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

Journal: BMC Cancer

Article Title: KRAS mutation promotes immune escape of lung adenocarcinoma via ZNF24/SLC7A5/PD-L1 axis

doi: 10.1186/s12885-025-14336-0

Figure Lengend Snippet: High expression of ZNF24, SLC7A5 and PD-L1 in NSCLC with KRAS mutation. A - B . The expression of all genes was detected by WB (A) and RT-qPCR (B). The expression of ZNF24, SLC7A5 and PD-L1 in NSCLC cell lines with KRAS mutation (H358 G12C , H2122 G12C , LLC mut ) was significantly higher than that in NSCLC cell lines with KRAS wild-type (Calu-3 wild ) and normal lung epithelial cells (Beas2B). C . IHC was used to detect the expression of ZNF24, SLC7A5 and PD-L1 in lung adenocarcinoma (LUAD) tissues. The expression levels of ZNF24, SLC7A5, and PD-L1 were markedly increased in lung adenocarcinoma tissues that had a KRAS mutation compared to those with a normal KRAS gene. Furthermore, the researchers also observed a positive correlation between the expression levels of ZNF24, SLC7A5, and PD-L1. D - E . The overexpression plasmid of KRAS G12C mutation was transfected into KRAS wild-type lung cancer cell line (Calu-3 wild ). The expression of ZNF24, SLC7A5 and PD-L1 was increased after transfection with the KRAS G12C mutant plasmid by WB ( D ) and RT-qPCR ( E ). The increase of genes caused by KRAS G12C mutation partially reversed by anti-p21Ras-scFv or Sotorasib. The experiments were repeated 3 times. Data are shown as means ± SD. P values were calculated with two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant

Article Snippet: For the experiment, primary antibodies specifically targeting ZNF24, SLC7A5, β-actin, and PD-L1 were obtained from Proteintech (11219-1-AP, USA, diluted at 1:1000), Santa Cruz (sc-374232, USA, diluted at 1:500), ZSGB-BIO (TA-09, China, diluted at 1:1000), and Abcam (ab82458, England, diluted at 1:1000) respectively.

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Transfection, Two Tailed Test

Journal: BMC Cancer

Article Title: KRAS mutation promotes immune escape of lung adenocarcinoma via ZNF24/SLC7A5/PD-L1 axis

doi: 10.1186/s12885-025-14336-0

Figure Lengend Snippet: The KRAS mutation upregulates PD-L1 expression through the ZNF24/SLC7A5 axis. A - D . WB was used to detect the expression of ZNF24, SLC7A5 and PD-L1 after inhibiting ZNF24 with shRNA in NSCLC cells with KRAS mutation. The protein expression levels of SLC7A5 and PD-L1 were decreased after inhibiting ZNF24. Overexpression of SLC7A5 partially reversed the decrease of PD-L1 caused by shZNF24, but had no effect on ZNF24. E - F . RT-qPCR was used to detect the expression of mRNA. The mRNA of PD-L1 was reduced after shZNF24 treatment. But shZNF24 had no significant effect on the mRNA expression of SLC7A5. Overexpression of SLC7A5 partially reversed the decrease of PD-L1 caused by shZNF24, but had no effect on ZNF24. The experiments were repeated 3 times. Data are shown as means ± SD. P values were calculated with two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant

Article Snippet: For the experiment, primary antibodies specifically targeting ZNF24, SLC7A5, β-actin, and PD-L1 were obtained from Proteintech (11219-1-AP, USA, diluted at 1:1000), Santa Cruz (sc-374232, USA, diluted at 1:500), ZSGB-BIO (TA-09, China, diluted at 1:1000), and Abcam (ab82458, England, diluted at 1:1000) respectively.

Techniques: Mutagenesis, Expressing, shRNA, Over Expression, Quantitative RT-PCR, Two Tailed Test

Journal: BMC Cancer

Article Title: KRAS mutation promotes immune escape of lung adenocarcinoma via ZNF24/SLC7A5/PD-L1 axis

doi: 10.1186/s12885-025-14336-0

Figure Lengend Snippet: DAPT can bind and inactivate ZNF24 by Molecular docking and SPR. A . DAPT could bind to ZNF24 through Autodock software. B - C . Affinity determination curve and fitting curve of ZNF24 protein with DAPT (3.906–62.5 µM). DAPT could specifically bind to ZNF24 within the concentration range of 3.906–62.5 µM. DAPT bound to the ZNF24 protein with KD values of 1.48 × 10 − 4 M. D - G . WB demonstrated that DAPT could downregulate the protein expression of SLC7A5 and PD-L1 in NSCLC cells with KRAS mutation compared to DMSO or PBS group. H - I . RT-qPCR displayed that DAPT downregulated the mRNA expression of PD-L1, but had no effect on the mRNA of SLC7A5 in NSCLC cells with KRAS mutation compared to DMSO or PBS group. The experiments were repeated 3 times. Data are shown as means ± SD. P values were calculated with two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant

