LAMB3 regulates ITGB4 to mediate metabolic reprogramming of lung cancer cells, thus promoting the proliferation and metastasis of cancer cells

LAMB3 regulates ITGB4 to mediate metabolic reprogramming of lung cancer cells, thus promoting the proliferation and metastasis of cancer cells | 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 LAMB3 regulates ITGB4 to mediate metabolic reprogramming of lung cancer cells, thus promoting the proliferation and metastasis of cancer cells Suting Chen, Limin Dong, Zhicheng Ouyang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7070948/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Non-small cell lung cancer (NSCLC) is a common lung cancer cell type. LAMB3 promotes the progress of cancer. However, the regulatory mechanism of LAMB3 on metabolic reprogramming of NSCLC is not clear. Methods RT-qPCR and western blot were used to detect the expression level of LAMB3 in NSCLC tissues and cells. The expression level of LAMB3 in A549 and H1299 cells was changed by transfection of related vectors. Cell counting kit-8 (CCK8) was used to detect the activity of NSCLC. Transwell was evaluated the migration and invasion of cancer cells. The metabolic level changes of NSCLC cells were detected by related kits. Results Our results showed that LAMB3 and ITGB4 were highly expressed in NSCLC tissues and cells. Knocking down LAMB3 inhibited the progress of NSCLC, including cell proliferation, metastasis and metabolic level. Overexpression of ITGB4 can reverse the effects of knocking down LAMB3 on tumor progression and metabolic reprogramming. Conclusion LAMB3 regulated ITGB4-mediated metabolic reprogramming of lung cancer cells, thus promoting the proliferation and metastasis of cancer cells. Biological sciences/Cancer Biological sciences/Cell biology Health sciences/Oncology Non-small cell lung cancer LAMB3 ITGB4 metabolic reprogramming metastasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights 1. LAMB3 is high expressed in pheochromocytoma. 2. Knocking down LAMB3 inhibits the progress of NSCLC. 3. LAMB3 regulates the reprogramming of glucose and lipid metabolism in lung cancer cells. 4. LAMB3 regulates ITGB4 to mediate the progress of lung cancer cells. Introduction In China, lung cancer is a highly malignant tumor, and more than 40% of lung cancer patients have no smoking history 1 . Early diagnosis and targeted therapy of lung cancer is an urgent problem to be solved at present 2 . Among them, non-small cell lung cancer (NSCLC) is the most common type of lung cancer 3 . Warburg effect is one of the tumor markers, mainly because tumor cells produce ATP through aerobic glycolysis to adapt to the changes of tumor microenvironment 4 . Therefore, more and more research focused on tumor metabolic reprogramming. Interestingly, tumor cells can also use basic nutrients including fatty acids and amino acids to survive 5 . The metabolic reprogramming of tumor cells and the mutual adjustment of microenvironment promote the metastasis of cancer. However, the molecular mechanism of metabolic reprogramming on cancer metastasis is still unclear in NSCLC. Laminin subunit β-3 (LAMB3) is one of the active components of basement membrane 6 . LAMB3 could promote the proliferation and metastasis of pancreatic ductal adenocarcinoma 7 . Similarly, LAMB3 promoted the metastasis of colorectal cancer and thus might be a potential therapeutic target 8 . We used GEPIA website to predict the expression of LAMB3 in TCGA database, and the results showed that LAMB3 was highly expressed in lung cancer. An interesting study found that LAMB3 can participate in the regulation of obesity and is related to the formation of body fat 9 . Then, whether LAMB3 is involved in metabolic regulation in NSCLC and thus promotes the progress of cancer cells remains to be verified. We predicted the possible downstream binding proteins of LAMB3 by STING and GeneMANIA, and found that it was mainly ITG family except LAM family. Among them, ITGB4 is highly expressed in LUSC and LUAD, and has physical interaction with LAMB3. ITGB4 can promote cancer in primary tumors of head and neck 10 . The abnormal expression of TIGB4 in NSCLC is closely related to acquired drug resistance of cancer cells 11 . Exciting, a study in breast cancer found that ITGB4 mediated metabolic reprogramming of tumor-related fibroblasts 12 . Our research mainly verified that LAMB3 up-regulates ITGB4 and mediates cell metabolic reprogramming in NSCLC cells, thus promoting the proliferation and metastasis of cancer cells. This study clarified the new regulatory mechanism of LRP1B in NSCLC and provided a new target for the diagnosis and treatment of NSCLC. Materials and Methods Collection of clinical samples The clinical tissue samples of this study are from 37 patients with NSCLC and their adjacent tissues who were surgically removed in Pingxiang people's hospital Hospital in 2021–2023. All patients have not received radiotherapy, chemotherapy and immunotherapy. All patients obtained written informed consent and were approved by the Pingxiang people's hospital Ethics Committee (No. 20210601). Research involving human participants was conducted following the ethical standards of the institution and the National Research Council. It adhered to the ethical standards of the 1964 Declaration of Helsinki and its subsequent amendments or similar. Immunohistochemistry (IHC) The tissue samples were embedded in paraffin and made into 5 µm slices. After pretreatment, the slices were incubated with anti-LAMB3 (ab97765, 1:1000, Abcam, UK) and anti-KI67 (ab15580, 1:1000, Abcam) overnight. Then it was incubated with peroxidase-coupled goat anti-mouse IgG for 2 h. Finally, the 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution and hematoxylin were used for double staining and observation. Cell culture and transfection Normal lung epithelial cells (BEAS-2) and lung cancer cell lines (A549, HCC827, H1299, H1299, H460) were purchased from Wuhan Procell Life Science and Technology Co., Ltd. (Wuhan, China). BEAS-2 cells were cultured in DMEM/F12 supplemented with fetal calf serum (FBS, 10%, Gibco, USA) and penicillin/Streptomycin (1%, Gibco). Lung cancer cell lines were cultured in the same RPMI 1640 medium which was treated in the same way. The culture conditions were 5% CO 2 and 37℃. shRNA, overexpression vector (OE) and negative control (NC) were designed and synthesized by Shanghai Sangon Biotechnology (Shanghai, China). All vectors were transfected with Lipofectamine 3000 reagent (Invitrogen, USA). Real-time fluorescence quantitative PCR (RT-qPCR) Total RNA was obtained from lung cancer tissues and cells by using TRIzol reagent (Invitrogen, USA). After 2 µg of total RNA reverse transcription treatment, the qPCR reaction was carried out using SYBR Green qPCR mix (Sangon, Shanghai). The primer sequence is (5'-3'): LAMB3-F-CCAAAGGTGCGACTGCAATG, R-AGTTCTTGCCTTCGGTGTGG; ITGB4-F-CACCTCCGTCTCCTCCCAC, R-GTTGGGGATGTTGAGCCGAT; β-actin-F-TTGCAGCTCCTTCGTTGCC, R-TTGCAGCTCCTTCGTTGCC. Western blot Protein was obtained from lung cancer cells by ProteoPrep® total protein extraction kit (Sigma-Aldrich, USA). Protein was transferred to membrane, sealed and incubated with anti-LAMB3 (ab97765, 1:2000, Abcam), anti-ITGB4 (ab197772, 1:2000, Abcam) and β-actin (ab8226, 1: 2000, Abcam). The next day, goat anti-mouse/rabbit IgG secondary antibody conjugated with horseradish peroxidase (HRP) was used for incubation. Finally, the protein bands were observed and recorded under the microscope. Cell counting kit-8 (CCK8) assays Lung cancer cells (2500 cells/well) were inoculated into a 48-well plate and cultured for 12 h. 10 µL of CCK8 (C0038, Beyotime, Shanghai) solution was added to each well for 2 h. Finally, the absorbance at 450 nm was quantified by microplate reader (Thermo Multiskan Spectrum, USA). Transwell assays Lung cancer cells were suspended in serum-free RPMI 1640 medium and then added to the upper chamber of transwell system (1×10 4 cells). RPMI 1640 medium containing 20% FBS was added to the lower chamber. Invasion detection needs to be pre-coated with matrigel in the upper chamber. After 24 h of culture at 37℃, the cells were fixed and stained with 0.1% crystal violet solution for 8 min. The cells were observed and photographed under a microscope. Determination of extracellular acidification rate (ECAR) Lung cancer cells (1.5×10 3 ) were inoculated in a 24-cell plate and cultured overnight. Subsequently, the cells were washed by PBS and cultured in an incubator without CO 2 at 37℃ for 60 min. Then, glucose, oligonucleotide and 2- deoxyglucose were added, and the extracellular flux analyzer (SeahorseXF24 FluxPak) was used to detect the extracellular acidification rate. Detection of triglyceride and cholesterol Lung cancer cells were collected into a 1.5 mL centrifuge tube, and 1 mL reagent (n-heptane: isopropanol = 1:1 volume ratio) was added for ice bath ultrasonic crushing. Cell supernatant was separated and collected by centrifugation. The triglyceride and cholesterol detection kit (ab65336, Abcam) were used for related detection. Lipid drop detection In order to label the lipid droplets in cells, we put the cells to be detected in an 8-hole slide and incubate them with Bodipy 493/503 (2 µM) (#72485, Sigma Aldrich) at 37℃ for 15 min. Then the fluorescently labeled lipid droplets were observed under a confocal microscope (Nikon, Tokyo, Japan). Animal model BALB/c nude mice (13–16 g, 4–6 weeks old) were purchased from Shanghai slack Experimental Animal Co., Ltd. to establish xenotransplantation tumor animal model. A549 cells (5 × 10 6 ) were injected subcutaneously into the armpit of each nude mouse. 30 days after tumor formation, mice were treated by spinal dislocation and the tumor was isolated. The animal experiment scheme conforms to ARRIVE guidelines and is reviewed and approved by Pingxiang people's hospital Ethics Committee (NO. 20230402). Statistical analysis All experiments were carried out independently for three times and statistically analyzed by using GraphPad Prism 8.0. All data = mean ± standard deviation (SD). Students' t test and one-way ANOVA were used to make significant analysis between the two groups and between multiple groups respectively. P value < 0.05 is considered statistically significant. Results LAMB3 was highly expressed in clinical tissues and cells of lung cancer First of all, it was predicted by GEPIA 2 database ( http://gepia2.cancer-pku.cn/#index ) that the LAMB3 in lung cancer was on the rise (Fig. 1 A). We further analyzed the cancer tissues of 37 patients with lung cancer and found that. Compared with the tissues adjacent to cancer, the expression of LAMB3 in lung cancer patients was increased observably (Fig. 1 B and 1 C). Then, we also found similar expression patterns in lung cancer cell lines (A549, HCC827, H1299, H1299, H460). Especially in A549 and H1299 cells, LAMB3 expression increased most significantly (Fig. 1 D and 1 E). LAMB3 promotes the malignant progress of lung cancer cells Then, we studied LAMB3 in A549 and H1299 cells. After transfection with sh-LAMB3, the levels of LAMB3 in cells were significantly decreased (Fig. 2 A). We also found that the cell activity, proliferation, migration and invasion ability decreased significantly after knocking down LAMB3 expression (Fig. 2 B- 2 D). We further explored LAMB3 in the mice model of lung cancer transplanted tumor. Inhibition of LAMB3 slowed down the tumor proliferation and diameter in mice (Fig. 3 A), and the expressions of KI67 and LAMB3 in tumor tissues of mice were significantly reduced after knocking down LAMB3 (Fig. 3 B and 3 C). LAMB3 regulates the metabolic reprogramming in lung cancer cells Interestingly, we also found that LAMB3 could regulate glucose and lipid metabolism in lung cancer cells. We found that after