the TMEM126A transmembrane domain mutant (Fig. 7 H, I). A rescue experiment demonstrated that JSH23 or TRAF6 knockdown attenuated the effects induced by TMEM126A silencing (Fig. 7 J, K), indicating that TMEM126A promoted TRFA6 degradation in vivo , thereby inhibiting P65 nuclear entry and NF-κB signalling pathway, leading to the inhibition of malignant progression of NSCLC. Discussion Our study, based on immunohistochemical analysis of 128 pathological specimens from patients with NSCLC, revealed that the expression of TMEM126A in cancer tissues was significantly lower than that in normal lung tissue. Furthermore, it is closely correlated with the degree of differentiation, tumour size, lymph node metastasis, and pTNM staging of NSCLC. This clinical discovery sparked interest in exploring the biological impact of TMEM126A in NSCLC. We validated TMEM126A expression in various lung cancer cell lines and freshly paired lung cancer and adjacent tissues and obtained experimental results consistent with the immunohistochemical conclusions. This suggests that TMEM126A may have translational value as a molecular marker for NSCLC and for predicting prognosis. Although we confirmed low TMEM126A expression in NSCLC, it remains unclear whether this is caused by promoter hypermethylation, transcriptional inhibition, or increased post-translational metabolic degradation. The Cancer Genome Atlas (TCGA) online database ( https://ualcan.path.uab.edu ) indicated that compared to that in normal lung tissue, the methylation level of the TMEM126A gene was significantly upregulated in NSCLC tissues ( P = 3.961500E-03), suggesting that low TMEM126A expression may be related to gene methylation. Next, we verified that TMEM126A, as an anti-cancer protein, inhibits the proliferation of lung cancer cells and promotes the PANoptotic process in lung cancer cells through in vitro and in vivo functional experiments. PANoptosis enables the eradication of cancer cells via various cell death pathways and has the potential to overcome diverse obstacles in cancer therapy, such as drug resistance and immune evasion. Understanding the mechanisms underlying the induction of PANoptosis in cancer cells and exploring potential therapeutic agents could lead to effective cancer treatments and improved patient outcomes. For example, He et al. utilised TCGA database to perform a comprehensive analysis of breast cancer characteristics and concluded that a high PANoptosis process is beneficial in reducing the incidence of breast cancer [ 37 ]. Studies have also shown that PANoptosis contributes to shaping the tumour microenvironment in patients with low-grade gliomas [ 38 ]. Furthermore, it has been shown to induce cell death in melanoma cell lines [ 39 ]. Lin et al . highlighted the significance of PPM1B-mediated YBX1 dephosphorylation and USP10-mediated deubiquitination in the regulation of PANoptosis and oxaliplatin sensitivity in gastric cancer cells [ 40 ]. Based on KEGG and GO enrichment analyses using RNA sequencing, TMEM126A was found to be closely associated with the NF-κB signalling pathway. Previous studies have indicated that the NF-κB signalling pathway plays a crucial role in regulating PANoptosis and inflammation in non-tumour diseases [ 41 – 44 ]. We identified that TMEM126A interacts with TRAF6, an upstream factor of the NF-κB pathway, thereby inhibiting P65 nuclear import and promoting PANoptosis in NSCLC. Notably, we found that TMEM126A enhanced autophagic activity, leading to an increase in TRAF6 metabolic degradation (Fig. 8 ). However, the pathways and underlying molecular mechanisms through which TMEM126A enhances autophagy, as well as the mechanism by which TMEM126A activates the NF-κB pathway upon binding to TRAF6, remain unclear. This is a limitation of the current study. This warrants further investigation in future studies. Conclusions Our findings imply that TMEM126A acts as a tumour suppressor by modulating proliferation and PANoptosis balance through the TRAF6/NF-κB signalling pathway and could serve as a potential target for targeted therapy in NSCLC. Abbreviations TMEM126A: the mitochondrial transmembrane protein-126A; TRAF6: tumour necrosis factor receptor-associated factor-6; CDK-4/6: cyclin-dependent kinase-4/6; CyclinD1: G1/S-specific cyclin-D1; Caspase-3: cysteinyl aspartate specific proteinase-3; BAX: Bcl-2 associated X protein; Bcl-2: B-cell lymphoma-2; p-RIPK-3: phosphorylated receptor-interacting serine/threonine protein kinase-3; p-MLKL: phosphorylated mixed lineage kinase domain-like protein; GSDMD: gasdermin D; p-TAK: phosphorylated TGF-beta-activated kinase-1; p-IKK: phosphorylated inhibitor of nuclear factor kappa-B kinase; p-IκB: phosphorylated NF-kappa-B inhibitor; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; CHX: cycloheximide; MG132: Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; 3-MA: 3-Methyladenine; P62: sequestosome-1/SQSTM-1; LC3: light chain-3; NC: negative control; EdU: 5-Ethynyl-2'-deoxyuridine; YO-PRO-1/PI: oxazole yellow/propidium iodide; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; shRNA: short-hairpin RNA; NSCLC: non-small-cell lung cancer; KEGG: Kyoto Encyclopedia of Genes and Genomes; PBS: phosphate-buffered saline; GO: Gene Ontology enrichment; NSCLC: non-small-cell lung cancer; IHC: immunohistochemistry; IP: immunoprecipitation; IB: immunoblotting; SDS: sodium dodecylsulphate; PAGE: polyacrylamide gel electrophoresis; PVDF: polyvinylidene difluoride. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of China Medical University (Approval Number: LS[2019]003) and was conducted in accordance with the principles of Helsinki. All patients provided informed consent. Animal experiments were conducted in accordance with the ethical standards for animal research at China Medical University (approval number: CMU20231358). Consent for publication All authors have approved the submission of the manuscript for publication and agree to be accountable for all aspects of the manuscript Competing interests The authors declare no conflict of interest. Funding This work was supported by the National Natural Science Foundation of Liaoning Province (grants 2022-MS-198 to Xuyong Lin) and the National Natural Science Foundation of China (grants 81902986 to Qiang Han, grants 82003119 to Xuezhu Rong). Author contributions Lai Wei, Ji Li, and Quanxiu Jin were responsible for designing the research studies, conducting experiments, acquiring, and analysing data. Huanyu Zhao, Yang Liu, Yuheng Feng, and Xueting Gan were tasked with conducting experiments and acquiring data. Xuezhu Rong, Qiang Han, and Xuyong Lin had the oversight of the study's conception and supervision, and authored the manuscript. All authors contributed to revising the draft versions and approving the final version of the manuscript. Acknowledgments We thank Dr. Hiroshi Kijima (Department of Pathology and Biological Sciences at Hiroshima University Graduate School of Medicine, Japan) for donating the LK2 cell line. Availability of data and materials All data generated or analysed during this study are included in this published article and its Supplementary Files. Uncropped original western blots used in this manuscript have been uploaded as “Supplemental Material”. 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Exosomes derived from FN14-overexpressing BMSCs activate the NF-kappaB signaling pathway to induce PANoptosis in osteosarcoma. Apoptosis. 30(3-4) , 880-893 (2025). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarymaterialsandmethods.docx Supplementary materials and methods Massspectrometry.xls Mass spectrometry SupplementaryFigureS1.tif Supplementary Figure S1. SupplementaryFigureLegend.docx Supplementary Figure Legends SupplementaryFigureS3.tif Supplementary Figure S3. RNAsequencing.xlsx RNA-sequencing WBoriginaldata.pdf WB original data SupplementaryFigureS2.tif Supplementary Figure S2. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Magnification, 400×, Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB-C: \u003c/strong\u003eImmunohistochemical analysis of TMEM126A expression in NSCLC tissues (B). Strong positive expression (+++) was observed in the bronchial epithelium (a) and alveolar epithelium (b), moderate positive expression (++) was observed in the highly differentiated adenocarcinoma (c) and squamous cell carcinoma (e), and negative expression (-) was observed in the poorly differentiated adenocarcinoma (d) and squamous cell carcinoma (f). Magnification, 400×; Scale bar, 50 μm. Statistical schematic diagram illustrating the differential expression of TMEM126A in adjacent lung and cancer tissues, as detected by immunohistochemistry (C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD-E:\u003c/strong\u003eWestern blotting was performed to detect the expression of TMEM126A in HBE cells and a panel of NSCLC cell lines (n = 7). GAPDH served as a loading control (D). The statistical bar chart (E) shows the relative expression levels of TMEM126A in various cell lines. NS: not significant; *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF-G:\u003c/strong\u003eProtein expression of TMEM126A, detected by western blotting, was downregulated in lung cancer tissues (11/12), with GAPDH serving as the loading control (F). Statistical scatter plot of grayscale values from the immunoblotting results (G).