Fig. 3

Circ0003998 functioned as a sponge for miR-143-3p. a FISH assay was used to detect the intracellular location of circ0003998; 18S and U6 were applied as positive controls in the cytoplasm and nucleus, respectively. Nuclei were stained with DAPI. b The intracellular location of circ0003998 was detected by separating the nuclear and cytoplasmic fractions of HCC cells and was assessed with nuclear control U6 and cytoplasmic control β-actin by qRT-PCR. c Anti-AGO2 RIP was performed in HepG2 cells. d RNA-pull down was performed in HepG2 cells using a circ0003998-specific probe and control probe, respectively; and the enrichment of miRNAs was detected by miRNA-seq. e FISH was performed to observe the co-localization between circ0003998 (red) and miR-143-3p (green) in HepG2 cells (magnification, × 400, scale bar, 25 μm). f-g The luciferase activities were detected in HepG2 cells and HEK293T cells after transfection with circ003998-WT or circ0003998-Mut and miR-143-3p mimics or miR-NC, respectively. h A schematic drawing showed the putative binding sites between miR-143-3p and circ0003998. i Relative expression of miR-143-3p in HCC tissues, PVTT tissues, and ANL tissues in cohort 1 (n = 25) were determined by qRT-PCR. j The relative expression of miR-143-3p was detected by qRT-PCR after over expression or silencing circ0003998 in HepG2 cells. k The relative expression of circ0003998 was detected by qRT-PCR after the transfection of miR-143-3p mimic or inhibitor. l-o Rescue transwell assays of migration and invasion were performed after transfection with indicated vectors, miR-143-3p mimic or inhibitors (magnification, × 100, scale bar, 100 μm). Data are presented as means ± SD; Student’s t-test was used. *p-value< 0.05, **p-value< 0.01, ***p-value< 0.001