silencing LAMB3, the glycolysis related enzymes HK and PFK1 in cells were decreased (Fig. 4 A). After knocking down LAMB3, the glycolysis of lung cancer cells was decreased (Fig. 4 B). Moreover, the triglyceride and cholesterol levels in the cells were decreased significantly after LAMB3 knockdown (Fig. 4 C). High expression of ITGB4 in lung cancer Similarly, it was found that the ITGB4 increased in lung cancer by GEPIA 2 database analysis (Fig. 5 A). In the tissues of lung cancer patients collected by our hospital, the expression of ITGB4 also was increased (Fig. 5 B and 5 C). LAMB3 regulates the progress of ITGB4-mediated lung cancer cells We tested the regulatory relationship by knocking down LAMB3 and overexpressing ITGB4 in lung cancer cells. As shown in the results of Fig. 6 A, the expressions of LAMB3 and ITGB4 in cells were decreased after knocking down LAMB3. At the same time, after transfection of OE-ITGB4, the inhibitory effect of sh-LAMB3 on ITGB4 was restored. However, the expression of LAMB3 was not influenced by the expression of ITGB4 (Fig. 6 A). Further verification showed that overexpression of ITGB4 was restored the inhibitory effect of sh-LAMB3 on the activity, proliferation, migration and invasion of lung cancer cells (Fig. 6 B- 6 D). LAMB3 regulates ITGB4 to mediate metabolic reprogramming Finally, we analyzed the influence of LAMB3/ITGB4 axis on glucose and lipid metabolism of lung cancer cells. Overexpression of ITGB4 partially reversed the inhibitory effect of silencing LAMB3 on glycolysis related enzymes HK and PFK1 in lung cancer cells (Fig. 7 A). Similarly, further detection of cell metabolic capacity showed that overexpression of ITGB4 restored the down-regulation effect of knocking out LAMB3 on glycolysis, triglyceride, cholesterol level and lipid droplet formation in lung cancer cells (Fig. 7 B- 7 D). Discussion Metabolic reprogramming of cancer is involved in driving and maintaining the malignant phenotype of cancer cells 13 . Metabolic reprogramming is very active in patients with lung cancer and shows heterogeneity 14 . Our research confirmed that LAMB3/ITGB4 axis could regulate the metabolic reprogramming of lung cancer cells, which had an impact on the progress of lung cancer cells (Fig. 8 ). This discovery provides a new potential target gene for therapeutic strategies targeting metabolic pathways in lung cancer treatment research. LAMB3 promotes tumorigenesis by encoding β2 subunit of trimeric basement membrane protein laminin 332 and interacting with other genes in cells 15 . A recent study found that targeting LAMB3 can inhibit the progress of MAPKi-resistant melanoma cells in vivo 16 . In the study of clinical samples of lung cancer patients, it was confirmed that the expression of LAMB3 was related to lymphatic metastasis 17 . We also found that knocking down the expression of LAMB3 could inhibit the malignant progress of tumor cells in lung cancer cell lines and transplanted tumor mice model. Interestingly, we also found that LAMB3' s regulation of lung cancer progression is related to metabolic reprogramming of cancer cells. By silencing LAMB3 in lung cancer cell line, it was found that the glycolytic ability and lipid metabolism level of the cell decreased. Therefore, we speculate that LAMB3 might regulate metabolic pathways and participate in regulating the progress of lung cancer. An exciting study shows that LAMB3 interacts with ITGB4 in colorectal cancer cells, up-regulating AKT pathway and thus enhancing the activity of tumor cells 18 . The high level of ITGB4 is related to the poor clinical prognosis of lung cancer. Knocking out ITGB4 can inhibit the proliferation and metastasis of NSCLC 19 . Our study also found that the ITGB4 increased significantly in NSCLC patients and cell lines. Interestingly, it was found that ITGB4 can activate PI3K/mTOR/SREBP1c signaling pathway and lead to reprogramming of lipid metabolism in hepatocellular carcinoma cells 20 . Similarly, we also confirmed in lung cancer cells that overexpression of ITGB4 can reverse the effects of silencing LAMB3 on proliferation, metastasis and metabolic reprogramming of NSCLC. However, there are some limitations in our research, and the effect of LAMB3 on the regulation and metastasis of glucose and lipid metabolism of cancer cells has not been verified in mice, which is also the focus of our further research. In a word, our study verified the regulation of LAMB3/ITGB4 pathway in the metabolic pathway of NSCLC. It is confirmed that LAMB3 participates in the regulation of glucose and lipid metabolism of lung cancer cells by regulating ITGB4, which mediates the development of lung cancer. It was also found that knocking down LAMB3 could inhibit the progress of lung cancer transplanted tumor in mice. In a word, our research provides new potential target genes for the development of targeted drugs for lung cancer. Declarations Conflicts of interest All authors agree with the presented findings, have contributed to the work, and declare no conflict of interest. Acknowledgments The authors express their gratitude to for his help for directing our article. CRediT author statement Conceptualization: Zhicheng Ouyang Methodology: Zhicheng Ouyang, Suting Chen Software: Suting Chen Validation: Suting Chen, Limin Dong Formal analysis: Suting Chen,Limin Dong Investigation: Suting Chen Resources: Suting Chen Data Curation: Suting Chen Writing - Original Draft: Limin Dong Writing - Review & Editing: Suting Chen Visualization: Suting Chen, Limin Dong Supervision: Zhicheng Ouyang Project administration: Zhicheng Ouyang Funding statement declaration None. Data availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 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Journal of Thoracic Oncology. 2022;17(12):1335-54. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7070948","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":517113460,"identity":"33c4333c-25bf-453d-ac3a-4b66a749f602","order_by":0,"name":"Suting Chen","email":"","orcid":"","institution":"Pingxiang People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Suting","middleName":"","lastName":"Chen","suffix":""},{"id":517113461,"identity":"e9ef371c-ef7c-4a5b-a81c-14edcde742ff","order_by":1,"name":"Limin Dong","email":"","orcid":"","institution":"Pingxiang People's 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04:38:06","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":64390,"visible":true,"origin":"","legend":"","description":"","filename":"02a3a20ad8d54ae5a68d22a1eff6e10d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/28aba92c28b300e87b4fac18.xml"},{"id":92045674,"identity":"cb4d9e0b-cb98-4f56-8caa-049e7ec32aa7","added_by":"auto","created_at":"2025-09-24 04:38:06","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74334,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/b344624efc0b0a1138a2c9d8.html"},{"id":92046075,"identity":"6ab1f8e9-8f78-41fd-bf6e-a74017fc8d45","added_by":"auto","created_at":"2025-09-24 04:46:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":725231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLAMB3 was highly expressed in clinical tissues and cells of lung cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) GEPIA 2 databasepredicted LAMB3 table map. (B) RT-qPCR was used to detect the expression of LAMB3 in tissues. (C) Western blot was used to detect the level of LAMB3 protein in tissues. (D) RT-qPCR was used to detect the expression of LAMB3 in cells. (E) The expression of LAMB3 in cells was detected by western blot. Data are the means ± SD for three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ns = no significance.\u003c/p\u003e","description":"","filename":"fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/adff219b7d1d2c200d5b0c15.jpg"},{"id":92045667,"identity":"b3593411-bae4-47a9-86f6-7776df027631","added_by":"auto","created_at":"2025-09-24 04:38:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2525060,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLAMB3 promotes the proliferation, migration and invasion of lung cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) RT-qPCR and western blot were used to detect the expression of LAMB3 in cells. (B) CCK8 evaluated the changes of cell activity. (C) EDU detected the changes of cell proliferation. (D) Transwell detected the level of cell migration and invasion. Data are the means ± SD for three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"fig2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/bbe5336544210e8b2d529919.jpg"},{"id":92045673,"identity":"35a10d6e-6cc2-419f-bf59-61309986c6f7","added_by":"auto","created_at":"2025-09-24 04:38:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1417703,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLAMB3 inhibits the growth of transplanted tumor in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of transplanted tumor of mice lung cancer and statistical analysis of tumor diameter (cm). (B) RT-qPCR was used to detect the expression of LAMB3. (C) The expression of LAMB3 and KI67 was detected by IHC. Data are the means ± SD for three independent experiments. ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/5a69b21e07bd540fa252a0b5.jpg"},{"id":92045670,"identity":"bd306e57-eb93-4ac6-91d2-c2353cf08882","added_by":"auto","created_at":"2025-09-24 04:38:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLAMB3 regulates the reprogramming of glucose and lipid metabolism in lung cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot was used to detect the expression levels of glycolysis related enzymes HK and PFK1. (B) The glycolytic ability in cells was analyzed by cell ECAR. (C) The contents of triglycerides and cholesterol in cells were detected by the kit. Data are the means ± SD for three independent experiments. *P\u0026lt;0.05, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/21d7c9ceeab72c2b2908897a.png"},{"id":92045672,"identity":"da6c8292-f9b9-4ab8-87e7-985da3b40580","added_by":"auto","created_at":"2025-09-24 04:38:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":592298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of ITGB4 was increased in lung cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) GEPIA 2 databasepredicted the expression map of ITGB4 in lung cancer. (A) RT-qPCR detected the expression of ITGB4 in tissues. (A) Western blot detected the expression of ITGB4 in tissues. Data are the means ± SD for three independent experiments. **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"fig5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/ae682d29a7656a234a720b08.jpg"},{"id":92046694,"identity":"bd98db95-c528-4548-b839-06725ac5302a","added_by":"auto","created_at":"2025-09-24 04:54:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2602160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLAMB3 regulates ITGB4 to mediate proliferation and metastasis of lung cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) RT-qPCR and western blot were used to detect the expression of LAMB3 in cells. (B) CCK8 evaluated the changes of cell activity. (C) EDU detected the changes of cell proliferation. (D) Transwell detected the level of cell migration and invasion. Data are the means ± SD for three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"fig6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/93dee9c57e789cf11e977c02.jpg"},{"id":92045671,"identity":"51bcede3-1352-49e6-835b-efafcdb923f1","added_by":"auto","created_at":"2025-09-24 04:38:05","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2004205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLAMB3 regulates ITGB4 to mediate metabolic reprogramming of lung cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot was used to detect the expression levels of glycolysis related enzymes HK and PFK1. (B) The