Fig 2: 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.

Fig 3: Validation of the expression of B7-H3 and STAT1 in meningiomas. (A) The expression of B7-H3 is displayed in different subtypes of meningiomas. The bar chart shows the relative density of B7-H3. The X axis marks the subtypes, and the Y axis represents the relative density; **p < 0.01, ***p < 0.001. (B) The expression of STAT1, phospho-STAT1 (Tyr701) and B7-H3 is displayed at different concentrations of LB-100 (10, 20, 40, and 80 uM). Two bar charts show the phosphorylation of STAT1 (Tyr701) and the relative density of B7-H3. The X axis marks different concentrations of LB-100, and the Y axis represents the relative density; *p < 0.05, ****p < 0.0001.

Fig 4: B7-H3-retargetedCAR-T cells suppressed chordoma tumor sphere formation. (A) Representative images of FACS examination of B7-H3 expression on primary chordoma cells isolated from two different clinical samples. (B) Representative images of B7-H3-retargeted CAR-T cells inhibited the tumor sphere formation of chordoma. Scale bars: 100 µm. **p < 0.01.

Fig 5: TNF-α-induced epithelial-to-mesenchymal transition regulates expression of immune checkpoint molecules in Hep3B cells. Reverse transcription-quantitative PCR analysis demonstrated (A) higher expression of PD-L1, PD-L2, CD73 and B7-H3, and (B) lower expression of VTCN1 and VISTA upon treatment with 20 ng/ml TNF-α for 72 h. n=3. *P<0.05, **P<0.01, ****P<0.001. (C) Western blot analysis demonstrated upregulation of PD-L1 and CD73 in Hep3B cells upon treatment with 20 ng/ml TNF-α for 72 h. n=3. *P<0.05, ****P<0.001 vs. control. GAPDH was used as the loading control. TNF-α, tumor necrosis factor-α; VTCN1, V-set domain-containing T-cell activation inhibitor 1; VISTA, V-domain immunoglobulin suppressor of T-cell activation; PD-L, programmed death receptor ligand.