Fig. 4

Circ_001680 binds to miR-340 to suppress the expression of BMI1. a Construction of BMI1 3′-UTR WT and mutant (Mut) luciferase reporter vectors. b The dual-luciferase reporter assays indicated that luciferase activity was decreased in CRC cells after cotransfection with the BMI1–3′-UTR-WT and miR-340 mimics. c The whole-cell lysates from SW480 and HCT116 cells were incubated with biotinylated probes against BMI1 or NC; after pull-down, endogenous BMI1 and miR-340 enrichments were detected by qRT-PCR. Results were presented as the percentage of pull-down to input. d The complex containing miR-340 and BMI1 in SW480 and HCT116 cells was immunoprecipitated by by anti-Ago2 using RIP (RNA immunoprecipitation) and followed by qRT-PCR analyses to detect miR-340 and BMI1 enrichment. All tests were carried out in triplicate. e, f qRT-PCR analysis of BMI1 in the indicated cells. g, h Protein expression of BMI1 in the indicated cells as determined by Western blotting. α-tubulin was used as a loading control. The protein expression levels were quantified by comparing the gray level of each band using Quantity One Software. i qRT-PCR analysis of circ_001680, miR-340 and BMI1 expression in the 25 fresh human CRC samples. j Spearman correlation analyses between BMI1 and circ_001680 expression (left), as well as BMI1 and miR-340 (right) expression in 25 fresh human CRC samples (p < 0.01).