glycolytic ability in cells was analyzed by cell ECAR. (C) The contents of triglycerides and cholesterol in cells were detected by the kit. (D) Immunofluorescence confocal detection of intracellular lipid droplets. Data are the means ± SD for three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"fig7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/c836fb256ced8bd7b10b3fe4.jpg"},{"id":92045693,"identity":"a21c0a59-2c51-44e4-b8fe-0c8f49c89f23","added_by":"auto","created_at":"2025-09-24 04:38:06","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":268223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of the mechanism by which LAMB3 regulates ITGB4 mediated metabolic reprogramming in lung cancer cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/7cf32e2c4559196166e178e5.jpg"},{"id":97369414,"identity":"c3b4389c-da59-41c8-aa1a-8c140bab98ba","added_by":"auto","created_at":"2025-12-03 16:24:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11142271,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/3dca7ca8-a759-4945-95a0-25cb9a409172.pdf"},{"id":92045679,"identity":"7aeaaa24-f599-4df7-bd29-92c2cb1198a4","added_by":"auto","created_at":"2025-09-24 04:38:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":833740,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7070948/v1/470263a3e3d8e8bce4423f93.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"LAMB3 regulates ITGB4 to mediate metabolic reprogramming of lung cancer cells, thus promoting the proliferation and metastasis of cancer cells","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. LAMB3 is high expressed in pheochromocytoma.\u003c/p\u003e\u003cp\u003e2. Knocking down LAMB3 inhibits the progress of NSCLC.\u003c/p\u003e\u003cp\u003e3. LAMB3 regulates the reprogramming of glucose and lipid metabolism in lung cancer cells.\u003c/p\u003e\u003cp\u003e4. LAMB3 regulates ITGB4 to mediate the progress of lung cancer cells.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eIn China, lung cancer is a highly malignant tumor, and more than 40% of lung cancer patients have no smoking history \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Early diagnosis and targeted therapy of lung cancer is an urgent problem to be solved at present \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Among them, non-small cell lung cancer (NSCLC) is the most common type of lung cancer \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Warburg effect is one of the tumor markers, mainly because tumor cells produce ATP through aerobic glycolysis to adapt to the changes of tumor microenvironment \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, more and more research focused on tumor metabolic reprogramming. Interestingly, tumor cells can also use basic nutrients including fatty acids and amino acids to survive \u003csup\u003e5\u003c/sup\u003e. The metabolic reprogramming of tumor cells and the mutual adjustment of microenvironment promote the metastasis of cancer. However, the molecular mechanism of metabolic reprogramming on cancer metastasis is still unclear in NSCLC.\u003c/p\u003e\u003cp\u003eLaminin subunit β-3 (LAMB3) is one of the active components of basement membrane \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. LAMB3 could promote the proliferation and metastasis of pancreatic ductal adenocarcinoma \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Similarly, LAMB3 promoted the metastasis of colorectal cancer and thus might be a potential therapeutic target \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. We used GEPIA website to predict the expression of LAMB3 in TCGA database, and the results showed that LAMB3 was highly expressed in lung cancer. An interesting study found that LAMB3 can participate in the regulation of obesity and is related to the formation of body fat \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Then, whether LAMB3 is involved in metabolic regulation in NSCLC and thus promotes the progress of cancer cells remains to be verified.\u003c/p\u003e\u003cp\u003eWe predicted the possible downstream binding proteins of LAMB3 by STING and GeneMANIA, and found that it was mainly ITG family except LAM family. Among them, ITGB4 is highly expressed in LUSC and LUAD, and has physical interaction with LAMB3. ITGB4 can promote cancer in primary tumors of head and neck \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The abnormal expression of TIGB4 in NSCLC is closely related to acquired drug resistance of cancer cells \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Exciting, a study in breast cancer found that ITGB4 mediated metabolic reprogramming of tumor-related fibroblasts \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur research mainly verified that LAMB3 up-regulates ITGB4 and mediates cell metabolic reprogramming in NSCLC cells, thus promoting the proliferation and metastasis of cancer cells. This study clarified the new regulatory mechanism of LRP1B in NSCLC and provided a new target for the diagnosis and treatment of NSCLC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCollection of clinical samples\u003c/h2\u003e\u003cp\u003eThe clinical tissue samples of this study are from 37 patients with NSCLC and their adjacent tissues who were surgically removed in Pingxiang people's hospital Hospital in 2021\u0026ndash;2023. All patients have not received radiotherapy, chemotherapy and immunotherapy. All patients obtained written informed consent and were approved by the Pingxiang people's hospital Ethics Committee (No. 20210601). Research involving human participants was conducted following the ethical standards of the institution and the National Research Council. It adhered to the ethical standards of the 1964 Declaration of Helsinki and its subsequent amendments or similar.