Article Snippet: For the experiment, primary antibodies specifically targeting ZNF24, SLC7A5, β-actin, and PD-L1 were obtained from Proteintech (11219-1-AP, USA, diluted at 1:1000), Santa Cruz (sc-374232, USA, diluted at 1:500), ZSGB-BIO (TA-09, China, diluted at 1:1000), and Abcam (ab82458, England, diluted at 1:1000) respectively.

Techniques: Software, Concentration Assay, Expressing, Mutagenesis, Quantitative RT-PCR, Two Tailed Test

Journal: BMC Cancer

Article Title: KRAS mutation promotes immune escape of lung adenocarcinoma via ZNF24/SLC7A5/PD-L1 axis

doi: 10.1186/s12885-025-14336-0

Figure Lengend Snippet: Inhibition of KRAS/ZNF24/SLC7A5 axis of tumor cells by co-culturing with CD8 + T cells can reduce PD-L1 expression. A - D . WB showed that inhibiting KRAS or ZNF24 of tumor cells by co-culturing with CD8 + T cells reduced the protein expression of PD-L1, and overexpression of SLC7A5 partially reversed the downregulation of PD-L1 caused by inhibiting ZNF24. E - F . RT-qPCR demonstrated that inhibiting KRAS or ZNF24 of tumor cells by co-culturing with CD8 + T cells decreased the mRNA levels of PD-L1, and overexpression of SLC7A5 partially reversed the mRNA downregulation of PD-L1 caused by inhibiting ZNF24. G . Immunofluorescence showed that inhibition of KRAS or ZNF24 of tumor cells by co-culturing with CD8 + T cells resulted in a decrease in the number of PD-L1 positive cells. Overexpression of SLC7A5 can partially reverse the number downregulation of PD-L1 positive cells caused by inhibition of ZNF24. The experiments were repeated 3 times. Data are shown as means ± SD. P values were calculated with two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant

Article Snippet: For the experiment, primary antibodies specifically targeting ZNF24, SLC7A5, β-actin, and PD-L1 were obtained from Proteintech (11219-1-AP, USA, diluted at 1:1000), Santa Cruz (sc-374232, USA, diluted at 1:500), ZSGB-BIO (TA-09, China, diluted at 1:1000), and Abcam (ab82458, England, diluted at 1:1000) respectively.

Techniques: Inhibition, Expressing, Over Expression, Quantitative RT-PCR, Immunofluorescence, Two Tailed Test

Journal: BMC Cancer

Article Title: KRAS mutation promotes immune escape of lung adenocarcinoma via ZNF24/SLC7A5/PD-L1 axis

doi: 10.1186/s12885-025-14336-0

Figure Lengend Snippet: Inhibiting the KRAS/ZNF24/SLC7A5 axis of tumor cells can increase the number and activity of CD8 + T cells in vitro. A - B . LDH experiments were utilized to evaluate the activity of CD8 + T cells. Inhibiting KRAS or ZNF24 could increase the activity of CD8 + T cells, which partially was reversed by over-expression of SLC7A5. C - D . The proliferation ability of tumor cells was tested through CCK-8. Inhibiting KRAS or ZNF24 reduced the proliferation ability of tumor cells. However, the proliferation ability of tumor cells in the shZNF24 + oveSLC7A5 group was increased respectively, compared to the ZNF24 group. E . The proportion of CD8 + T cells was evaluated by flow cytometry. The number of CD8 + T cells was increased by repressing KRAS or ZNF24 of tumor cells, which was partially reversed by over-expression of SLC7A5. F . The proportion of CD8 + PD-1 + T cells was evaluated by flow cytometry. The number of CD8 + PD-1 + T cells was decreased by repressing KRAS or ZNF24 of tumor cells, which was partially reversed by over-expression of SLC7A5. The experiments were repeated 3 times. Data are shown as means ± SD. P values were calculated with two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant

Article Snippet: For the experiment, primary antibodies specifically targeting ZNF24, SLC7A5, β-actin, and PD-L1 were obtained from Proteintech (11219-1-AP, USA, diluted at 1:1000), Santa Cruz (sc-374232, USA, diluted at 1:500), ZSGB-BIO (TA-09, China, diluted at 1:1000), and Abcam (ab82458, England, diluted at 1:1000) respectively.

