Fig. 8

CircRYK stabilizes VLDLR mRNA by interacting with HuR. (A) The motif of HuR. (B) RBPmap was used to predict the locations of HuR binding sites within the 3’UTR of the VLDLR gene. The vertical blue lines at the bottom display the HuR binding sites in the 3’UTR of VLDLR mRNA. (C) The HuR protein interacts with VLDLR mRNA, as shown by the RIP assay. (D-E) qRT‒PCR was utilized to measure the rate of VLDLR mRNA degradation in HuR-overexpressing or knockdown U87 cells at various time points. (F) Western blotting was utilized to determine the expression of the VLDLR protein in GBM cells transfected with si-NC, si-HuR, vector, or HuR overexpression plasmids. (G) RIP experiments revealed that HuR and VLDLR mRNA coprecipitated in U87 cells when circRYK was knocked down or overexpressed. (H) In GBM cells, RNA pulldown tests using the biotin-labelled VLDLR 3’UTR were carried out. The circRYK overexpression or shcircRYK plasmids were transfected into GBM cells. (I-J) Measurement of the rate at which VLDLR mRNA is degraded in U87 cells after transfection with different plasmids or short interfering RNAs. (K-L) The level of VLDLR in GBM cells was assessed after transfection with circRYK overexpression plasmids containing both wild-type and mutant variants. circ-mut-miR denoted a mutation in the miR-330-5p binding region, whereas circ-mut-Δ248-299 denoted a truncated version of the circRYK sequence from position 248 to 299. The term “circ-mut-(miR + Δ248–299)” was used to refer to both the mutant miRNA binding site and the truncated section of the sequence. (M-N) Transwell and clonogenicity assays were employed to verify GBM progression in U87 and U87-GSC cells under different treatment conditions. Scale bar, 100 μm. Each experiment was performed three times, and the results are displayed as the mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)