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eThe tissue samples were embedded in paraffin and made into 5 \u0026micro;m slices. After pretreatment, the slices were incubated with anti-LAMB3 (ab97765, 1:1000, Abcam, UK) and anti-KI67 (ab15580, 1:1000, Abcam) overnight. Then it was incubated with peroxidase-coupled goat anti-mouse IgG for 2 h. Finally, the 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution and hematoxylin were used for double staining and observation.\u003c/p\u003e\n\u003ch3\u003eCell culture and transfection\u003c/h3\u003e\n\u003cp\u003eNormal lung epithelial cells (BEAS-2) and lung cancer cell lines (A549, HCC827, H1299, H1299, H460) were purchased from Wuhan Procell Life Science and Technology Co., Ltd. (Wuhan, China). BEAS-2 cells were cultured in DMEM/F12 supplemented with fetal calf serum (FBS, 10%, Gibco, USA) and penicillin/Streptomycin (1%, Gibco). Lung cancer cell lines were cultured in the same RPMI 1640 medium which was treated in the same way. The culture conditions were 5% CO\u003csub\u003e2\u003c/sub\u003e and 37℃.\u003c/p\u003e\u003cp\u003eshRNA, overexpression vector (OE) and negative control (NC) were designed and synthesized by Shanghai Sangon Biotechnology (Shanghai, China). All vectors were transfected with Lipofectamine 3000 reagent (Invitrogen, USA).\u003c/p\u003e\n\u003ch3\u003eReal-time fluorescence quantitative PCR (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was obtained from lung cancer tissues and cells by using TRIzol reagent (Invitrogen, USA). After 2 \u0026micro;g of total RNA reverse transcription treatment, the qPCR reaction was carried out using SYBR Green qPCR mix (Sangon, Shanghai). The primer sequence is (5'-3'): LAMB3-F-CCAAAGGTGCGACTGCAATG, R-AGTTCTTGCCTTCGGTGTGG; ITGB4-F-CACCTCCGTCTCCTCCCAC, R-GTTGGGGATGTTGAGCCGAT; β-actin-F-TTGCAGCTCCTTCGTTGCC, R-TTGCAGCTCCTTCGTTGCC.\u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cp\u003eProtein was obtained from lung cancer cells by ProteoPrep\u0026reg; total protein extraction kit (Sigma-Aldrich, USA). Protein was transferred to membrane, sealed and incubated with anti-LAMB3 (ab97765, 1:2000, Abcam), anti-ITGB4 (ab197772, 1:2000, Abcam) and β-actin (ab8226, 1: 2000, Abcam). The next day, goat anti-mouse/rabbit IgG secondary antibody conjugated with horseradish peroxidase (HRP) was used for incubation. Finally, the protein bands were observed and recorded under the microscope.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell counting kit-8 (CCK8) assays\u003c/h2\u003e\u003cp\u003eLung cancer cells (2500 cells/well) were inoculated into a 48-well plate and cultured for 12 h. 10 \u0026micro;L of CCK8 (C0038, Beyotime, Shanghai) solution was added to each well for 2 h. Finally, the absorbance at 450 nm was quantified by microplate reader (Thermo Multiskan Spectrum, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTranswell assays\u003c/h3\u003e\n\u003cp\u003eLung cancer cells were suspended in serum-free RPMI 1640 medium and then added to the upper chamber of transwell system (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells). RPMI 1640 medium containing 20% FBS was added to the lower chamber. Invasion detection needs to be pre-coated with matrigel in the upper chamber. After 24 h of culture at 37℃, the cells were fixed and stained with 0.1% crystal violet solution for 8 min. The cells were observed and photographed under a microscope.\u003c/p\u003e\n\u003ch3\u003eDetermination of extracellular acidification rate (ECAR)\u003c/h3\u003e\n\u003cp\u003eLung cancer cells (1.5\u0026times;10\u003csup\u003e3\u003c/sup\u003e) were inoculated in a 24-cell plate and cultured overnight. Subsequently, the cells were washed by PBS and cultured in an incubator without CO\u003csub\u003e2\u003c/sub\u003e at 37℃ for 60 min. Then, glucose, oligonucleotide and 2- deoxyglucose were added, and the extracellular flux analyzer (SeahorseXF24 FluxPak) was used to detect the extracellular acidification rate.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDetection of triglyceride and cholesterol\u003c/h2\u003e\u003cp\u003eLung cancer cells were collected into a 1.5 mL centrifuge tube, and 1 mL reagent (n-heptane: isopropanol\u0026thinsp;=\u0026thinsp;1:1 volume ratio) was added for ice bath ultrasonic crushing. Cell supernatant was separated and collected by centrifugation. The triglyceride and cholesterol detection kit (ab65336, Abcam) were used for related detection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eLipid drop detection\u003c/h2\u003e\u003cp\u003eIn order to label the lipid droplets in cells, we put the cells to be detected in an 8-hole slide and incubate them with Bodipy 493/503 (2 \u0026micro;M) (#72485, Sigma Aldrich) at 37℃ for 15 min. Then the fluorescently labeled lipid droplets were observed under a confocal microscope (Nikon, Tokyo, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAnimal model\u003c/h2\u003e\u003cp\u003eBALB/c nude mice (13\u0026ndash;16 g, 4\u0026ndash;6 weeks old) were purchased from Shanghai slack Experimental Animal Co., Ltd. to establish xenotransplantation tumor animal model. A549 cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) were injected subcutaneously into the armpit of each nude mouse. 30 days after tumor formation, mice were treated by spinal dislocation and the tumor was isolated. The animal experiment scheme conforms to ARRIVE guidelines and is reviewed and approved by Pingxiang people's hospital Ethics Committee (NO. 20230402).