Fig. 6: HuR Inhibitory peptide suppresses cervical cancer progression in vitro and in vivo by blocking circTICRR-HuR interaction.

A The schematic domain structure of HuR. B In vitro RNA pull-down assay depicting the circTICRR-HuR interaction from HeLa cells validated by western blot after incubation with full-length, or truncated3XFlag-tagged recombinant HuR protein. C Upper panel: the schematic structure of the SLGY (60-63 aa), F247/Y249, and F287/F289 residues of the HuR protein crucial for interacting with circTICRR. Lower panel: the inhibitory peptides sequence containing the core amino acids of HuR. D Biotin-labeled peptide pull-down assay revealing the interaction of Control peptide (CTL) (30 μmol/L) or HIP (30 μmol/L) with circTICRR in HeLa cells. E In vitro RNA pull-down assay depicting the circTICRR/HuR interaction from HeLa cells validated by western blot after incubation with full-length, or mutant F287A/F289A of 3XFlag-tagged recombinant HuR protein. F Confocal images showing the distribution of synthesized inhibitory peptide (HIP-3: 30 μmol/L) or control peptide (CTL-3: 30 μmol/L) after incubation with SiHa and CaSki cells for 48 h. The nuclei were stained with DAPI (blue), while the membrane was stained with DiI (red). Scale bar, 20 μm. G Protein pulled down by control oligo or circTICRR probe with HuR antibody was detected by western blot. Cell viability (H) was determined by CCK-8 assay, cell apoptosis (I) was measured by Flow Cytometry in the SiHa and CaSki cells treated with HIP-3 or CTL-3. J SiHa and CaSki cells treated with HIP-3 or CTL-3 for 72 h were incubated with the autophagy inhibitor CQ or Baf-A1 for another 6 h. Then the autophagy-related protein LC3B I/II was monitored by Western blotting. K SiHa and CaSki cells transfected with mRFP-GFP-LC3B for 24 h were incubated with HIP-3 or CTL-3 for another 48 h. The images were captured by confocal microscopy. Scale bar, 20 μm. Images of tumors L, tumor volume and weight M, images of H&E and apoptotic cells (N) in the xenografts treated with HIP-3 or CTL-3 after collection of the tumors (n = 5 for each group). Scale bar, 100 μm. Data are representative of at least three independent experiments and presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.