Fig. 4: Circ235 functions as a sponge for miR-330-5p in Bca cells.

A RIP was carried out to assess circ235 pulled down by anti-AGO2 antibody or IgG in T24 cells. B Possible miRNAs binding to circ235 were predicted using CircInteractome, and top scores are listed in the diagram C, D The luciferase activity of 293T or T24 cells transfected with wild-type mimics, or controls, was investigated using a dual-luciferase reporter assay employing a psiCHECK-2 vector. The hRluc/fluc ratio was calculated to indicate luciferase activity. E Transfected T24 cells with the aforementioned mimics or controls were assessed for the capacity to migrate using a transwell assay. Scale bar = 25 μm. F Statistical diagram of the migratory capabilities of T24 cells that underwent transfection with the aforementioned mimics or controls in the transwell assay. G Circ235 and miR-330-5p were predominantly co-localized in Bca cells’ cytoplasm based on RNA FISH. Scale bar = 10 μm. H, I Biotin-labeled probes for wild-type or mutant miR-330-5p were applied to pull down RNA from Bca T24 or UMUC3 cell lines, and their relative input expression was detected by qRT-PCR with β-actin serving as the control. J The schematic illustrates the luciferase reporter vectors of circ-CCNY in both wild-type and mutant forms, revealing the presence of binding sites that are complementary to miR-330-5p within the circ-CCNY structure. K To determine the relative activity levels, luciferase activity intensity was measured in cells co-transfected with either miR-330-5p or NC mimics and the luciferase reporter vectors of circ235 wild or mutant type. Representative images (L) and statistical diagrams (N) of colony formation assays were implemented using three sets of T24 or UMUC3 cells (sh-NC, sh-circ235, and sh-circ235+miR-330-5p inhibitor). The addition of a miR-330-5p inhibitor partially rescued the proliferation of Bca cells infected with circ235-shRNA using lentiviral tools. Representative images (M) and statistical diagrams (O) of transwell tests were used to evaluate migratory capabilities of three sets of T24 and UMUC3 cells (sh-NC, sh-circ235, and sh-circ235+miR-330-5p inhibitor). The addition of a miR-330-5p inhibitor still partially rescued the migration of Bca cells infected with circ235-shRNA using lentiviral tools. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.