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments were carried out independently for three times and statistically analyzed by using GraphPad Prism 8.0. All data\u0026thinsp;=\u0026thinsp;mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Students' \u003cem\u003et\u003c/em\u003e test and one-way ANOVA were used to make significant analysis between the two groups and between multiple groups respectively. \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eLAMB3 was highly expressed in clinical tissues and cells of lung cancer\u003c/h2\u003e\u003cp\u003eFirst of all, it was predicted by GEPIA 2 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia2.cancer-pku.cn/#index\u003c/span\u003e\u003cspan address=\"http://gepia2.cancer-pku.cn/#index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) that the LAMB3 in lung cancer was on the rise (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We further analyzed the cancer tissues of 37 patients with lung cancer and found that. Compared with the tissues adjacent to cancer, the expression of LAMB3 in lung cancer patients was increased observably (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Then, we also found similar expression patterns in lung cancer cell lines (A549, HCC827, H1299, H1299, H460). Especially in A549 and H1299 cells, LAMB3 expression increased most significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eLAMB3 promotes the malignant progress of lung cancer cells\u003c/h2\u003e\u003cp\u003eThen, we studied LAMB3 in A549 and H1299 cells. After transfection with sh-LAMB3, the levels of LAMB3 in cells were significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We also found that the cell activity, proliferation, migration and invasion ability decreased significantly after knocking down LAMB3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe further explored LAMB3 in the mice model of lung cancer transplanted tumor. Inhibition of LAMB3 slowed down the tumor proliferation and diameter in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), and the expressions of KI67 and LAMB3 in tumor tissues of mice were significantly reduced after knocking down LAMB3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eLAMB3 regulates the metabolic reprogramming in lung cancer cells\u003c/h2\u003e\u003cp\u003eInterestingly, we also found that LAMB3 could regulate glucose and lipid metabolism in lung cancer cells. We found that after silencing LAMB3, the glycolysis related enzymes HK and PFK1 in cells were decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). After knocking down LAMB3, the glycolysis of lung cancer cells was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, the triglyceride and cholesterol levels in the cells were decreased significantly after LAMB3 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eHigh expression of ITGB4 in lung cancer\u003c/h2\u003e\u003cp\u003eSimilarly, it was found that the ITGB4 increased in lung cancer by GEPIA 2 database analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the tissues of lung cancer patients collected by our hospital, the expression of ITGB4 also was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eLAMB3 regulates the progress of ITGB4-mediated lung cancer cells\u003c/h2\u003e\u003cp\u003eWe tested the regulatory relationship by knocking down LAMB3 and overexpressing ITGB4 in lung cancer cells. As shown in the results of Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the expressions of LAMB3 and ITGB4 in cells were decreased after knocking down LAMB3. At the same time, after transfection of OE-ITGB4, the inhibitory effect of sh-LAMB3 on ITGB4 was restored. However, the expression of LAMB3 was not influenced by the expression of ITGB4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Further verification showed that overexpression of ITGB4 was restored the inhibitory effect of sh-LAMB3 on the activity, proliferation, migration and invasion of lung cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eLAMB3 regulates ITGB4 to mediate metabolic reprogramming\u003c/h2\u003e\u003cp\u003eFinally, we analyzed the influence of LAMB3/ITGB4 axis on glucose and lipid metabolism of lung cancer cells. Overexpression of ITGB4 partially reversed the inhibitory effect of silencing LAMB3 on glycolysis related enzymes HK and PFK1 in lung cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Similarly, further detection of cell metabolic capacity showed that overexpression of ITGB4 restored the down-regulation effect of knocking out LAMB3 on glycolysis, triglyceride, cholesterol level and lipid droplet formation in lung cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMetabolic reprogramming of cancer is involved in driving and maintaining the malignant phenotype of cancer cells \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Metabolic reprogramming is very active in patients with lung cancer and shows heterogeneity \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Our research confirmed that LAMB3/ITGB4 axis could regulate the metabolic reprogramming of lung cancer cells, which had an impact on the progress of lung cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This discovery provides a new potential target gene for therapeutic strategies targeting metabolic pathways in lung cancer treatment research.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLAMB3 promotes tumorigenesis by encoding β2 subunit of trimeric basement membrane protein laminin 332 and interacting with other genes in cells \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. A recent study found that targeting LAMB3 can inhibit the progress of MAPKi-resistant melanoma cells \u003cem\u003ein vivo\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In the study of clinical samples of lung cancer patients, it was confirmed that the expression of LAMB3 was related to lymphatic metastasis \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We also found that knocking down the expression of LAMB3 could inhibit the malignant progress of tumor cells in lung cancer cell lines and transplanted tumor mice model. Interestingly, we also found that LAMB3' s regulation of lung cancer progression is related to metabolic reprogramming of cancer cells. By silencing LAMB3 in lung cancer cell line, it was found that the glycolytic ability and lipid metabolism level of the cell decreased. Therefore, we speculate that LAMB3 might regulate metabolic pathways and participate in regulating the progress of lung cancer.