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/01605d05cb79675087bef145.png"},{"id":91715296,"identity":"558808cd-1f68-41f2-8e70-b649a1fdb13c","added_by":"auto","created_at":"2025-09-19 13:14:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4007984,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTMEM126A promoted the PANoptosis of lung cancer cells and suppressed their proliferation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B:\u003c/strong\u003eWestern blotting was conducted in H460 and H1299 cells to detect the transfection efficiency of the TMEM126A plasmid with gradient overexpression, using GAPDH as the loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC-F: \u003c/strong\u003eEdU proliferation assay demonstrated that overexpression of TMEM126A in H460 and H1299 cells inhibited cell proliferation (C, D). Magnification, 400×; Scale bar, 50 μm. Results of the EdU assay are presented in a statistical bar chart (E-F, mean ± standard deviation, n = 3). **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG:\u003c/strong\u003eOverexpression of TMEM126A significantly decreased the expression of cell proliferation-related proteins CDK4, CDK6, and CyclinD1, with GAPDH serving as the loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH-K: \u003c/strong\u003eOverexpression of TMEM126A in H460 and H1299 cells. YO-PRO-1/PI fluorescence double-staining revealed that TMEM126A overexpression promoted PANoptosis in tumour cells (H-I). Magnification, 200×; Scale bar, 100 μm. The experimental results of YO-PRO-1/PI staining are presented as a statistical bar chart (mean ± standard deviation, n = 3) (J, K). *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL:\u003c/strong\u003eWestern blotting results showed that ectopic TMEM126A expression promotes the upregulation of apoptosis-related (C-caspase-3, BAX), necroptosis-related (p-RIP3 and p-MLKL), and pyroptosis-related (GSDMD-N) proteins while downregulating the expression of anti-apoptotic proteins (BCL-2). GAPDH served as the loading control.\u003c/p\u003e\n\u003cp\u003eC-caspase-3, cleaved caspase-3; GSDMD-FL, GSDMD full-length; p-RIP3, p-MLKL, phosphorylated RIP3 and MLKL.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/cf4356a83d7f13a628cc33f4.png"},{"id":91715843,"identity":"15878c0a-6ba4-402c-975d-2855d2ec8648","added_by":"auto","created_at":"2025-09-19 13:22:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3842268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTMEM126A knockdown attenuated PANoptosis in lung cancer cells, thereby inhibiting their proliferation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B:\u003c/strong\u003eWestern blotting was performed to assess the efficiency of TMEM126A knockdown using two independent short hairpin RNAs in A549 and H1299 cells, with GAPDH as the loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC-F:\u003c/strong\u003eThe EdU proliferation assay indicated that knocking down TMEM126A enhances the proliferation capability of lung cancer cells (C-D, magnification, 400´; scale bar, 50 μm), which are presented in a statistical bar chart (E-F, mean ± standard deviation, n = 3). *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG:\u003c/strong\u003eFollowing the knockdown of TMEM126A, expression of the cell proliferation-related proteins CDK4, CDK6, and CyclinD1 was significantly upregulated, with GAPDH serving as the loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH-K:\u003c/strong\u003eAfter silencing TMEM126A in A549 and H1299 cells, YO-PRO-1/PI fluorescence double-staining assay results indicated that TMEM126A silencing inhibited tumour cell apoptosis (H-I). Magnification, 200×; Scale bar: 100 μm. The experimental results of YO-PRO-1/PI staining are presented as a statistical bar chart (mean ± standard deviation, n = 3) (J, K). *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL: \u003c/strong\u003eSilencing TMEM126A downregulated apoptosis-related (cleaved-caspase-3, BAX), necroptosis-related (p-RIP3 and p-MLKL), and pyroptosis-related (GSDMD-N) proteins while upregulating the expression of anti-apoptotic proteins (BCL-2). GAPDH served as the loading control.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/6ca3321cfb92ed64051d0277.png"},{"id":91715292,"identity":"4d0a620b-a311-43d7-955b-9a9ae02f00e0","added_by":"auto","created_at":"2025-09-19 13:14:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4294457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTMEM126A was verified as a negative regulator of the NF-κB signalling pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B: \u003c/strong\u003eGO enrichment analysis (A) and KEGG signalling pathway analysis (B) based on RNA sequencing results indicated that TMEM126A is closely associated with the NF-κB signalling pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC: \u003c/strong\u003eOverexpression of TMEM126A downregulated the expression of key proteins p-TAK1, p-IKK, and p-IκBα in the NF-κB signalling pathway in H460 and H1299 cells. GAPDH served as the loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD-E: \u003c/strong\u003eImmunofluorescence staining (D) and nuclear-cytoplasmic separation assay (E) demonstrated that TMEM126A overexpression reduced the level of P65 nuclear translocation. Magnification, 400×; scale bar, 25 μm. β-Tubulin and Lamin B served as the loading controls for cytoplasm and nucleus, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF-G: \u003c/strong\u003eH1299 cells and A549 cells were transfected with shRNA-control or shRNA-TMEM126A, and 36 h later, the culture medium was replaced with the media containing DMSO or JSH23 (10 μM) for 12 h. The P65 nuclear import level was detected by a nuclear-cytosolic separation assay and immunoblotting.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/0b44bb61c66132b0a03e9e2d.png"},{"id":91715842,"identity":"0081c0a1-2ed9-45ac-918f-ecc0ba2349c5","added_by":"auto","created_at":"2025-09-19 13:22:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4809259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRAF6, an upstream factor of NF-κB signalling pathway, was identified to interact with TMEM126A.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eMass spectrometry analysis and Coomassie Brilliant Blue-R250 staining identified the interaction between TRAF6 and TMEM126A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB-E: \u003c/strong\u003eCell lysates isolated from H460 cells transfected with Myc-TMEM126A or from H1299 cells were subjected to a co-immunoprecipitation assay with Myc-tag monoclonal antibody and TRAF6 antibody, respectively, with IgG serving as a control group; the precipitates were analysed by immunoblotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF-G: \u003c/strong\u003eSchematic diagram of plasmids with various TMEM126A point mutations constructed based on a molecular docking model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH:\u003c/strong\u003eImmunofluorescence experiments revealed that TMEM126A and TRAF6 co-localised in H460 and H1299 cells. Magnification, 400×; scale bar, 25 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI-J: \u003c/strong\u003eH460 and H1299 cells were co-transfected with Flag-TRAF6, Myc-TMEM126A, or TMEM126A mutant plasmid, and cell lysates were subjected to immunoprecipitation with a FLAG monoclonal antibody. TMEM126A was detected by immunoblotting using an anti-Myc antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK:\u003c/strong\u003eWestern blotting assay indicated that wild-type TMEM126A upregulated the phosphorylation levels of TAK, IKK, and IκBα involved in NF-κB signalling pathway, whereas TMEM126A mutant-1 (i.e. transmembrane-domain mutations) abrogated the effect. GAPDH served as the loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL: \u003c/strong\u003eCell lysates from H460 and H1299 cells co-transfected with shRNA-TMEM126A and shRNA-TRAF6 were subjected to salvage experiments for the detection of phosphorylation levels of key factors in the NF-κB signalling pathway; TRAF6 silencing abrogated the changes of key proteins mediated by TMEM126A knockdown.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/c1e788803e52942641a7dad0.png"},{"id":91715297,"identity":"c1648376-f382-476b-ab37-2dd8aaf5e988","added_by":"auto","created_at":"2025-09-19 13:14:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4130891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTMEM126A promoted the metabolic degradation of TRAF6 via the autophagy pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-D: \u003c/strong\u003eWestern blotting results indicated that the protein expression of TRAF6 was upregulated following the overexpression of TMEM126A, using GAPDH as the loading control (A-B), while no significant change was observed in the mRNA levels detected by RT-qPCR (C-D). Data from a representative experiment were plotted as the mean of three replicates plus the standard deviation (NS: not significant).