Journal: bioRxiv

Article Title: Extracellular vesicle-derived miR-425-5p (miR-425) activates astrocytes in the brain to promote breast cancer brain metastasis via the novel miR-425-ZNF24-CCL8 signaling axis

doi: 10.1101/2025.06.05.658130

Figure Lengend Snippet: A) Predicted miR-425 binding sites within the CREB1, BCOR, and ZNF24 3’-UTRs from TargetScan. B) ZNF24 mRNA expression is significantly decreased in astrocytes overexpressing miR-425. CREB1 and BCOR mRNA expression is unchanged as measured by RT-qPCR. C) ZNF24 protein expression is decreased in astrocytes transfected with miR-425 mimic as indicated by western blot analysis. D) miR-425 suppresses ZNF24 3’-UTR activity as measured by dual luciferase reporter assay. E) Ectopic expression of ZNF24 significantly decreases CCL8 and VEGFA mRNA expression, while KITLG is unchanged. mRNA expression measured by RT-qPCR. F) Ectopic expression of ZNF24 suppresses CCL8 protein expression, but not SCF as indicated by western blot analysis. G) ZNF24 knockdown significantly increases CCL8 and VEGFA mRNA expression, but not KITLG. mRNA expression as indicated by RT-qPCR. H) ZNF24 knockdown increases CCL8 and SCF protein expression as measured by western blot analysis. Fold change was calculated in Panels B, D, E, and G. Student’s t- test was used in Panels B, D, E, and G. N = 3 experimental replicates unless otherwise indicated.

Article Snippet: ZNF24 immunofluorescent antibody (Bioss; bs18508R-FITC) was used at a 1:50 dilution, GFAP antibody (Cell Signaling; 3656) was used at 1:50 dilution, and DAPI antibody at 1:1000 dilution on 10 μm sections of mouse brains with brain metastases.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Activity Assay, Luciferase, Reporter Assay, Knockdown

Journal: bioRxiv

Article Title: Extracellular vesicle-derived miR-425-5p (miR-425) activates astrocytes in the brain to promote breast cancer brain metastasis via the novel miR-425-ZNF24-CCL8 signaling axis

doi: 10.1101/2025.06.05.658130

Figure Lengend Snippet: A) Human astrocytes activated by a cocktail of IL-1α, TNFα, and C1q. Representative 20x images. Scale bar indicates 100 µm. B) Validation of GFAP mRNA expression in activated astrocytes as indicated by RT-qPCR. C) Activated astrocytes transfected with ZNF24 plasmid have significantly reduced GFAP expression as indicated by GFAP IF. Representative 20x images. Scale bar indicates 100 µm. D) Validation of GFAP reduction and ZNF24 overexpression with ectopic expression of ZNF24. mRNA expression measured by RT-qPCR. E) ZNF24 binding to the CCL8 promoter in two regions (−69 and -205 bases upstream of the TSS) measured by ChIP-qPCR. F) Ectopic expression of ZNF24 suppresses CCL8 promoter activity as indicated by dual luciferase reporter assays. G-H) ZNF24 and CCL8 mRNA expression remains unchanged in breast cancer cells transfected with control or miR-425 mimic. mRNA expression measured by RT-qPCR. Fold change was calculated in Panels B, D-H. Student’s t -test was used in Panels A-H. N = 3 experimental replicates unless otherwise indicated.

Article Snippet: ZNF24 immunofluorescent antibody (Bioss; bs18508R-FITC) was used at a 1:50 dilution, GFAP antibody (Cell Signaling; 3656) was used at 1:50 dilution, and DAPI antibody at 1:1000 dilution on 10 μm sections of mouse brains with brain metastases.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Over Expression, Binding Assay, ChIP-qPCR, Activity Assay, Luciferase, Control

Journal: bioRxiv

Article Title: Extracellular vesicle-derived miR-425-5p (miR-425) activates astrocytes in the brain to promote breast cancer brain metastasis via the novel miR-425-ZNF24-CCL8 signaling axis

doi: 10.1101/2025.06.05.658130

Figure Lengend Snippet: A) Increased EV-miR-425 expression in mouse serum from the SKBR3-Luc-miR-425 group compared to the control group. miR-425 expression measured by RT-qPCR (N = 5 per group). B) SKBR3-Luc-miR-425 group mouse serum has significantly higher levels of mouse CCL8 as measured by ELISA (N = 7 per group). C) No significant difference in mouse SCF levels in SKBR3-Luc-miR-425 or control mice serum as measured by ELISA (N = 5 per group). D-E) GFAP H-score and intratumoral astrocytes are significantly higher in brain metastases from the SKBR3-Luc-miR-425 mouse group than the control in mouse brain sections as measured by IHC (N = 5 per group). F) Brain metastases from the SKBR3-Luc-miR-425 group have significantly increased Ki-67 positive cells as measured by IHC (N = 5 per group). G) Tumor-adjacent and infiltrative astrocytes in brain metastases from the SKBR3-Luc-miR-425 group have decreased ZNF24 staining measured by IHC (N = 5 per group). H) Representative IHC images at 20x magnification. Scale bar indicates 100 µm. I) Co-staining IF of ZNF24 and GFAP in mouse brain sections containing brain metastases. Representative images at 20x magnification. Scale bar indicates 100 µm. J) Schematic of described mechanism by which breast cancer-derived EV-miR-425 activates astrocytes by suppressing ZNF24, increasing CCL8, and thereby promoting BCBM. Fold change was calculated in Panel A. Student’s t -test used in Panels A-G, and I.

Article Snippet: ZNF24 immunofluorescent antibody (Bioss; bs18508R-FITC) was used at a 1:50 dilution, GFAP antibody (Cell Signaling; 3656) was used at 1:50 dilution, and DAPI antibody at 1:1000 dilution on 10 μm sections of mouse brains with brain metastases.

Techniques: Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Derivative Assay