Fig 1: LRIG2 was a direct target of miR-182. a The binding site of LRIG2 and miR-182 was predicted by TargetScan. b, c The relative luciferase activity in EC cells was detected by dual-luciferase reporter assay. d The mRNA expression of LRIG2 in EC tissues was examined by RT-qPCR. e Western blot was used to detect the protein expression of LRIG2 in EC tissues. f The negative relationship between miR-182 and LRIG2 was discovered in EC tissues (r = - 0.3122, P < 0.01). g The positive relationship between circ_0001776 and LRIG2 was observed in EC tissues (r = 0.5728, P < 0.01). h LRIG2 expression was determined after EC cells transfected with anti-NC or anti-miR-182. i The protein expression of LRIG2 in EC cells was detected by western blot. j LRIG2 expression in EC cells transfected with anti-NC or anti-miR-182 was measured by western blot. k, l LRIG2 expression in EC cells transfected with vector, circ_0001776, circ_0001776 + miR-NC or circ_0001776 + miR-182 was examined by western blot. **P < 0.01
Fig 2: ADAM17 mediates the shedding of the ectodomain of LRIG2 (sLRIG2) (A) WB analyses the expression of sLRIG2 in the supernatant and FLAG, ADAM17 in the cytoplasm of U87-LRIG2 and GL261-LRIG2 cells treated with four different concentrations of ADAM17’s activator (PMA 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL). (B) WB analyses the protein expression of sLRIG2 in the supernatant and FLAG and ADAM17 in the intracellular of LRIG2 overexpressed GBM cells transfected with two different siRNA targeting ADAM17. (C) WB analyses the protein expression level of sLRIG2 in the supernatant and FLAG in the intracellular of LRIG2 overexpressed GBM cells treated with ADAM17’s activator (PMA 200 ng/mL) or ADAM17’s inhibitor (TAPI-1 100 ng/mL) or PMA (200 ng/mL) and TAPI-1 (100 ng/mL). GBM, glioblastoma; WB, Western blot.
Fig 3: High LRIG2 is correlated with higher glioma TAM density and shorter survival. LRIG family members’ expression level in glioma cells and the infiltration of immune cells from the SCEA datasets *P<0.05,**P<0.01.(A) and GEO datasets (GSE131928; GSE135437) (B). Unpaired Student’s t-test. Error bar=SD. (C) GO and KEGG pathway enrichment analysis of LRIG2-related gene sets in glioma from the TCGA datasets. (D) Representative IHC images of the infiltration of immune cell subsets in LRIG2 high (H-SCORE=6)and low expression (H-SCORE<6) tumors. Top panel: scale bar, 400 µm. Bottom panel: scale bar, 80 µm. (E) Correlation analysis between the protein expression(D) of LRIG2 and the TAM marker CD68 and CD206 in clinical glioma tissue microarray (n=88, H-SCORE). Pearson’s correlation test. (F) Kaplan-Meier survival curves for correlation between mRNA and protein expression of LRIG2 and survival of glioma patients in the CGGA dataset and clinical glioma tissue microarray. Log-rank test. (G) Kaplan-Meier survival analysis for the correlation between infiltration degree of immune cell subsets in glioma microenvironment and survival of glioma patients in the CGGA dataset and clinical glioma tissue microarray, log-rank test. CGGA, Chinese Glioma Genome Atlas; KEGG, Kyoto Encyclopedia of Genes and Genomes; ns, not significant; TAM, tumor-associated microglia/macrophage; TCGA, The Cancer Genome Atlas.
Fig 4: Targeting LRIG2 expression/secretion synergizes with anti-CD47 in anti-GBM therapeutics. (A) Treatment scheme for evaluating in vivo efficacy of targeting LRIG2 combined with anti-CD47 and TAPI-1. (B) and (C) Representative luciferase images of GL261shScr cells (B) or GL261shLRIG2-1 (C) derived tumors in mice treated with IgG control, anti–CD47, TAPI-1, or TAPI-1and anti-CD47 antibody n=8). (D, E) Effect of monotherapy and combination anti–CD47 and TAPI-1 on tumor-bearing (GL261shScr cells/GL261shLRIG2-1 cells) mice(n=8/9). Kaplan-Meier survival curves for each treatment group Significance level was calculated by log-rank analysis. *P<0.05, **p<0.01, ***p<0.001. (F) Western blotting of sLRIG2 in the serum of tumor-bearing mice from different groups (GL261shScr-IgG, GL261shLRIG2-1 IgG, GL261shLRIG2-1 anti-CD47, GL261shLRIG2-1 TAPI-1, GL261shLRIG2-1-anti-CD47+TAPI-1). (G) Graphic analysis of M2-likes TAM(CD45+CD11b+F4/80+CD206+) and M1-likes TAM(CD45+CD11b+F4/80+CD11c+) cells density in the control group (GL261-shScr), LRIG2 knockdown group (GL261shLRIG2-1), LRIG2 knockdown group treated with anti–CD47 antibody (GL261shLRIG2-1 CD47), LRIG2 knockdown group treated with TAPI-1 (GL261shLRIG2-1 TAPI-1), LRIG2 knockdown group treated with anti-CD47 antibody and TAPI-1(GL261shLRIG2-1 CD47 TAPI-1). (Mean±SD; n=4). *P<0.05, **p<0.01, ***p<0.001.One-way ANOVA with Bonferroni correction. (H) Proposed schematic of LRIG2 mediates GBM immune escape. ANOVA, analysis of variance; GBM, glioblastoma.
Fig 5: U87 and U251 cell lines were transfected with si-LRIG2 or si-NC, then treated with 100 ng/ml EGF or DDH2O for 24 h. (A) The protein expression level of EGFR, p-EGFR and VEGF-A were assessed by western blot assay. (B) The densitometry data of western blotting. **P<0.01 vs. the si-NC + DDH2O group. ##P<0.01 vs. the si-LRIG2 + DDH2O group. Results are representative of three different experiments. LRIG2, leucine-rich repeats and immunoglobulin-like domains 2; EGFR, epidermal growth factor receptor; VEGF-A, vascular endothelial growth factor A; si-LRIG2, LRIG2 siRNA; si-NC, negative control siRNA.
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