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE-F:\u003c/strong\u003eNegative correlation was seen between TMEM126A and TRAF6 protein levels in HBE and a panel of NSCLC cell lines (n = 7). Gray values were measured using the ImageJ software and analysed using Spearman statistics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG-J: \u003c/strong\u003eOverexpression of TMEM126A accelerated the degradation of TRAF6.\u003cstrong\u003e \u003c/strong\u003eThe TMEM126A plasmid and the corresponding empty vector were transfected into H460 and H1299 cells, respectively. After 40 h, the cells were treated with cycloheximide (CHX, 532.5 nM) and the cell lysates were subjected to immunoblotting at various time points. GAPDH served as the loading control. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK-L: \u003c/strong\u003eAutophagy inhibitor 3-MA could eliminate the degradation-promoting effect of TMEM126A on TRAF6 at both the exogenous and endogenous levels. Myc-TMEM126A and Flag-TRAF6 were co-transfected, or TMEM126A alone was transfected into H460 and H1299 cells; after 36 h, MG132 (20 μM), 3-MA (5 μM), and DMSO were added to each group, and the proteins were harvested for immunoblotting detection after 12 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM-N:\u003c/strong\u003eThe impact of bidirectional regulation of TMEM126A on autophagy-related markers (LC3 and P62) using immunoblotting. GAPDH was used as a loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eO: \u003c/strong\u003e3-MA eliminated the autophagy-promoting effect of TMEM126A overexpression. The TMEM126A plasmid and empty vector were transfected into H460 and H1299 cells. After 36 h, 3-MA (5 μM) and DMSO were added to each group, respectively. The proteins were collected for immunoblotting after 12 h.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/5df89d615e8d14c48e66cd1e.png"},{"id":91715848,"identity":"3887dc7a-9ec2-4c9c-be13-b92229fc7d59","added_by":"auto","created_at":"2025-09-19 13:22:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9758031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTMEM126A suppressed the growth of transplanted tumours in nude mice \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eFlowchart of tumorigenesis in nude mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB-G:\u003c/strong\u003eGross anatomical images of tumour tissues from the nude mouse tumorigenesis experiment (B). Wild-type TMEM126A significantly inhibited tumour growth in terms of both volume (C) and weight (D), whereas the transmembrane-domain mutant (TMEM126A-mut-1) failed to exert the effect. TMEM126 knockdown promoted the growth of transplanted tumours; NF-κB inhibitor JSH23 (3 mg/kg) abrogated the effect (E-G).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH-K:\u003c/strong\u003eRepresentative IHC images of transplanted tumours. Wild-type TMEM126A downregulated the expression of TRAF6 and weakened the nuclear translocation of P65, whereas the transmembrane domain mutant lacked this effect (H-I). JSH23 treatment eliminated the effects of TMEM126A knockdown.\u003c/p\u003e\n\u003cp\u003eData was presented as mean ± SD. NS, not significant. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/bf477be3c512ca01a9ee2e07.png"},{"id":91715308,"identity":"bdd7499d-911f-4be1-9b31-c537fba2e145","added_by":"auto","created_at":"2025-09-19 13:14:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2947449,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of molecular mechanism of TMEM126A- modulating PANoptosis and Proliferation in NSCLC.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/0cc2cca53d9ec1b92823d005.png"},{"id":91717825,"identity":"95c18d72-a89a-4398-9ad8-257b72ae44a5","added_by":"auto","created_at":"2025-09-19 13:38:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":38981134,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/912a887d-a46d-420d-b1a1-eebd2e05623a.pdf"},{"id":91715291,"identity":"223db0e7-a11d-44d2-97bc-3a1594e831c0","added_by":"auto","created_at":"2025-09-19 13:14:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22094,"visible":true,"origin":"","legend":"Supplementary materials and methods","description":"","filename":"Supplementarymaterialsandmethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/0e225f7cadd0add157d2029b.docx"},{"id":91715840,"identity":"e0051c1d-e604-4b0a-9d28-462f5ab00bcf","added_by":"auto","created_at":"2025-09-19 13:22:24","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":100352,"visible":true,"origin":"","legend":"\u003cp\u003eMass spectrometry\u003c/p\u003e","description":"","filename":"Massspectrometry.xls","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/a221ea77ffbfc40229fb0fb9.xls"},{"id":91715301,"identity":"6d430e62-5daf-48a3-a274-8dbf19961695","added_by":"auto","created_at":"2025-09-19 13:14:25","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4429072,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure S1.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/30de426bfe8e2ee1fb5648e7.tif"},{"id":91715289,"identity":"4e299b38-3eb0-4a17-83bd-a5689783ba1c","added_by":"auto","created_at":"2025-09-19 13:14:25","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18656,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure Legends\u003c/p\u003e","description":"","filename":"SupplementaryFigureLegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/4f7c418cad66cf4307ecd9fe.docx"},{"id":91715305,"identity":"88e3e8be-522a-44f0-8ae0-22a031909644","added_by":"auto","created_at":"2025-09-19 13:14:25","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":5408028,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure S3.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/5e378e44d3795126ed1ec60f.tif"},{"id":91715314,"identity":"6091d2a9-55b0-46ab-a6cb-7c5ff658b4bf","added_by":"auto","created_at":"2025-09-19 13:14:25","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":15686356,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-sequencing\u003c/p\u003e","description":"","filename":"RNAsequencing.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/890c14fa5c7306b0b668acf9.xlsx"},{"id":91715854,"identity":"9696878e-35c3-4024-bbfb-4c1575a504e8","added_by":"auto","created_at":"2025-09-19 13:22:25","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":36707166,"visible":true,"origin":"","legend":"\u003cp\u003eWB original data\u003c/p\u003e","description":"","filename":"WBoriginaldata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/a295ee71d4cd36576cbf3c0a.pdf"},{"id":91715318,"identity":"fb9933d1-0897-4128-bf53-8df612d41f23","added_by":"auto","created_at":"2025-09-19 13:14:25","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":3222452,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure S2.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7527532/v1/1599b023675d3ca224cdcd52.tif"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Molecular Mechanism of TMEM126A Modulating PANoptosis and Proliferation via TRAF6/NF-κB Signalling Pathway in Non-Small Cell Lung Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGrowth kinetics-related factors, such as the proliferation capacity and PANoptosis rate of tumour cells, determine the growth speed and malignant progression. PANoptosis is a newly proposed inflammatory programmed cell death pathway characterised by the key features of apoptosis, pyroptosis, and/or necroptosis. Briefly be described as follows: Caspase-8, released from the apoptotic complex, increases mitochondrial outer membrane permeability by activating Bid, which in turn releases cytochrome C and promotes the release of Caspase-3 and Caspase-7, ultimately inducing apoptosis. In contrast, the RIPK-1/3 complex facilitates the phosphorylation of MLKL, leading to necroptosis. Additionally, Gasdermin D (GSDMD) is cleaved into an N-terminal pore-forming domain (GSDMD-N) and a C-terminal self-inhibitory domain by Caspase-1. GSDMD-N binds to phospholipids in the cell membrane, creating pores that ultimately trigger inflammatory responses and induce cell death [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Currently, PANoptosis has garnered significant attention as a potential target for tumour therapeutic intervention [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Autophagy is a physiological process that transports damaged, denatured, and aging proteins and organelles from cells to lysosomes for degradation, thereby playing an important role in maintaining cellular homeostasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Research has indicated that autophagy disorders can lead to physiological abnormalities in cellular metabolism, inflammatory regulation, DNA damage, and other signalling pathways, and are closely related to PANoptosis [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the specific molecular mechanisms regulating autophagy and PANoptosis remain to be fully elucidated.