Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: TIPS construction and validation. ( a ): Based on 302 TIDEGs, the training set is screened using univariate Cox analysis to identify 18 prognosis-related genes. Unifactorial analysis of TIDEGs in the training set. ( b ): Three TICPGs are identified using multivariate Cox analysis, including KLRK1 (β=-0.303), LTB (β=-0.562), and RBP4 (β = 0.161). The risk score formula is as follows: risk score=(-0.303×KLRK1)+(-0.562×LTB)+(0.161×RBP4). ( c ): Survival curve analysis of TIPS in the high-risk group vs. low-risk group in the training set, ROC curves, distribution of risk scores, distribution of survival statuses, and heatmap of TICPGs. The area under the curve (AUCs) of TIPS at 1, 3, and 5 years are 0.7, 0.704, and 0.739, respectively. ( d ): Heatmap of TIPS in the test set of high-risk group vs. low-risk group survival curve analysis, ROC curve, risk score distribution, survival status distribution, TICPGs heat map. In the test and full sets ( n = 158), the high- and low-risk groups are divided based on optimal cutoff value; KM curves showed that the prognosis of the low-risk group was significantly better ( p < 0.05). ROC analysis shows the AUCs in the test set at 1, 3, and 5 years of 0.737, 0.64, and 0.647, respectively. ( e ): TIPS in the full set of high-risk group vs. low-risk group survival curve analysis, ROC curve, risk score distribution, survival status distribution, TICPGs heat map. Corresponding AUCs in the full set are 0.713, 0.677, and 0.711, respectively. ( f ): Full set of univariate analysis of TIPS vs. traditional clinical indicators. ( g - i ): ROC curve of the AUC values in TIPS at 1, 3, and 5 years. (j): Full set of multifactorial analyses of TIPS versus STAGE, T versus N staging. DEGs, differentially expressed genes; EMT, epithelial-mesenchymal transition; ROC, receiver operating characteristic; TIPS, TME immune-related genes prognostic signature; TIDEGs, TME immune-related DEGs; TME, tumor microenvironment.

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Biomarker Discovery

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: Effects of RBP4 overexpression or knockdown on TSCC cell proliferation, migration, and invasion. ( a ): Differential analysis of KLRK1, LTB, and RBP4 expression in tumor tissues vs. paired paracancerous normal tissues in the TCGA-TSCC dataset. ( b ): In the GSE13601 ( p = 5e-06), GSE78060 ( p = 0.082), and GSE31056 ( p = 1e-05) datasets, the RBP4 expression in tumor samples vs. paired paracancerous normal tissue samples. RBP4 differential expression analysis in tumor samples relative to paraneoplastic normal tissue samples in GSE13601 , GSE78060 , and GSE31056 datasets. The left panel shows the volcano plots of DEGs; the right panel shows the box plots of RBP4 expression levels in tumor samples versus paraneoplastic normal samples. ( c ): Immunohistochemistry observation of RBP4 expression in TSCC and paraneoplastic tissues. The upper panel shows the expression of RBP4 in TSCC tissues. Scale bar: 200 μm. The lower panel shows the expression of RBP4 in paraneoplastic tissues. Scale bar: 50 μm. ( d - g ): Overexpression or knockdown of RBP4 in CAL27 and SCC-15 cells using lentivirus and validated by qRT-PCR with western blotting experiments. ( h ): CCK-8 method is used to analyze the effect of changes in RBP4 levels on the proliferative ability of TSCC cells. ( i ): In vivo experiments validate the effect of changes in RBP4 expression levels on the TSCC cell proliferation rate. ( j ): Effects of changes in RBP4 expression levels on TSCC cell migration ability were observed by scratch assay. ( k ): Effects of changes in RBP4 expression levels on TSCC migration invasion ability were observed by Transwell assay. DEGs, differentially expressed genes; TCGA, The Cancer Genome Atlas Program; TSCC, tongue squamous cell carcinoma. (*Compared with NC, * p < 0.05, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Over Expression, Knockdown, Migration, Expressing, Quantitative Proteomics, Immunohistochemistry, Quantitative RT-PCR, Western Blot, CCK-8 Assay, In Vivo, Wound Healing Assay, Transwell Assay