\u003c/p\u003e\u003cp\u003eAn exciting study shows that LAMB3 interacts with ITGB4 in colorectal cancer cells, up-regulating AKT pathway and thus enhancing the activity of tumor cells \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The high level of ITGB4 is related to the poor clinical prognosis of lung cancer. Knocking out ITGB4 can inhibit the proliferation and metastasis of NSCLC \u003csup\u003e19\u003c/sup\u003e. Our study also found that the ITGB4 increased significantly in NSCLC patients and cell lines. Interestingly, it was found that ITGB4 can activate PI3K/mTOR/SREBP1c signaling pathway and lead to reprogramming of lipid metabolism in hepatocellular carcinoma cells \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Similarly, we also confirmed in lung cancer cells that overexpression of ITGB4 can reverse the effects of silencing LAMB3 on proliferation, metastasis and metabolic reprogramming of NSCLC. However, there are some limitations in our research, and the effect of LAMB3 on the regulation and metastasis of glucose and lipid metabolism of cancer cells has not been verified in mice, which is also the focus of our further research.\u003c/p\u003e\u003cp\u003eIn a word, our study verified the regulation of LAMB3/ITGB4 pathway in the metabolic pathway of NSCLC. It is confirmed that LAMB3 participates in the regulation of glucose and lipid metabolism of lung cancer cells by regulating ITGB4, which mediates the development of lung cancer. It was also found that knocking down LAMB3 could inhibit the progress of lung cancer transplanted tumor in mice. In a word, our research provides new potential target genes for the development of targeted drugs for lung cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree with the presented findings, have contributed to the work, and declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their gratitude to for his help for directing our article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT author statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Zhicheng Ouyang\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethodology: Zhicheng Ouyang, Suting Chen\u003c/p\u003e\n\u003cp\u003eSoftware: Suting Chen\u003c/p\u003e\n\u003cp\u003eValidation: Suting Chen, Limin Dong\u003c/p\u003e\n\u003cp\u003eFormal analysis: Suting Chen,Limin Dong\u003c/p\u003e\n\u003cp\u003eInvestigation: Suting Chen\u003c/p\u003e\n\u003cp\u003eResources: Suting Chen\u003c/p\u003e\n\u003cp\u003eData Curation: Suting Chen\u003c/p\u003e\n\u003cp\u003eWriting - Original Draft: Limin Dong\u003c/p\u003e\n\u003cp\u003eWriting - Review \u0026amp; Editing: Suting Chen\u003c/p\u003e\n\u003cp\u003eVisualization: Suting Chen, Limin Dong\u003c/p\u003e\n\u003cp\u003eSupervision: Zhicheng Ouyang\u003c/p\u003e\n\u003cp\u003eProject administration: Zhicheng Ouyang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 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Journal of Thoracic Oncology. 2022;17(12):1335-54.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Non-small cell lung cancer, LAMB3, ITGB4, metabolic reprogramming, metastasis","lastPublishedDoi":"10.21203/rs.3.rs-7070948/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7070948/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cdiv id=\"ASec1\" class=\"AbstractSection\"\u003e\u003cdiv class=\"Heading\"\u003eBackground\u003c/div\u003e\u003cp\u003eNon-small cell lung cancer (NSCLC) is a common lung cancer cell type. LAMB3 promotes the progress of cancer. However, the regulatory mechanism of LAMB3 on metabolic reprogramming of NSCLC is not clear.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"ASec2\" class=\"AbstractSection\"\u003e\u003cdiv class=\"Heading\"\u003eMethods\u003c/div\u003e\u003cp\u003eRT-qPCR and western blot were used to detect the expression level of LAMB3 in NSCLC tissues and cells. The expression level of LAMB3 in A549 and H1299 cells was changed by transfection of related vectors. Cell counting kit-8 (CCK8) was used to detect the activity of NSCLC. Transwell was evaluated the migration and invasion of cancer cells. The metabolic level changes of NSCLC cells were detected by related kits.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"ASec3\" class=\"AbstractSection\"\u003e\u003cdiv class=\"Heading\"\u003eResults\u003c/div\u003e\u003cp\u003eOur results showed that LAMB3 and ITGB4 were highly expressed in NSCLC tissues and cells. Knocking down LAMB3 inhibited the progress of NSCLC, including cell proliferation, metastasis and metabolic level. Overexpression of ITGB4 can reverse the effects of knocking down LAMB3 on tumor progression and metabolic reprogramming.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"ASec4\" class=\"AbstractSection\"\u003e\u003cdiv class=\"Heading\"\u003eConclusion\u003c/div\u003e\u003cp\u003eLAMB3 regulated ITGB4-mediated metabolic reprogramming of lung cancer cells, thus promoting the proliferation and metastasis of cancer cells.\u003c/p\u003e\u003c/div\u003e","manuscriptTitle":"LAMB3 regulates ITGB4 to mediate metabolic reprogramming of lung cancer cells, thus promoting the proliferation and metastasis of cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 04:37:59","doi":"10.21203/rs.3.rs-7070948/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"012e3ecd-3916-46e8-bcf5-77c5226064c2","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54949468,"name":"Biological sciences/Cancer"},{"id":54949469,"name":"Biological sciences/Cell biology"},{"id":54949470,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-12-03T05:08:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 04:37:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7070948","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7070948","identity":"rs-7070948","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}