\u003c/p\u003e\u003cp\u003eThe mitochondrial transmembrane protein TMEM126A (also known as OPA7), with a molecular weight of approximately 25 kDa, is crucial for the proper assembly and functionality of mitochondrial complex I and plays a significant role in maintaining mitochondrial function. TMEM126A comprises 195 amino acids, and its primary functional domains contain four transmembrane structures and four topological domains [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Current studies have mainly focused on the close association between TMEM126A mutations and autosomal recessive optic nerve atrophy [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, Kim EC and his colleagues discovered that in mouse bone marrow cells and macrophages, TMEM126A, in conjunction with its ligand CD137L and in synergy with TLR4 signalling, upregulates the expression of genes related to antigen presentation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, TMEM126A is considered to play a regulatory role in the immune response. However, research on TMEM126A and its association with tumours remains limited. To date, only one study has revealed that silencing TMEM126A facilitates the generation of ROS and depolarisation of the mitochondrial membrane potential, subsequently promoting extracellular matrix remodelling, epithelial-mesenchymal transition, and metastasis in breast cancer cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The expression pattern and potential impact of TMEM126A on the malignant progression of lung cancer, as well as the underlying molecular mechanisms, remain unclear.\u003c/p\u003e\u003cp\u003eIn this study, we aimed to investigate the expression of TMEM126A in NSCLC and demonstrate its vital role of TMEM126A in PANoptosis, autophagy, and cancer cell proliferation \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, thereby providing an experimental basis for developing small-molecule targeted therapeutic drugs for NSCLC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatients and samples\u003c/h2\u003e\u003cp\u003eThis study was approved by the Ethics Committee of China Medical University (Approval Number: LS[2019]003) and conducted in accordance with the principles of the Helsinki Declaration. A total of 128 clinical lung cancer tissue samples and 12 pairs of fresh tumours and paired adjacent tissues involved in the study were obtained from the Department of Pathology, First Hospital of China Medical University (2019\u0026ndash;2023). None of the patients had undergone radiation therapy or chemotherapy prior to surgery, and all signed informed consent forms. Based on the latest World Health Organization histological classification criteria for lung cancer (2021 edition) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], 77 cases were defined as adenocarcinoma and 51 were squamous cell carcinoma. The average age of the patients with lung cancer was 60 years. According to the UICC/AJCC TNM staging criteria (2023 edition) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], we categorised 52 cases into stages I and II, while 76 cases were classified as stage III.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunohistochemical staining\u003c/h3\u003e\n\u003cp\u003eThe experimental process is described in detail in the Supplementary Materials and Methods. Two pathologists scored the TMEM126A staining intensity and positivity rate based on the following criteria: staining intensity- 0 (uncoloured), 1 (light yellow), 2 (yellow), and 3 (dark yellow-brown); and positivity rate- 0 (1\u0026ndash;25%), 1 (26\u0026ndash;50%), 2 (51\u0026ndash;75%), and 3 (76\u0026ndash;100%). Finally, the total score for each slide was obtained by multiplying the scores for staining intensity and positivity rate. As the expression score of TMEM126A was typically\u0026thinsp;\u0026gt;\u0026thinsp;6 in most adjacent cancer tissues, we defined a score of \u0026ge;\u0026thinsp;6 as positive and \u0026lt;\u0026thinsp;6 as negative. For P65 and TRAF6, nuclear staining scores (\u0026ge;\u0026thinsp;4 points) and cytosolic staining scores (\u0026ge;\u0026thinsp;6) were considered as positive expression.\u003c/p\u003e\n\u003ch3\u003eCell culture and immunofluorescence\u003c/h3\u003e\n\u003cp\u003eThe HBE cell line was purchased from the American Type Culture Collection (#AC338600, ATCC, Manassas, VA, USA). The LK2 cell line was kindly donated by Dr. Hiroshi Kijima from the Department of Pathology and Biological Sciences, Hiroshima University Graduate School of Medicine, Japan. Other NSCLC cell lines, including H1299 (#SCSP-589), A549 (#TCHu150), H460 (#SCSP-584), SK-MES-1 (SCSP-5010), HCC827 (SCSP-538), and H1975 (SCSP-597) were obtained from the cell bank of the Shanghai Institute of Biological Sciences (Shanghai, China). All cell lines were identified through short tandem repeat (STR) DNA analysis and were cultured in RPMI 1640 medium containing 10% foetal bovine serum and 1% penicillin-streptomycin at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The immunofluorescence assay process is described in detail in the Supplementary Materials and Methods.\u003c/p\u003e\n\u003ch3\u003ePlasmids, short-hairpin RNA (shRNA), reagents and transfection\u003c/h3\u003e\n\u003cp\u003eThe information about plasmids and shRNA were listed in the Supplementary and Materials and Methods. Cells were transfected with Lipofectamine 8000 reagent (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. MG132 (#474790), cycloheximide (CHX; #C7698), and JSH23 (#481408-M) were purchased from Sigma-Aldrich (Shanghai, China).\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein extraction, western blotting, nuclear and cytoplasmic protein separation experiment, immunoprecipitation (IP) and mass spectrometry analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProtein extraction, western blotting and \u003cem\u003enuclear and cytoplasmic protein separation experiment\u003c/em\u003e procedures, as well as the primary antibodies used in this study were described in detail in the Supplementary and Materials and Methods. For IP, cells were lysed using immunoprecipitation lysis buffer (P0013J, Beyotime Biotechnology) and centrifuged at 4\u0026deg;C and 12000 rpm for 20 min to collect the supernatant. Subsequently, 40 \u0026micro;l of protein A/G agarose magnetic beads (P2012, Beyotime Biotechnology) was added, and the setup was sealed at 4\u0026deg;C for 2\u0026ndash;4 h, followed by centrifugation at 1000 rpm for 5 min, aspiration of the supernatant, and addition of the antibody or IgG control at 4\u0026deg;C overnight. The next day, 40 \u0026micro;l of magnetic beads was added to collect the immunocomplex. The magnetic beads were washed thrice with the prepared IP lysis buffer; the beads were retained, 40 \u0026micro;l of 2\u0026times; loading buffer was added, boiled in 100\u0026deg;C boiling water for 10 min, and subjected to immunoblotting and mass spectrometry analysis.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eqRT-PCR procedure was described in detail in the Supplementary and Materials and Methods. The primer sequences are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimers for real-time (RT)-qPCR\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003ePrimer sequences (5\u0026prime;\u0026rarr;3\u0026prime;)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTMEM126A\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;- GCTTCCAGAAGCAGAAAGGAATC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;- CTGGCTGCTAGACCACCATT \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTRAF6\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5- CTGCAAAGCCTGCATCATAA \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;- GGGGACAATCCATAAGAGCA \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;- GGACCTGACCTGCCGTCTAG \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;- GTAGCCCAGGATGCCCTTGA \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e5-Ethynyl-2\u0026prime;-deoxyuridine (EdU) staining and Oxazole yellow/ Propidium iodide (YO- PRO-1/PI) staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEdU staining and YO-PRO-1/PI staining assays were performed according to the manufacturer protocols. The procedures were described in detail in the Supplementary and Materials and Methods.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSubcutaneous xenograft tumour experiment\u003c/h2\u003e\u003cp\u003eAnimal experiments were conducted in accordance with the ethical standards for animal research at China Medical University (approval number: CMU20231358). The maximum tumour size permitted by the ethics committee was 2000 mm\u003csup\u003e3\u003c/sup\u003e, and none of the tumours in our study exceeded this size. The formula for calculating tumour volume was as follows: length \u0026times; width\u003csup\u003e2\u003c/sup\u003e/2. Tumour tissue was partially fixed with 4% paraformaldehyde, embedded in paraffin, and serially sectioned for H\u0026amp;E and immunohistochemical staining.