Techniques: Activity Assay, In Vitro, Over Expression, CCK-8 Assay, Flow Cytometry, Two Tailed Test

Journal: BMC Cancer

Article Title: KRAS mutation promotes immune escape of lung adenocarcinoma via ZNF24/SLC7A5/PD-L1 axis

doi: 10.1186/s12885-025-14336-0

Figure Lengend Snippet: Inhibiting the KRAS/ZNF24/SLC7A5 axis can inhibit the growth of transplanted tumors and promote tumor cells apoptosis. A . The model of C57BL/6 mouse transplantation tumor was established by using LLC cells. B - D . Compared with the PBS group, inhibiting KRAS (by RGD-KGH-R1-scFv) or ZNF24 (by DAPT) repressed the growth of transplanted tumors, and the final tumor weight was also significantly lower than that of the PBS group. E - F . Histological morphology was observed via HE. Spotty necrosis was seen in inhibiting KRAS or ZNF24 group. But in the PBS group, extensive necrosis was observed (Bottom right corner). The mitotic figures in the PBS group were significantly higher than those in inhibiting KRAS or ZNF24 group. G - H . IHC was used to detect the number of Ki-67 + and CD34 + cells. The results showed that the number of positive cells in the KRAS or ZNF24 inhibition group was significantly lower than that in the PBS. The experiments were repeated 3 times. Data are shown as means ± SD. P values were calculated with two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant

Article Snippet: For the experiment, primary antibodies specifically targeting ZNF24, SLC7A5, β-actin, and PD-L1 were obtained from Proteintech (11219-1-AP, USA, diluted at 1:1000), Santa Cruz (sc-374232, USA, diluted at 1:500), ZSGB-BIO (TA-09, China, diluted at 1:1000), and Abcam (ab82458, England, diluted at 1:1000) respectively.

Techniques: Transplantation Assay, Inhibition, Two Tailed Test

Journal: BMC Cancer

Article Title: KRAS mutation promotes immune escape of lung adenocarcinoma via ZNF24/SLC7A5/PD-L1 axis

doi: 10.1186/s12885-025-14336-0

Figure Lengend Snippet: Inhibiting the KRAS/ZNF24/SLC7A5 axis can increase the number of CD8 positive cells and reduce the number of PD-L1 positive tumor cells. IHC was used to detect the relative markers of immune escape. The number of PD-L1 + and PD-1 + cells in inhibiting KRAS or ZNF24 group was smaller than the PBS group. Inhibiting KRAS or ZNF24 group has more CD8 + T cells than PBS group. However, the number of CD4 + cells was no significant difference between the three group. At the same time, the expression of CD34 and Ki-67 was lower in the DAPT and RGD-KGH-R1-scFv group compared to the PBS group. The experiments were repeated 3 times. Data are shown as means ± SD. P values were calculated with two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: For the experiment, primary antibodies specifically targeting ZNF24, SLC7A5, β-actin, and PD-L1 were obtained from Proteintech (11219-1-AP, USA, diluted at 1:1000), Santa Cruz (sc-374232, USA, diluted at 1:500), ZSGB-BIO (TA-09, China, diluted at 1:1000), and Abcam (ab82458, England, diluted at 1:1000) respectively.

Techniques: Expressing, Two Tailed Test

Journal: BMC Cancer

Article Title: KRAS mutation promotes immune escape of lung adenocarcinoma via ZNF24/SLC7A5/PD-L1 axis

doi: 10.1186/s12885-025-14336-0

Figure Lengend Snippet: Schematic diagram of the mechanism in KRAS mutation promoted immune escape of lung adenocarcinoma via ZNF24/SLC7A5/PD-L1 axis. KRAS mutation upregulated PD-L1 expression through the MEK/ERK/ZNF24/SLC7A5 pathway. ZNF24 could upregulate the level of PD-L1 through SLC7A5. ZNF24 and SLC7A5 promoted PD-L1 expression in the form of protein complexes. DAPT and RGD-KGH-R1-scFv reduced PD-L1 expression induced by KRAS mutation and reprogram immunosuppressive tumor microenvironment to enhance CD8 + T cell activity through inhibiting ZNF24 or KRAS respectively

Article Snippet: For the experiment, primary antibodies specifically targeting ZNF24, SLC7A5, β-actin, and PD-L1 were obtained from Proteintech (11219-1-AP, USA, diluted at 1:1000), Santa Cruz (sc-374232, USA, diluted at 1:500), ZSGB-BIO (TA-09, China, diluted at 1:1000), and Abcam (ab82458, England, diluted at 1:1000) respectively.

Techniques: Mutagenesis, Expressing, Activity Assay