Hsa_circ_0058495-mediated IGF2BP2 ubiquitination and m6A modification of MEKK1 promote the progression of PDAC

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with dismal clinical outcomes. We identified hsa_circ_0058495 as significantly upregulated in PDAC tissues and PDAC cell-derived exosomes, where it contributes to tumor proliferation and invasion. The molecular mechanisms underlying its oncogenic function, however, remain incompletely understood. Methods: Differential circRNA expression was profiled by RNA sequencing. The functional role of hsa_circ_0058495 and its molecular interactions were interrogated through Western blotting, RT-qPCR, co-immunoprecipitation, RNA pull-down, and RNA immunoprecipitation assays. Confocal microscopy and PET/CT imaging were employed to delineate its biological effects in vitro and in vivo. Results: Hsa_circ_0058495 was enriched in PDAC-derived exosomes and stabilized IGF2BP2 by preventing TRIM25-mediated ubiquitination and attenuating autophagy-dependent degradation. Stabilized IGF2BP2 enhanced the stability of MEKK1 mRNA, leading to sustained ERK1/2 phosphorylation and consequent promotion of PDAC cell proliferation and invasion. Moreover, exosomal hsa_circ_0058495 facilitated M2 macrophage polarization, thereby fostering an immunosuppressive tumor microenvironment. Conclusions: Hsa_circ_0058495 promotes PDAC progression by stabilizing IGF2BP2 and activating the MEKK1-ERK signaling cascade, while exosomal transfer of hsa_circ_0058495 drives M2 macrophage polarization to reinforce tumor-associated immunosuppression. These findings establish hsa_circ_0058495 as a pivotal regulator of PDAC progression and underscore its potential utility as both a diagnostic biomarker and a therapeutic target.

Keywords: M2 polarization; PDAC; circRNA; m6A modification; ubiquitination.

Figure 1

Hsa_circ_0058495 is upregulated in PDAC. (A-B) Volcano plots of circRNAs differentially expressed in tumor and adjacent tissues in PDAC of GSE79634 and GSE69362 datasets. (C) Heatmap showing the levels of circRNAs in PDAC tumor and normal tissues derived exosomes detected by high-throughput sequencing. (D) Venn diagram showing the overlapping circRNAs in PDAC tissues and exosomes. (E-F) Results of RT-qPCR showing the level of hsa_circ_0058495 in HPDE6C7, PANC1, BxPC3, and SW1990 cells and cell-derived exosomes. (G-H) Results of RT-qPCR showing the level of hsa_circ_0058495 in tumor and adjacent tissues and serum from PDAC patients and healthy individuals. (I) Results of FISH assay showing the abundance of hsa_circ_0058495 in PDAC tissues and adjacent tissues. Hsa_circ_0058495 (red) and nuclei (blue) were shown. Scale bar, 100 µm. (J) Boxplot showing the level of RHBDD1 mRNA in the tumor and normal tissues of PDAC in TCGA datasets. (K) Kaplan-Meier survival analysis showing the correlation between the level of RHBDD1 mRNA and patient's overall survival time of PDAC in TCGA datasets. ns, no significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2

