Fig 2: Blockade of PD-L1 and B7-H3 markedly abrogates the aggressive tumor behavior caused by miR-326 downregulation.A–F T cells were co-cultured with miR-NC- or miR-326-overexpressed H1975 cells, and PD-L1/B7-H3 blocking antibody was added. Cytokines in the supernatant were measured by ELISA (n = 3). G TNF-a, IFN-?, IL-10, and IL-1ß expression of CD8+ T cells after miR-326 overexpression and/or PD-L1/B7-H3 antibody incubation by flow cytometry (n = 3). H Transwell assay of control and miR-326-overexpressed cells, and PD-L1/B7-H3 blocking antibody was added. I, J Histogram of the number of migrated cells (n = 3). All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way (I, J) or two-way (A–G) ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 versus NC inhibitor. ##P < 0.01, ###P < 0.001 versus miR-326 inhibitor. Scale bar, 200 µM.

Fig 3: Overexpression of miR-326 enhances TNF-a and IFN-? expression of infiltrated CD8+ T cells and prevents metastasis by targeting PD-L1 and B7-H3 on cancer cells.5 × 105 miR-326 overexpressed LLC cells or empty vector control LLC cells were subcutaneously injected into C57BL/6 mice. MiR-326 antagomir or NC antagomir were intratumor injected when the tumor diameter reached 0.5 cm. The mice were sacrificed 28 days after inoculation. A Tumor volume (n = 7). B Tumor weight (n = 6). C Pulmonary metastasis nodule calculated by hematoxylin–eosin staining. (n = 6) D, E PD-L1, B7-H3 expression, and CD8+ T cells infiltration were determined by IHC staining in tumor tissues. F–I PD-L1, B7-H3, TNF-a, and IFN-? were detected by qRT-PCR in tumor sites (n = 3). J TNF-a and IFN-? expression of infiltrated CD8+ T cells by flow cytometry (n = 3). All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way (B, C and F–I) or two-way (A) ANOVA with Tukey’s post hoc test or Student’s t test (J). *P < 0.05, ***P < 0.001 versus LV-NC. #P < 0.05, ##P < 0.01, ###P < 0.001 versus LV-miR-326. Scale bar, 200 µM.

Fig 4: B7-H3 interacts with FN. (A) TCGA database analysis of B7-H3 and the correlation coefficient of FN’s two genes. (B, C), The interaction of B7-H3 with FN in 786-O and ACHN cells detected by co-IP assay. (D, E) The expression of FN in the B7-H3-KO and control 786-O and ACHN cells assayed by western blotting. (F) The expression of FN in the B7-H3-KO and control ACHN cells isolated from immunodeficient mice as assayed by immunohistochemical staining. After 6 weeks of injection, tumor tissues were collected and immunohistochemical methods were used to detect FN expression levels in tumor tissues. Values are expressed as means ± SD (t-tests, n = 3). *P < 0.05, **P < 0.01 B7-H3, control group vs. B7-H3-KO group. Scale bars: 25 µm (F).

Fig 5: PD-L1, ICOSLG, and B7-H3 are the direct target genes of miR-326.A Software (miRBase, PicTar, and TargetScan) prediction of the miR-326-targeted genes. B–D 293T cells were transfected with luciferase reporter plasmid containing PD-L1/B7-H3/ICOSLG wild-type or mutant-type 3' UTR and miR-NC (n = 3). E Schematic diagram of the binding sites between miR-326 and the target genes (PD-L1, B7-H3, and ICOSLG) and their mutant sequence. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with two-way ANOVA with Tukey’s post hoc test (B–D). ***P < 0.001 versus 3' UTR + miR-NC. ###P < 0.001 versus 3' UTR + miR-326.