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe results of immunohistochemistry experiments were analysed using the IBM SPSS Statistics 27 (IBM, Armonk, NY, USA) system for chi-square tests and \u003cem\u003eP\u003c/em\u003e-value statistics. The other experiments were quantitatively analysed using ImageJ software 18.0, and the remaining experimental results were subjected to \u003cem\u003et\u003c/em\u003e-test using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). The experiments were performed in triplicate, and statistical significance was set at \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eTMEM126A was under-expressed both in tissue specimens and NSCLC cell lines\u003c/h2\u003e\u003cp\u003eTo explore the expression pattern of TMEM126A in NSCLC, we first performed immunofluorescence staining and immunohistochemistry (IHC) of NSCLC tissues (n\u0026thinsp;=\u0026thinsp;128) and cell lines (n\u0026thinsp;=\u0026thinsp;3; H1299, A549, H460). The results indicated that TMEM126A is mainly located in the cytoplasm and that its expression is strongly positive in the normal bronchial and alveolar epithelia (29/40, 72.5%) while being weakly positive or negative in adenocarcinoma and squamous cell carcinoma (59/128, 46.1%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Statistical analysis showed that low expression of TMEM126A was significantly and positively correlated with tumour size (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021), poor differentiation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), positive lymph node metastasis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.035), and advanced TNM stage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), though not related to the patient's sex (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.155), age (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.725), or histological classification (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.539) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We selected a normal bronchial epithelial cell line (HBE) and a panel of NSCLC cell lines (n\u0026thinsp;=\u0026thinsp;7) to detect the expression of TMEM126A. Immunoblotting results indicated that the expression of TMEM126A in multiple NSCLC cell lines (6/7) was significantly lower than that in HBE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). In addition, we measured TMEM126A protein expression in 12 pairs of fresh NSCLC tissue samples and adjacent normal specimens. TMEM126A expression in the NSCLC tissues was significantly lower than in adjacent tissues (11/12, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). Therefore, our preliminary results demonstrated a low expression pattern of TMEM126A in NSCLC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAssociation between TMEM126A expression and clinicopathological characteristics in NSCLC.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinicopathological characteristics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTMEM126A\u003c/p\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTMEM126A\u003c/p\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP\u003c/p\u003e\u003cp\u003e(two-side)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27(46.55%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31(53.45%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e2.039\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003efemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e42(60.00%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e28(40.00%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.155\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e35(52.24%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e32(47.76%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.157\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e34(55.74%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27(44.26%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.725\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor diameter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;3 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e43(64.18%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e24(35.82%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e5.971\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;3 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26(42.62%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35(57.38%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.021\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHistological type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAC*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e40(51.95%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e37(48.05%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.298\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCC\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e29(56.86%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22(43.14%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.593\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDifferentiation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWell-moderate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e44(75.86%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14(24.14%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e20.576\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e25(35.71%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e45(64.29%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLymph node metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e40(63.49%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23(36.51%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e4.588\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e29(44.62%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e36(55.38%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.035\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNM classification\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅠ- Ⅱ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13(25.00%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e39(75.00%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e29.451\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅢ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e56(73.68%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20(26.32%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e*: Adenocarcinoma, #: Squamous cell carcinoma\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePotential binding sites predicted by the molecular docking model for the interaction between TMEM126A and TRAF6.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTMEM126AResidues\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTRAF6\u003c/p\u003e\u003cp\u003eResidues\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDistance(A)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSpecific Interactions\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA:Lys 158\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB:Gln 263\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1x hb to B:Gln 263\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA:Ile 149\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB:Arg 314\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1x clash to B:Arg 314\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA:Trp 148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB:Glu 321\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1x hb to B:Glu 321\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA:Arg 64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB:Cys 218\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1x hb to B:Cys 218\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA:Ser 52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB:Asn 256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1x hb to B:Asn 256\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA:Leu 48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB:Arg 255\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1x clash to B:Arg 255\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eEctopic expression of TMEM126A inhibited the proliferation ability and promoted PANoptosis in NSCLC cells\u003c/h2\u003e\u003cp\u003eAn imbalance between the survival and death of tumour cells is crucial for determining tumour growth and malignant progression. Therefore, we investigated whether TMEM126A plays a regulatory role in the survival and PANoptosis of lung cancer cells. We selected H460 and H1299 cells, which exhibit low and moderate expression of TMEM126A, respectively, for TMEM126A plasmid gradient transfection. Western blotting results demonstrated the transfection efficiency and effectiveness of the subsequent functional experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). EdU staining results demonstrated that TMEM126A overexpression significantly inhibited the proliferation