Hsa_circ_0058495 interacts with IGF2BP2 and upregulates the expression of IGF2BP2. (A) Schematic illustration showing the genomic localization of hsa_circ_0058495 and the back splice junction detected by Sanger sequencing. (B) Agarose gel electrophoresis analysis of PCR product amplified by divergent and convergent primers of hsa_circ_0058495 from gDNA and cDNA of BxPC3. (C) BxPC3 cells were treated with actinomycin D (5 µg/mL) for 0, 2, 4, 8, 12, and 24 hours, respectively. RT-qPCR was used to detect the level of hsa_circ_0058495 and RHBDD1 linear mRNA at the indicated times. (D) RNA was treated with RNase R (2 U/μg) at 37 ℃ for 15 minutes, and RT-qPCR was used to detect the level of hsa_circ_0058495 and GAPDH linear mRNA. (E) Nuclear-cytoplasmic fractionation RNA was extracted from BxPC3 and PANC1, and RT-qPCR was used to detect the level of hsa_circ_0058495 in nuclear and cytoplasm. (F) FISH assay showing the subcellular localization of hsa_circ_0058495 in BxPC3 and PANC1 cells. Hsa_circ_0058495 (red) and nuclei (blue) were shown. Scale bar, 20 µm. (G) RNA pull-down assay was performed by biotin-labeled hsa_circ_0058495 probe for the back splicing region and antisense probe was used as control. The level of IGF2BP2 pulled down was determined by immunoblotting, with β-actin as loading controls of input. (H) Schematic illustration of the domains contained by IGF2BP2 and shortened expressing vectors. (I) 293T cells were transfected with Flag-IGF2BP2 or Flag-RRM1-2, Flag-KH1-2, Flag-KH3-4, the cell lysates were analyzed by RNA immunoprecipitation (with anti-Flag) and RT-qPCR was used to detect the level of hsa_circ_0058495 being immunoprecipitated. (J) The secondary structure of hsa_circ_0058495 predicted by RNAfold website. (K) RNA pull-down assay was performed by biotin-labeled probes of three fragments of hsa_circ_0058495. The level of IGF2BP2 pulled down was determined by immunoblotting, with β-actin as loading control of input. (L-M) BxPC3 cells were transfected with siRNA against hsa_circ_0058495 or plasmid expressing hsa_circ_0058495 for 24 hours. The level of IGF2BP2 mRNA was detected by RT-qPCR, and the expression of IGF2BP2 protein was detected by immunoblotting. (N) Fluorescence in situ hybridization (FISH) by cy3-labeled hsa_circ_0058495 probe and immunofluorescence (IF) by anti-IGF2BP2 were performed in BxPC3 cells. Hsa_circ_0058495 (red), IGF2BP2 (green), and nuclei (blue) were shown. Scale bar, 20 µm. ns, no significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3

Hsa_circ_0058495 stabilizes IGF2BP2 protein from both autophagy-lysosome and ubiquitin-proteasome mediated degradation. (A-C) BxPC3 cells were transfected with plasmid expressing hsa_circ_0058495 or empty vector, siRNA against hsa_circ_0058495 or si-NC (negative control) for 24 hours. BxPC3 cells were treated with cycloheximide (CHX) (40 μg/mL) for 0, 2, 4, 6, and 8 hours. Subsequently, IGF2BP2 protein level was determined by immunoblotting, and the half-life of IGF2BP2 protein was calculated. (D) BxPC3 cells were treated with MG132 (10 μM), Bafilomycin A1 (BafA1, 1 μM), 3-methyladenine (3-MA, 5 mM) for 12 hours, or rapamycin (Rapa, 1 μM) for 4 hours. IGF2BP2 protein was detected by immunoblotting, with GAPDH as loading control. (E-G) BxPC3 cells were transfected with plasmid expressing hsa_circ_0058495 or empty vector in the presence or absence of rapamycin (1 μM) for 4 hours (E), with siRNA against hsa_circ_0058495 or si-NC in the presence or absence of 3-MA (5 mM) for 24 hours (F) or MG132 (10 μM) for 12 hours (G). IGF2BP2 was determined by immunoblotting, with GAPDH as loading control. (H-I) BxPC3 cells were transfected with siRNA against hsa_circ_0058495 or si-NC, plasmid expressing hsa_circ_0058495 or empty vector for 24 hours. IGF2BP2, LC3I/II, and p62 proteins were determined by immunoblotting, with GAPDH as loading controls. (J-K) BxPC3 cells with stable transfection of RFP-GFP-LC3 lentivirus were then transfected with plasmid expressing hsa_circ_0058495 or empty vector, siRNA against hsa_circ_0058495 or si-NC for 24 hours. Rapamycin (1 μM) was used to activate the process of autophagy and the fluorescence intensity was detected with confocal microscopy. Scale bar, 5 µm. ns, no significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4

