Fig. 3

Circ_001680 targets miR-340 and inhibits its transcriptional regulatory activity. a Graphical illustration showing the predicted position of the circ_001680 target on the miR-340 sequence. b Luciferase activity assay showing that circ_001680 influences the luciferase activity of miR-340 3’UTRs in SW480 and HCT116 cells. c qRT-PCR analysis of miR-340 in the indicated lentivirus-infected cells. d qRT-PCR analysis of circ_001680 and miR-340 expression in 43 human colorectal cancer tissues (left); correlation analysis showing that the expression of circ_001680 is negatively correlated with miR-340 (right). e The co-localization between circ_001680 (red) and miR-340 (green) was observed (arrowheads) by fluorescence in situ hybridization in SW480 and HCT116 cells. Cell nuclei were counterstained with DAPI (blue). Scale bar, 5 μm. f According to the RNA pull-down flowchart (left), whole-cell lysates from SW480 and HCT116 cells were incubated with biotinylated probes against circ_001680 or NC; after pull-down, endogenous circ_001680 and miR-340 enrichments were detected by qRT-PCR. Results were presented as the percentage of pull-down to input. g According to the flowchart outlining the experimental procedures (left), The complex containing circ_001680 and miR-340 in indicated cells was immunoprecipitated by anti-Ago2 using RIP (RNA immunoprecipitation) assay and followed by qRT-PCR analyses to detect circ_001680 and miR-340 enrichment (right). h, i Overexpression of miR-340 inhibits the cell growth and migration capacities of SW480 cells induced by circ_001680, as determined by a colony formation assay (h) and a transwell assay (i) in SW480 cells. j Image of the subcutaneous tumor mode. SW480-vector, SW480-circ_001680, SW480-circ_001680 inhibitor and SW480-circ_001680 + miR-340 cells were subcutaneously injected into nude mice separately (n = 6/group). k The tumor volume among the four indicated groups at the indicated time points. l H&E (upper) and Ki-67 immunohistochemistry (IHC, lower) assays were adapted to detect the tumor section. m Statistical analysis of Ki-67-positive cells. All tests were carried out in triplicate. The error bars represent the means ± SDs from three independent experiments. *p < 0.05**, p < 0.01, ***p < 0.005