of NSCLC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-F). Immunoblotting was performed to assess the expression of cell proliferation-related proteins after transfecting a TMEM126A-overexpressing plasmid. The results indicated that, compared to those in the control group, ectopic TMEM126A expression led to a decrease in the expression levels of the cell cycle-dependent kinases CDK4 and CDK6, as well as the cell cycle protein CyclinD1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). In contrast, YO-PRO-1/PI staining indicated that, compared to that in the control group, the number of YO-PRO-1-positive cells significantly increased in TMEM126A-overexpressed group, suggesting that TMEM126A promoted the PANoptosis of tumour cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-K). Accordingly, immunoblotting analysis revealed that overexpression of TMEM126A elevated the expression of apoptosis-related proteins, such as cleaved-caspase-3 and Bax, while suppressing the expression of the anti-apoptotic protein Bcl-2. Additionally, it led to an increase in the phosphorylation levels of necrotic apoptosis-related proteins, including phosphorylated RIPK3 and phosphorylated MLKL, and a significant upregulation of the activated form of the pyroptosis-related protein GSDMD-N (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTMEM126A knockdown promoted proliferation and weakened PANoptosis of NSCLC cells\u003c/h2\u003e\u003cp\u003eWe selected A549 and H1299 cells (with relatively high and moderate TMEM126A expression, respectively) for the knockdown experiments to demonstrate the biological role of TMEM126A in NSCLC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). The EdU staining assay showed that downregulation of TMEM126A expression promoted the proliferation of cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F), and correspondingly upregulated the expression levels of CDK4 and CDK6 proteins, as well as CyclinD1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). YO-PRO-1/PI staining results showed that TMEM126A silencing decreased the number of YO-PRO-1-positive cells, indicating the inhibition of PANoptosis in NSCLC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-K). Immunoblotting analysis revealed that compared to those in the control group, TMEM126A knockdown resulted in the downregulation of apoptosis-related protein (cleaved-caspase-3, Bax) expression, necrotic apoptosis-related protein (phosphorylated RIPK3 and phosphorylated MLKL) expression, and cell death-related protein (GSDMD-N) expression, and the upregulation of anti-apoptotic protein (Bcl-2) expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Thus, our results suggested that TMEM126A is an anti-oncogenic protein that inhibits proliferation and promotes the PANoptosis of NSCLC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eTMEM126A could be a negative regulator of the NF-κB signalling pathway\u003c/h2\u003e\u003cp\u003eFor exploring the potential molecular mechanisms by which TMEM126A exerts its anti-cancer effect on the malignant progression of NSCLC, we first transfected small interfering RNA sequences into A549 cells to knock down the expression of TMEM126A; RNA-sequencing, Gene Oncology enrichment (GO), and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses indicated that TMEM126A is closely related to the NF-κB signalling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). NF-κB signalling pathway plays a crucial role in numerous physiological and pathological processes of eukaryotic organisms, including inflammation, cell proliferation, transformation, tumorigenesis, and apoptosis inhibition. The primary mechanism of NF-κB activation involves the phosphorylation of the IKK complex, consisting of two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ). This phosphorylation leads to the degradation of IκBα, thereby releasing NF-κB (P65), which is then transported into the nucleus and binds to specific DNA sequences to initiate transcription [\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition, TNF receptor-associated factor-6 (TRAF6) interacts with the activated form of mitogen-activated protein kinase (phosphorylated TAK1) and activates the NF-κB pathway [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Hence, we initially performed immunoblotting to detect the impact of TMEM126A on the changes in the expression levels of key proteins in the NF-κB signalling pathway, both in H460 and H1299 cells. Results indicated that the phosphorylation levels of TAK1, IKK, and IκB-α decreased upon TMEM126A gradient overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Therefore, we conducted immunofluorescence and nuclear-cytoplasmic protein separation assays to detect the effect of TMEM126A on the nuclear-cytoplasmic distribution of P65, and found ectopic TMEM126A expression to significantly reduce the level of P65 nuclear translocation in H460 and H1299 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). Notably, we utilised the NF-κB signalling pathway inhibitor JSH23 (a P65 nuclear entry inhibitor) and discovered that it abrogates the promoting effect of TMEM126A knockdown on P65 nuclear import (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G), indicating that TMEM126A plays a crucial role in inhibiting NF-κB signalling pathway. We further explored whether TMEM126A exerts its biological behaviour in inhibiting the malignant progression of lung cancer by suppressing the activity of the NF-κB signalling pathway. We silenced TMEM126A in H1299 and A549 cells treated with JSH23 and DMSO as the experimental and the control groups, respectively. EdU staining experiments indicated that JSH23 could eliminate the tumour cell proliferation promoted by TMEM126A knockdown (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, B) as well as the upregulation of protein expression of CDK4, CDK6, and CyclinD1 (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). In contrast, YO-PRO-1/PI staining demonstrated that JSH23 reversed the biological effect of TMEM126A knockdown on PANoptosis inhibition (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD, E) as well as its regulatory effects on the expression levels of cleaved caspase-3, Bax, Bcl-2, p-RIPK3, p-MLKL, and GSDMD-N proteins (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF). In summary, we concluded that TMEM126A exerts its biological function by inhibiting cell proliferation and promoting PANoptosis, thereby suppressing the NF-κB signalling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eInteraction of TRAF6 in the NF-κB pathway with TMEM126A\u003c/h2\u003e\u003cp\u003eWe further explored the specific molecular mechanisms by which TMEM126A regulates the NF-κB signalling pathway. Mass spectrometry and co-immunoprecipitation were used to identify the tumour necrosis factor receptor-associated factor 6 (TRAF6), which interacts with TMEM126A (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-E). Accumulating research has shown that TRAF6 plays a pivotal role in activating the NF-κB pathway, and regulating apoptosis and autophagy [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The molecular docking model between the proteins was validated (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hdock.phys.hust.edu.cn\u003c/span\u003e\u003cspan address=\"https://hdock.phys.hust.edu.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the results revealed that TMEM126A and TRAF6 share six binding sites, as listed in Table-3 and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G, suggesting a strong binding affinity between them. Immunofluorescence analysis revealed that they co-localised in the cytoplasm of H1299 and H460 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). The structural basis of this interaction was explored next. Based on the six binding sites identified by molecular docking, three mutants were designed; the first involved mutations at positions 48, 52, and 64 within the transmembrane domain, the second involved mutations at positions 148, 149, and 158 in the topological domain, and the third involved mutations at all six aforementioned positions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Immunoprecipitation results indicated that when mutations occurred at positions 48, 52, and 64, TMEM126A was unable to bind TRAF6, suggesting that TMEM126A interacts with TRAF6 primarily through its transmembrane domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, J). Since TRAF6 is recognised as an upstream factor in the NF-κB signalling pathway, we further investigated whether TMEM126A inhibits the NF-κB signalling pathway via TRAF6. We transfected wild-type and mutant TMEM126A plasmids, respectively, into H460 and H1299 cells, and explored the regulatory effects of TMEM126A on proteins related to the NF-κB signalling pathway and on the malignant phenotype. Immunoblotting revealed that wild-type TMEM126A could gradually downregulate the phosphorylation levels of TAK, IKK, and IκB α in a dose-dependent manner, whereas mutant TMEM126A (i.e., unable to bind to TRAF6) could not (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). EdU and YO-PRO-1/PI staining assays confirmed that the TMEM126A with a transmembrane-domain mutation was unable to exert its anti-cancer effects by inhibiting cell proliferation (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-D) and downregulating the proliferation-related proteins (CDK4, CDK6, CyclinD1, Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE); promotion of PANoptosis (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eF-I) and upregulation of PANoptosis-related proteins (cleaved caspase-3, Bax, Bcl2, p-RIPK3, p-MLKL, and GSDMD-N, Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eJ) were also abrogated. Finally, a rescue assay indicated that TRAF6 silencing could abrogate the regulatory effect mediated by TMEM126A on the expression levels of p-TAK1, p-IKΚ, and p-IκB α proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). In summary, we concluded that TMEM126A exerts a key anti-cancer effect by binding to TRAF6 through its transmembrane domain, thereby inhibiting the activity of the NF-κB signalling pathway and modulating the proliferative ability and PANoptosis process of NSCLC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eTMEM126A promoted the metabolic degradation of TRAF6 via autophagy\u003c/h2\u003e\u003cp\u003eWe found that the overexpression of TMEM126A significantly downregulated the protein expression of TRAF6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Subsequently, we conducted RT-qPCR to determine that TMEM126A had no significant regulatory effect on TRAF6 mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Spearman's analysis of western blotting results of HBE and a panel of lung cancer cell lines (n\u0026thinsp;=\u0026thinsp;7) revealed a negative correlation between TMEM126A and TRAF6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F, R\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.786, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028). The results indicated that TMEM126A downregulated TRAF6 expression at the post-transcriptional level. Notably, we overexpressed TMEM126A in H460 and H1299 cells and introduced cycloheximide (CHX) at various time points. The findings indicated that ectopic TMEM126A expression accelerated the degradation of TRAF6 and shortened its half-life (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-J). Protein degradation pathways are known to depend on two systems, namely the ubiquitin-proteasome degradation system and the autophagy-lysosome pathway degradation system. Therefore, we opted to utilise the proteasome inhibitor MG132 and the autophagosome inhibitor 3-methyladenine (3-MA), respectively, to investigate the specific reasons behind the degradation of TRAF6 by TMEM126A at both exogenous and endogenous levels. Results indicated that the inclusion of 3-MA eradicated the downregulation of TRAF6 expression facilitated by TMEM126A overexpression, whereas MG132 did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, L). This suggested that the degradation of TRAF6 by TMEM126A occurred primarily through the autophagic lysosomal pathway. We conducted further experiments on the correlation between TMEM126A expression and autophagy. Western blotting results indicated that TMEM126A overexpression in H460 and H1299 cells promoted the transformation of the autophagy-related protein LC3 (I to II, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM), and downregulated P62 expression, whereas TMEM126A silencing exerted the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN). Transmission electron microscopy revealed that TMEM126A overexpression increased the number of autophagosomes in lung cancer cells (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA, B). Immunofluorescence analysis revealed that TMEM126A treatment increased the number of autophagic spots (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC-F), demonstrating that TMEM126A plays a crucial role in activating autophagic activity in NSCLC. To further confirm the possibility of TMEM126A degrading TRAF6 through the promotion of autophagy, we reported that TMEM126A-mediated TRAF6 degradation disappeared upon the addition of 3-MA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO), thereby suggesting that TMEM126A facilitates the metabolic degradation of TRAF6 by activating the autophagy pathway. In summary, TMEM126A promotes the autophagic degradation of TRAF6, thereby inhibiting the downstream NF-κB signalling pathways. This in turn could suppress cell proliferation, promote PANoptosis, and lead to the suppression of malignant progression in NSCLC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eTMEM126A suppressed the growth of transplanted tumours in nude mice in vivo\u003c/h2\u003e\u003cp\u003eThe above-mentioned experimental results were next validated at the cellular level. To ensure the rigor and effectiveness of our findings, we conducted experiments \u003cem\u003ein vivo\u003c/em\u003e to validate our results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). A nude mouse xenograft experiment demonstrated that overexpression of wild-type TMEM126A in H460 cells significantly suppressed both the volume and weight of the transplanted tumours compared to that in the control group. Conversely, transfection with mutant TMEM126A, which has a transmembrane domain mutation preventing it from binding TRAF6, had no such effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-D). On the other hand, knocking down the expression of TMEM126A in A549 significantly promoted the growth of transplanted tumours, whereas the addition of NF-κB signalling pathway inhibitor (JSH23, 3 mg/kg) or TRAF6 knockdown blocked the effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-G). Subsequently, we embedded the transplanted tumours in paraffin, prepared sections, and conducted haematoxylin-eosin and immunohistochemical staining. The experimental results revealed that TRAF6 expression was downregulated in H460 cells transfected with wild-type TMEM126A and that the nuclear import level of P65 decreased. However, this phenomenon was not observed in the group transfected with the TMEM126A transmembrane domain mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, I). A rescue experiment demonstrated that JSH23 or TRAF6 knockdown attenuated the effects induced by TMEM126A silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ, K), indicating that TMEM126A promoted TRFA6 degradation \u003cem\u003ein vivo\u003c/em\u003e, thereby inhibiting P65 nuclear entry and NF-κB signalling pathway, leading to the inhibition of malignant progression of NSCLC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study, based on immunohistochemical analysis of 128 pathological specimens from patients with NSCLC, revealed that the expression of TMEM126A in cancer tissues was significantly lower than that in normal lung tissue. Furthermore, it is closely correlated with the degree of differentiation, tumour size, lymph node metastasis, and pTNM staging of NSCLC. This clinical discovery sparked interest in exploring the biological impact of TMEM126A in NSCLC. We validated TMEM126A expression in various lung cancer cell lines and freshly paired lung cancer and adjacent tissues and obtained experimental results consistent with the immunohistochemical conclusions. This suggests that TMEM126A may have translational value as a molecular marker for NSCLC and for predicting prognosis. Although we confirmed low TMEM126A expression in NSCLC, it remains unclear whether this is caused by promoter hypermethylation, transcriptional inhibition, or increased post-translational metabolic degradation. The Cancer Genome Atlas (TCGA) online database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ualcan.path.uab.edu\u003c/span\u003e\u003cspan address=\"https://ualcan.path.uab.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) indicated that compared to that in normal lung tissue, the methylation level of the \u003cem\u003eTMEM126A\u003c/em\u003e gene was significantly upregulated in NSCLC tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.961500E-03), suggesting that low TMEM126A expression may be related to gene methylation.\u003c/p\u003e\u003cp\u003eNext, we verified that TMEM126A, as an anti-cancer protein, inhibits the proliferation of lung cancer cells and promotes the PANoptotic process in lung cancer cells through \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e functional experiments. PANoptosis enables the eradication of cancer cells via various cell death pathways and has the potential to overcome diverse obstacles in cancer therapy, such as drug resistance and immune evasion. Understanding the mechanisms underlying the induction of PANoptosis in cancer cells and exploring potential therapeutic agents could lead to effective cancer treatments and improved patient outcomes. For example, He \u003cem\u003eet al.