Fig. 4

circHMGCS1–016 regulates the miR-1236-3p/GAL-8 and CD73 axis in the ICC cells. A. The overlapped differentiated proteins were shown. For differentially expressed proteins (left panel), the GO and KEGG analysis were performed; B. Western blot for CD73 and GAL-8 proteins in modified RBE and QBC939 cells; C. Putative binding site of miR-1236-3p with respect to GAL-8 and CD73 via StarBase v3.0. D. The luciferase activity of pLG3-CD73 or GAL-8 in the 293 T cells co-transfected with miR-1236-3p. Data are representative of 3 independent tests (** p < 0.01, *** p < 0.001, n.s. p > 0.05); E. The levels of CD73 or GAL-8 proteins were determined by western blot in the ICC cells with different miR-1236-3p or circHMGCS1–016 expression; F. The level of GAL-8 in the supernatant from ICC cells with different miR-1236-3p or circHMGCS1–016 expression was determined; Data are representative of 3 independent tests (*** p < 0.001, n.s. p > 0.05); G. The level of sCD73 in the supernatant from ICC cells with different miR-1236-3p or circHMGCS1–016 expression was determined; Data are representative of 3 independent tests(*** p < 0.001); H. The level of adenosine concentration in the supernatant from ICC cells with different miR-1236-3p or circHMGCS1–016 expression was determined; Data are representative of 3 independent test; I and J. A co-culture showed the supernatant from ICC cells overexpressing circHMGCS1–016 inhibited the CD4⁺ and CD8⁺ T cell proliferation; Data are representative of 3 independent tests (*** p < 0.001, n.s. p > 0.05); K. Chemokine chips and ELISA were employed to determine the different chemokines in the supernatant between RBE-control, RBE-circHMGCS1–016 and RBE-shmiR-1236-3p groups