Fig. 4: miR-361-5p inhibited the sonic hedgehog signaling pathway and stemness.

a Schematic illustration of the target miRNA candidate screening process. b, c Expression of miR-361-5p was quantified by real-time PCR in HT-29 (b) and HCT 116 cells (c). The cells were co-cultured with F. nucleatum or treated with 1 mM metformin for 24 h; unpaired t test. d The predicted binding sequences for miR-361-5p within the human GLI1 3′ UTR. Seed sequences are highlighted. e Luciferase activity was measured in HCT 116 cells transfected with miR-316-5p mimics or control mimics, miR-316-5p inhibitors or control inhibitors for 72 h. The luciferase reporters expressing wild-type or mutant human GLI1 3′ UTRs were used; unpaired t test. f, g Real-time PCR (f) and western blotting (g) were performed in HT-29 cells to detect the expression levels of SHH, GLI1, and NANOG after transfection with miR-361-5p mimics or inhibitors for 48 h; unpaired t test. h, i Real-time PCR (h) and western blotting (i) were performed in HCT 116 cells to detect the expression levels of SHH, GLI1, and NANOG after transfection with miR-361-5p mimics or inhibitors for 48 h; unpaired t test. j, k Tumorspheres were observed by light microscopy in HT-29 (j) and HCT 116 (k) cells to detect the tumorsphere formation capability after transfection with miR-361-5p mimics; the number of the tumorspheres were quantified in the right panel. Scale bar, 400 μm; unpaired t test. n.s., P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.