\u003c/em\u003e utilised TCGA database to perform a comprehensive analysis of breast cancer characteristics and concluded that a high PANoptosis process is beneficial in reducing the incidence of breast cancer [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Studies have also shown that PANoptosis contributes to shaping the tumour microenvironment in patients with low-grade gliomas [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Furthermore, it has been shown to induce cell death in melanoma cell lines [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Lin \u003cem\u003eet al\u003c/em\u003e. highlighted the significance of PPM1B-mediated YBX1 dephosphorylation and USP10-mediated deubiquitination in the regulation of PANoptosis and oxaliplatin sensitivity in gastric cancer cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBased on KEGG and GO enrichment analyses using RNA sequencing, TMEM126A was found to be closely associated with the NF-κB signalling pathway. Previous studies have indicated that the NF-κB signalling pathway plays a crucial role in regulating PANoptosis and inflammation in non-tumour diseases [\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. We identified that TMEM126A interacts with TRAF6, an upstream factor of the NF-κB pathway, thereby inhibiting P65 nuclear import and promoting PANoptosis in NSCLC. Notably, we found that TMEM126A enhanced autophagic activity, leading to an increase in TRAF6 metabolic degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). However, the pathways and underlying molecular mechanisms through which TMEM126A enhances autophagy, as well as the mechanism by which TMEM126A activates the NF-κB pathway upon binding to TRAF6, remain unclear. This is a limitation of the current study. This warrants further investigation in future studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings imply that TMEM126A acts as a tumour suppressor by modulating proliferation and PANoptosis balance through the TRAF6/NF-κB signalling pathway and could serve as a potential target for targeted therapy in NSCLC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTMEM126A: the mitochondrial transmembrane protein-126A; TRAF6: tumour necrosis factor receptor-associated factor-6; CDK-4/6: cyclin-dependent kinase-4/6; CyclinD1: G1/S-specific cyclin-D1; Caspase-3: cysteinyl aspartate specific proteinase-3; BAX: Bcl-2 associated X protein; Bcl-2: B-cell lymphoma-2; p-RIPK-3: phosphorylated receptor-interacting serine/threonine protein kinase-3; p-MLKL: phosphorylated mixed lineage kinase domain-like protein; GSDMD: gasdermin D; p-TAK: phosphorylated TGF-beta-activated kinase-1; p-IKK: phosphorylated inhibitor of nuclear factor kappa-B kinase; p-I\u0026kappa;B: phosphorylated NF-kappa-B inhibitor; DAPI: 4\u0026apos;,6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; CHX: cycloheximide; MG132: Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; 3-MA: 3-Methyladenine; P62: sequestosome-1/SQSTM-1; LC3: light chain-3; NC: negative control; EdU: 5-Ethynyl-2\u0026apos;-deoxyuridine; YO-PRO-1/PI: oxazole yellow/propidium iodide; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; shRNA: short-hairpin RNA; NSCLC: non-small-cell lung cancer; KEGG: Kyoto Encyclopedia of Genes and Genomes; PBS: phosphate-buffered saline; GO: Gene Ontology enrichment; NSCLC: non-small-cell lung cancer; IHC: immunohistochemistry; IP: immunoprecipitation; IB: immunoblotting; SDS: sodium dodecylsulphate; PAGE: polyacrylamide gel electrophoresis; PVDF: polyvinylidene difluoride.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of China Medical University (Approval Number: LS[2019]003) and was conducted in accordance with the principles of Helsinki. All patients provided informed consent. Animal experiments were conducted in accordance with the ethical standards for animal research at China Medical University (approval number: CMU20231358).\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eAll authors have approved the submission of the manuscript for publication and agree to be accountable for all aspects of the manuscript\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of Liaoning Province (grants 2022-MS-198 to Xuyong Lin) and the National Natural Science Foundation of China (grants 81902986 to Qiang Han, grants 82003119 to Xuezhu Rong).\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eLai Wei, Ji Li, and Quanxiu Jin were responsible for designing the research studies, conducting experiments, acquiring, and analysing data. Huanyu Zhao, Yang Liu, Yuheng Feng, and Xueting Gan were tasked with conducting experiments and acquiring data. Xuezhu Rong, Qiang Han, and Xuyong Lin had the oversight of the study\u0026apos;s conception and supervision, and authored the manuscript. All authors contributed to revising the draft versions and approving the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe thank Dr. Hiroshi Kijima (Department of Pathology and Biological Sciences at Hiroshima University Graduate School of Medicine, Japan) for donating the LK2 cell line.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its Supplementary Files. Uncropped original western blots used in this manuscript have been uploaded as \u0026ldquo;Supplemental Material\u0026rdquo;. Additional details are available upon request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKarki R, \u003cem\u003eet al\u003c/em\u003e. ADAR1 restricts ZBP1-mediated immune response and PANoptosis to promote tumorigenesis. \u003cem\u003eCell Reports.\u003c/em\u003e \u003cstrong\u003e37(3)\u003c/strong\u003e, 109858 (2021).\u003c/li\u003e\n \u003cli\u003eZhu P, Ke ZR, Chen JX, Li SJ, Ma TL \u0026amp; Fan XL. Advances in mechanism and regulation of PANoptosis: Prospects in disease treatment. \u003cem\u003eFrontiers in Immunology.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1120034. (2023).\u003c/li\u003e\n \u003cli\u003eShi C, \u003cem\u003eet al\u003c/em\u003e. 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STING agonist diABZI induces PANoptosis and DNA mediated acute respiratory distress syndrome (ARDS). \u003cem\u003eCell Death Dis.\u003c/em\u003e \u003cstrong\u003e13(3)\u003c/strong\u003e, 269 (2022).\u003c/li\u003e\n \u003cli\u003eZhao D, \u003cem\u003eet al\u003c/em\u003e. Copper exposure induces inflammation and PANoptosis through the TLR4/NF-kappaB signaling pathway, leading to testicular damage and impaired spermatogenesis in Wilson disease. \u003cem\u003eChem Biol Interact.\u003c/em\u003e \u003cstrong\u003e396\u003c/strong\u003e, 111060 (2024).\u003c/li\u003e\n \u003cli\u003eWang L, Huang Y, Zhang X, Chen W \u0026amp; Dai Z. Exosomes derived from FN14-overexpressing BMSCs activate the NF-kappaB signaling pathway to induce PANoptosis in osteosarcoma. \u003cem\u003eApoptosis.\u003c/em\u003e \u003cstrong\u003e30(3-4)\u003c/strong\u003e, 880-893 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"TMEM126A, TRAF6, NF-κB signalling pathway, autophagy, PANoptosis, NSCLC","lastPublishedDoi":"10.21203/rs.3.rs-7527532/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7527532/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe mitochondrial transmembrane protein-126A (TMEM126A) is crucial for the accurate assembly of mitochondrial complex I and plays a significant role in preserving mitochondrial function. We aimed to investigate the expression of TMEM126A in non-small cell lung cancer (NSCLC), its biological impact on the malignant progression of NSCLC, and its underlying molecular mechanisms. Here we show TMEM126A was underexpressed in NSCLC and was closely correlated with clinicopathological factors and poor prognosis. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e functional experiments validated the vital tumour-suppressing roles of TMEM126A in inhibiting cell proliferation and promoting PANoptosis and autophagy. Mechanistically, TMEM126A was identified to interact with and facilitate the autophagy-mediated degradation of TRAF6 via its own transmembrane domain, thereby suppressing the NF-κB signalling pathway and weakening the proliferation of NSCLC. In conclusion, TMEM126A plays a significant inhibitory role in NSCLC malignant progression, which provides experimental evidence to support the development of small-molecule inhibitors.\u003c/p\u003e","manuscriptTitle":"Molecular Mechanism of TMEM126A Modulating PANoptosis and Proliferation via TRAF6/NF-κB Signalling Pathway in Non-Small Cell Lung Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-19 13:14:19","doi":"10.21203/rs.3.rs-7527532/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ec6a8f34-a52f-4f3b-abe7-8beda7a57289","owner":[],"postedDate":"September 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54597680,"name":"Biological sciences/Cancer/Lung cancer"},{"id":54597681,"name":"Biological sciences/Cell biology/Cell death"}],"tags":[],"updatedAt":"2026-04-02T14:57:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-19 13:14:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7527532","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7527532","identity":"rs-7527532","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}