FIG 4.

circPIKfyve functions as a miRNA sponge of miR-21-3p. (A) Schematic illustration of circPIKfyve-wt and circPIKfyve-mut sequence cloned into pmirGLO luciferase reporter vectors. (B) The relative luciferase activities were detected in EPC after cotransfection with circPIKfyve-wt or circPIKfyve-mut and miR-21-3p mimic or NC. (C and D) The concentration gradient (C) and time gradient (D) experiments of miR-21-3p mimics were conducted. (E and F) circPIKfyve downregulated GFP expression. EPC were cotransfected with empty vector, circPIKfyve-wt or circPIKfyve-mut and miR-21-3p mimics, or NC. The fluorescence intensity and GFP expression were evaluated with an enzyme-labeling instrument and Western blotting, respectively. (G) The Ago2-RIP assay was executed in MIC after transfection with miR-21-3p mimics and NC, followed by qPCR to detect cirPIKfyve expression levels. (H and I) RNA pulldown assay was executed in MIC, followed by qPCR to detect the enrichment of miR-21-3p and circPIKfyve. (J) The MS2-RIP assay was executed in MIC after transfection with pcDNA3.1-MS2, pcDNA3.1-MS2-circPIKfyve, and pcDNA3.1-MS2-circPIKfyve-mut, followed by qPCR to detect the enrichment of miR-21-3p. All data represent the means ± SE from three independent triplicate experiments. *, P < 0.05; **, P < 0.01.