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: Mechanism study of RBP4 affecting TSCC cell proliferation, migration, and invasion. ( a ): Bioinformatic analysis of the effect of RBP4 on the PI3K/Akt/mTOR signaling pathway. ( b ): Western blotting to detect the effect of changes in the expression level of RBP4 on the activation level of the PI3K/Akt/mTOR pathway in TSCC cells. ( c ): After addition of 740 Y-P (0.2 mM), western blotting detects the alteration of the effect of RBP4 on the PI3K/Akt/mTOR pathway. ( d ): CCK-8 method is used to analyze the changes in the effect of RBP4 on the proliferative capacity of TSCC cells after the addition of 740 Y-P (0.2 mM). ( e - f ): To explore the mechanism by which changes in RBP4 levels affect TSCC cell migration and invasion, we determine the changes in Snail expression in RBP4 overexpressing and RBP4 knockdown TSCC cells. qRT-PCR and western blotting are used to detect the effect of RBP4 on the expression level of Snail and EMT-related proteins. ( g ): PI3K agonist treatment reverses the effects of RBP4 overexpression on Snail/E-cadherin/N-cadherin/Vimentin regulation. Western blotting to detect the changes of the effect of RBP4 on the PI3K/Akt/mTOR pathway after the addition of 740 Y-P (0.2 mM). ( h ): PI3K agonist treatment enhances cell migration and invasion, suggesting that RBP4 suppresses the TSCC malignant phenotype by inhibiting the PI3K/Akt/mTOR-Snail axis. Scratch assay to detect the changes of the effect of RBP4 on the migration ability of TSCC cells after the addition of 740 Y-P (0.2 mM). Changes in the effect of TSCC cell migration ability. ( i ): Transwell assay to detect changes in the effect of RBP4 on TSCC cell invasion ability after addition of 740 Y-P (0.2 mM). TSCC, tongue squamous cell carcinoma. (*Compared with NC, * p < 0.05, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Migration, Western Blot, Expressing, Activation Assay, CCK-8 Assay, Knockdown, Quantitative RT-PCR, Over Expression, Wound Healing Assay, Transwell Assay

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: RBP4 in TSCC cells affects macrophage phenotypic transformation. To investigate the association between RBP4 expression levels and tumor-infiltrating macrophage phenotypes in TSCC tissues, we perform a correlation bioconfidence analysis using the GEO database. ( a ): In the GSE13601 dataset, the left graph represents the distribution of macrophage M1 polarization in the subgroup of tumor samples versus paracancerous samples, and the right graph represents the distribution of macrophage M1 polarization in the subgroup of samples with high and low RBP4 expression in tumor tissues. ( b ): In the GSE31056 dataset, the left graph represents the distribution of macrophage M1 polarization in the subgroup of tumor samples versus paracancerous samples, and the right panel represents the distribution of macrophage M1 polarization in the subgroup of samples with high and low tumor tissue RBP4 expression. Significance is calculated using the Wilcoxon rank-sum test. ( c ): CD86 and RBP4 expression levels are determined using immunohistochemical staining of paraneoplastic and TSCC tissues. ( d ): Flow cytometry is performed to detect phenotypic changes in macrophages co-cultured with TSCC. TSCC, tongue squamous cell carcinoma. (*Compared with NC, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Transformation Assay, Expressing, Immunohistochemical staining, Staining, Flow Cytometry, Cell Culture

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: RBP4 promotes macrophage M1 polarization by activating the TLR4/NF-κB pathway. ( a ): KEGG Bar Charts of human recombinant RBP4 protein-treated M0 macrophage cells after transcriptome sequencing. ( b ): Correlation analysis of RBP4 and TLR4 protein expression using Spearman and Pearson methods. ( c ): Western blotting assay to detect the effect of human recombinant RBP4 on macrophage TLR4/NF-KB pathway and the change of the effect of RBP4 action after the addition of TAK242. ( d , e ): qRT-PCR and western blotting are used to detect the effect of the human recombinant RBP4 protein on the macrophage phenotype and the effect of RBP4 action after the addition of TAK242. ( f , g ): Western blotting experiments are used to detect the effects of changes in RBP4 expression levels on the macrophage TLR4/NF-κB pathway in a Transwell co-culture system. ( h ): Western blotting experiments are used to detect the effects of tumor-secreted RBP4 on the macrophage TLR4/NF-κB pathway after the addition of TAK242. Changes in activation level. ( i ): Western blotting experiments are used to detect the effect of tumor-secreted RBP4 on macrophage phenotype after addition of TAK242 in Transwell co-culture system. TAK242 treatment blocks RBP4 overexpression induced by TSCC cells in the macrophage TLR4/p-NF-κB upregulation and M1-type polarization. KEGG, Kyoto Encyclopedia of Genes and Genomes; TSCC, tongue squamous cell carcinoma. (*Compared with Control, * p < 0.05, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Recombinant, Sequencing, Expressing, Western Blot, Quantitative RT-PCR, Co-Culture Assay, Activation Assay, Over Expression, Control

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: Effect of RBP4 on macrophage phenotype. ( a ): Animals were grouped according to the injection of different cells, and the tumor volume, growth curve, and mass were measured and recorded. ( b ): The levels of CD86 and CD206 cell infiltration in the sections of transplanted tumors from each group are analyzed using immunofluorescence staining. ( c ): Changes in the activation level of the TLR4/NF-KB pathway in each group of transplanted tumor tissues are detected using Western blotting. ( d ): Mechanistic diagram of the effects of changes in RBP4 levels on the proliferation, migration, and invasion abilities of TSCC cells and the effects of tumor-secreted RBP4 on the phenotype of TAMs. TAMs, tumor-associated macrophages; TSCC, tongue squamous cell carcinoma. (*Compared with RAW264.7 + Control, * p < 0.05, ** p < 0.01).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Injection, Immunofluorescence, Staining, Activation Assay, Western Blot, Migration, Control