Fig 2: IGFBP3 is targeted by miR-515-5p. (A) The binding sites between IGFBP3 and miR-515-5p were predicted by starbase. (B) The interaction between IGFBP3 and miR-515-5p was confirmed by dual-luciferase reporter assay (n=3). (C and D) IGFBP3 mRNA and protein expression were detected by qRT-PCR and Western blot analyses, respectively, in 16HBE cells transfected with miR-NC, miR-515-5p, anti-NC, or anti-miR-515-5p (n=3). (E and F) 16HBE cells were divided into 6 groups: con, CSE, CSE + si-NC, CSE + si-circ_0040929, CSE + si-circ_0040929 + anti-NC, or CSE + si-circ_0040929 + anti-miR-515-5p. The mRNA and protein levels of IGFBP3 were determined by qRT-PCR and Western blot analyses, respectively (n=3). (G) IGFBP3 expression was examined by ELISA assay in serum samples of non-smoker (n=22), smokers (n=22), and COPD patients (n=22). *P<0.05, ***P<0.001.

Fig 3: Cre/loxP-mediated cd274/PD-L1 deletion in activated-HSC/myofibroblasts suppresses ICC growth in mice(A) SB murine ICC cells were implanted into the livers of PD-L1+/+Cre (control) and PD-L1F/FCre mice by portal vein injection. SB implantation led to smaller tumors in PD-L1F/FCre mice than in PD-L1+/+Cre mice. *p < 0.05 by t test, n = 6, 7.(B) Isolated SB ICC tumors were subjected to IF for aSMA. The average aSMA IF density was reduced in SB tumors of PD-L1F/FCre mice than in SB tumors of PD-L1+/+Cre mice. *p < 0.05 by t test, n = 4 tumors. Scale bar: 50 µm.(C) WB performed with tumor lysates revealed that the protein levels of aSMA, CTGF, IGFBP3, and thrombospondin-2 were all reduced in SB tumors of PD-L1F/FCre mice than in those of PD-L1+/+Cre mice. *p < 0.05, **p < 0.01 by t test, n = 5, 6.(D) SB tumors were subjected to double IF for aSMA and PD-L1. The average PD-L1 expression level was much lower in PD-L1F/FCre myofibroblasts (arrowheads) than in PD-L1+/+Cre myofibroblasts (arrows). ****p < 0.0001 by t test, n = 23, 17. Scale bar, 50 µm.(E) A schematic presentation of this study demonstrating two distinct mechanisms by which PD-L1 protects TßRI mRNA and TßRII protein of HSCs. Together, PD-L1 stabilizes TGF-ß receptors and promotes HSC myofibroblastic activation. PM, plasma membrane. All data are represented as mean ± SD.

Fig 4: KANK1 inhibits AKT signaling by upregulating IGFBP3 in osteosarcoma cells.a–d MNNG/HOS cells were infected with the indicated shRNAs for 72 h. Cells were used for RNA-seq analysis a, d. GO (b) and KEGG pathway enrichment analyses (c) were performed. e, f U-2OS, MG63, and MNNG/HOS cells were infected with the indicated shRNAs. After 72 h, cells were harvested for western blot (e) and qRT-PCR analysis (f). Data are presented as mean ± SD (n = 3). **P < 0.01; ***P < 0.001. g, h MG63 and MNNG/HOS cells were transfected with the indicated plasmids. After 48 h, cells were harvested for western blotting (g) and qRT-PCR analysis (h). Data are shown as mean ± SD (n = 3). **P < 0.01; ***P < 0.001. i Western blot analysis of MG63 and MNNG/HOS cells infected with the indicated shRNAs for 72 h. j Western blot analysis of MG63 and MNNG/HOS cells transfected with the indicated constructs for 72 h. k Western blot analysis of MG63 and MNNG/HOS cells infected with the indicated shRNAs for 72 h.

Fig 5: A schematic representation of TRAIP as a key prognostic DEG between CTCs and metastatic lesions in osteosarcoma.TRAIP targets KANK1 for degradation, activating the IGFBP3/AKT signaling pathway and promoting osteosarcoma cell proliferation and invasion.