Fig. 5

CircRYK acts as a miRNA sponge of miR-330-5p to upregulate VLDLR expression. (A-B) RIP and qRT‒PCR methods were used to assess the binding of circRYK to the AGO2 protein. (C) U87 cells that overexpress circRYK were mixed with a circRYK-specific probe and a negative control (NC) probe to perform the circRIP experiment. The possible miRNAs linked with circRYK were investigated using qRT‒PCR assays.(D) qRT‒PCR was used to confirm the copy numbers of miR-330-5p. (E) Potential binding site sequences between the wild-type (wt) and mutant-type (mut) variants of circRYK and miR-330-5p. (F) Experiments using dual-luciferase reporter assays were conducted to determine the association between circRYK and miR-330-5p. (G) With Pearson’s correlation, the relationship between the expression levels of circRYK and miR-330-5p was examined (r=-0.4013, p = 0.0039). (H) The colocalization of circRYK and miR-330-5p in transfected GBM cells was investigated using FISH. (I) qRT‒PCR was utilized to confirm the expression of miR-330-5p in treated GBM cells. (J) The biological targets of miR-330-5p may include two mRNAs, as shown by a Venn diagram. (K) Conceptual representation of the binding sites between the wild-type (wt) or mutant (mut) VLDLR 3’UTR and miR-330-5p. (L-M) A luciferase reporter assay in GBM cells was utilized to confirm the functional connection between miR-330-5p and VLDLR. Scale bar, 100 μm. Each experiment was performed thrice, and the results are displayed as the mean ± SD (***P < 0.001)