Fig 2: RPN2 is a direct target of miR-422a. (A) A schematic diagram of the seed sequence of miR-422a that matches the RPN2 3′UTR and the design of wild-type or mutant RPN2 3′UTR constructs including reporters. (B) Changes in RPN2 expression in LN229 and U87 cells transfected with miR-NC or miR-422a mimic were detected by western blotting. GAPDH was used as a loading control. The relative protein quantification was performed with ImageJ software. **P<0.01 vs. miR-NC. (C) Luciferase reporter assays with LN229 and U87 cells following co-transfection with the WT or MT RPN2 3′UTR of RPN2 and miR-422a mimic or miR-NC. **P<0.01 vs. miR-NC. (D) A RIP assay was conducted to detect the enrichment level of RPN2 in IgG or Ago2 immunoprecipitation complexes in LN229 and U87 cells transfected with miR-422a or miR-NC. **P<0.01 vs. miR-NC. (E) RPN2 expression levels in five normal controls and 29 GBM patients. **P<0.01 vs. Normal. RPN2, ribophorin II; miR-422a, microRNA-422a; 3′UTR, 3′-untranslated region; NC, negative control; WT, wild-type; MT, mutant; RIP, RNA immunoprecipitation.

Fig 3: WEE2-AS1 stabilizes RPN2 protein by preventing CUL2-mediated ubiquitin-proteasome degradation. A qRT-PCR assays showing the relative expression in GBM cells transfected with sh-NC or sh-WEE2-AS1. Data represent the mean ± SD from at least three independent experiments. B Western blot assays showing RPN2 protein levels in GBM cells transfected with sh-NC or sh-WEE2-AS1. Western blot assays showing RPN2 protein level in GBM cells (C) transfected with ov-NC or ov-WEE2-AS1, treated with the protein synthesis inhibitor cycloheximide (CHX, 25 μg/mL) at the indicated time, and (D) transfected with sh-NC or sh-WEE2-AS1, treated with the proteasome inhibitor MG132 (5 μmol/L) at the indicated time. E Co-IP assays showing the RPN2 ubiquitination levels in GBM cells transfected with sh-NC or sh-WEE2-AS1. UB, ubiquitination. F Top, the potential ubiquitination sites of RPN2 predicted via BDM-PUM (http://bdmpub.biocuckoo.org/) and the UbiBrowser database (http://ubibrowser.ncpsb.org/). Bottom, Co-IP assays showing the RPN2 ubiquitination level after transfection with Flag-tagged wild-type or mutant RPN2 KR vectors. KR, mutation of lysine (K) to arginine G Co-IP assays showing the RPN2 ubiquitination level in GBM cells co-transfected with sh-NC or sh-WEE2-AS1 and Flag-tagged wild-type or K322 mutant RPN2 KR vectors. H Top, Crystal structure of RPN2 proteins with K322. Bottom, conservation ability of the K322 ub site on the RPN2 protein. I Silver staining assays showing the proteins that interacted with RPN2, which were identified by co-IP/mass spectrometry; arrows indicate CUL2 protein bands. J Immunofluorescence staining experiments showing the colocalization of RPN2 and CUL2 in GBM cells. Scale bar, 25 μm. K Western blot assays showing CUL2 and RPN2 protein levels in GBM cells transfected with si-NC or si-CUL2. L Co-IP assays showing the RPN2 ubiquitination levels in GBM cells transfected with si-NC or si-CUL2. M Co-IP assays showing the intensity of the interaction between RPN2 and CUL2 in GBM cells transfected with ov-NC or ov-WEE2-AS1 and treated with MG132 at the indicated times. The statistical significance is shown as follows: ns>0.05. N Western blot assays showing CUL2 and RPN2 protein levels in GBM cells transfected with sh-NC or sh-WEE2-AS1.

Fig 4: WEE2-AS1 interacts with RPN2 protein and contributes to GBM malignant progression by activating the PI3K-AKT signaling pathway. A GSVA showing differences in hallmark biological pathways between the high and low RPN2 samples in the TCGA GBM cohort. Scatter plots were used to visualize these differences in pathways. The size of the circle indicates the size of the fold change (FC), and the color indicates the statistical significance of the difference. The red color indicates statistical significance, and blue indicates statistical insignificance. B Correlations between RPN2 and the enrichment scores of cancer hallmark pathways in the TCGA GBM cohort. C GSEA showing that classical carcinogenic pathways involved in tumor pathogenesis were significantly enriched in the high RPN2 expression group in the TCGA GBM dataset. D Western blot analysis showing the interaction between WEE2-AS1 and RPN2 in (top) LN229 GBM cells and (bottom) GSC20 GSCs. E RIP-qPCR assays showing the relative enrichment of WEE2-AS1 detected by RPN2 antibody. Data represent the mean ± SD from at least three independent experiments. F RNA FISH-immunofluorescence (FISH-IF) assays showing the colocalization of WEE2-AS1 and RPN2 in GBM cells. G Secondary structure of WEE2-AS1 predicted by the RNAfold WebServer. H RNA pull-down assay by in vitro transcribed biotinylated RNAs corresponding to different fragments of WEE2-AS1 in LN229 and GSC20 cells. Western blot assays showing the phosphorylation levels of AKT in LN229 GBM cells transfected with (I) sh-NC or sh-WEE2-AS and (J) si-NC or si-RPN2. K CCK-8 assays showing the proliferation ability of GBM cells cotransfected with ov-NC or ov-WEE2-AS1 and siRPN2 as indicated. L Representative tumor sphere formation images of GSCs cotransfected with ov-NC or ov-WEE2-AS1 and si-RPN2 as indicated; scale bar, 200 µm. The quantification histogram represents the average sphere diameter. Data represent the mean ± SD from at least three independent experiments. M Representative Transwell migration and invasion assays showing the migration and invasion ability of GBM cells cotransfected with ov-NC or ov-WEE2-AS1 and siRPN2 as indicated; scale bar, 200 µm. The quantification histogram represents the relative cell numbers. Data represent the mean ± SD from at least three independent experiments. N Western blot assays showing E-cadherin, N-cadherin, CD44, RPN2, AKT and p-AKT protein levels in GBM cells cotransfected with ov-NC or ov-WEE2-AS1 and siRPN2 as indicated. The statistical significance is shown as *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: RPN2 is a target of miR-378e. (a) StarBase database predicted the complementary sequences between miR-378e and RPN2. (b-e) The target association between miR-378e and RPN2 in T98 and U251 cells was validated by luciferase reporter assay and RIP assay. **P < 0.01, ***P < 0.001 compared with miR-NC group