Fig 2: CircMTO1/miR-541-5p/ZIC1 axis inhibited HCC tumor growth in vivo.A Nude mice were subcutaneously injected with (a.) PBS, (b.) cholesterol-conjugated negative control (NC), (c.) cholesterol-conjugated si-circMTO1, (d.) cholesterol-conjugated inhibitor (e.) cholesterol-conjugated si-circMTO1 + cholesterol-conjugated inhibitor. After five injections, tumors were dissected and imaged. B Before injection, the tumor volume was measured and the tumor growth curve plotted. C Tumor weight was calculated on the day the mice were killed. Data represents mean ± SD (n = 5 per group). D–F Expression levels of circMTO1, miR-541-5p, and ZIC1 in xenograft tumors were measured using RT-qPCR. G H&E staining revealed the structure of xenograft tumors. Scale bar; 40 µm. H Changes in ZIC1, ß-catenin, c-myc, cyclin D1, E-cadherin, N-cadherin, Vimentin, and MMP2 expression in xenograft tumors were detected through IHC staining. Scale bar; 20 µm. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Isolation and identification of brown pre-adipocytes from interscapular brown adipose tissue (iBAT). (A) Schematic of procedure for the isolation, culture and differentiation of interscapular brown adipocytes (iBACs). The hallmarks (UCP1 and ZIC1) of iBACs were detected by immunohistochemistry (IHC) and immunofluorescence (IF) to detect the purity of the cells. Focusing on the induction period, we improved the components and the duration of induction time. (B) Hematoxylin and eosin staining of fetal interscapular adipose tissue (upper panel) revealed the presence of some dispersed cells with a multilocular aspect, characteristic of brown adipocytes. IHC staining of iBAT samples confirmed the positive result for the brown fat marker UCP1 (lower panel). Scale bar, 50 µm (left), 25 µm (right). (C) IF staining of ZIC1 on BACs before and after differentiation confirmed the purity of BACs. Scale bar, 50 µm.

Fig 4: CircMTO1 inhibited the malignant progression of HCC via miR-541-5p/ZIC1 by regulating Wnt/ß-catenin signaling and EMT.A–C SMMC-7721 and HepG2 cells were transfected with miR-541-5p mimics or miR-541-5p inhibitor. A-C ß-catenin, cyclin D1, c-myc, E-cadherin, N-cadherin, Vimentin, and MMP2 mRNA and protein expression were detected through RT-qPCR and western blot, respectively. D–F SMMC-7721 and HepG2 cells were transfected with si-circMTO1 or co-transfected with si-circMTO1 and miR-541-5p inhibitor D–F, ß-catenin, cyclin D1, c-myc, E-cadherin, N-cadherin, Vimentin, and MMP2 mRNA and protein expression were detected through RT-qPCR and western blot, respectively. Data were presented as mean ± SD of three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Modulation of ZIC1 and the effect on the indicated downstream mediators of Wnt/ß-catenin signaling and EMT.A–D SMMC-7721 and HepG2 cells were transfected with ZIC1 siRNAs or ZIC1-overexpressing plasmid (OE-ZIC1). A, B Analysis of ZIC1 expression via RT-qPCR. C, D Analysis of ZIC1 expression via western blot. Vector: the empty pcDNA3.1(+) plasmid. E, F ß-catenin, cyclin D1, c-myc, E-cadherin, N-cadherin, Vimentin, and MMP2 mRNA expression were detected via RT-qPCR after transfection with si-ZIC1. G, H ß-catenin, cyclin D1, c-myc, E-cadherin, N-cadherin, Vimentin, and MMP2 mRNA expression were detected via RT-qPCR after transfection with OE-ZIC1. Data were presented as mean ± SD of three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001.