Hsa_circ_0058495 stabilizes IGF2BP2 by hindering TRIM25-mediated ubiquitination. (A) 293T cells were transfected with Flag-IGF2BP2 along with over-0058495 or over-NC and the cell lysates were analyzed by immunoprecipitation (with anti-Flag) and IB. (B) 293T cells were transfected with Flag-KH3-4 along with over-0058495 or over-NC and the cell lysates were analyzed by immunoprecipitation (with anti-Flag) and IB. (C) 293T cells were transfected with Flag-TRM29 or Flag-PML, Flag-RSF1, Flag-TRIM25 along with HA-IGF2BP2 and the cell lysates were analyzed by co-immunoprecipitation (with anti-HA) and IB. (D) 293T cells were transfected with Flag-IGF2BP2 and HA-TRIM25 or empty vector with HA tag. The cell lysates were analyzed by co-immunoprecipitation (with anti-Flag) and IB. (E) 293T cells were transfected with HA-TRIM25 or empty vector. IGF2BP2 and TRIM25 proteins were detected by immunoblotting, with α-tubulin as loading control. (F) 293T cells were transfected with Flag-IGF2BP2 or shortened expressing vectors Flag-RRM1-2, Flag-KH1-2, Flag-KH3-4 along with HA-TRIM25. The cell lysates were analyzed by Co-immunoprecipitation (with anti-Flag) and IB. (G) 293T cells were transfected with Flag-IGF2BP2 or shortened expressing vectors Flag-RRM1-2, Flag-KH1-2, Flag-KH3-4 along with HA-TRIM25, in the presence of over-0058495 or over-NC. The cell lysates were analyzed by co-immunoprecipitation (with anti-Flag) and IB. (H-I) Immunofluorescence (IF) assay was performed to determine the localization of IGF2BP2 and TRIM25 in BxPC3 and PANC1 cells. TRIM25 (green), IGF2BP2 (red) and nuclei (blue) are shown. Scale bar, 20 µm. (A)-(D) and (F)-(G): twenty-four hours after transfection, the cells were treated with 10 µM MG132 for 12 h before harvest.

Figure 5

Hsa_circ_0058495 promotes the proliferation and invasion of PDAC cells in vitro and in vivo. (A) BxPC3 cells were transfected with siRNA against hsa_circ_0058495 or plasmid expressing hsa_circ_0058495 for 24 hours. EdU assay was performed to assess the proliferation ability of cells. Proliferated cells (red) and nuclei (blue) were shown. Counts of total cells and proliferated cells are shown in the column. Scale bar, 40 µm. (B) The same transfected cells were fixed and CCK-8 assay was applied to determine the growth rate of cells. (C) The same transfected cells were fixed and wound healing assay was used to determine invasion ability. The cell invasion rate was calculated and shown in the column. Scale bar, 80 µm. (D) Imaging of PET/CT for KPC mice injected exosomes overexpressed hsa_circ_0058495 or not through tail veins. (E) Tumor sizes of KPC mice. (F) H&E staining for tumor tissues from KPC mice. Scale bar, 1000 µm. (G-H) IHC staining of IGF2BP2 and Ki67 protein for tumor tissues from KPC mice injected exosomes overexpressed hsa_circ_0058495 or not through tail veins. Scale bar, 200 µm. ns, no significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 6

Hsa_circ_0058495/IGF2BP2 promotes the activation of the ERK pathway by stabilizing MEKK1 mRNA in an m6A-dependent manner. (A-B) PANC1 and BxPC3 cells were transfected with siRNA against IGF2BP2 or plasmid expressing IGF2BP2 for 24 hours. The level of MEKK1 mRNA was determined by RT-qPCR. (C-D) PANC1 and BxPC3 cells were transfected with plasmid expressing IGF2BP2 or empty vectors for 24 hours, then treated with actinomycin D (5 μg/mL) for 0, 2, 4, and 6 hours. The half-life of MEKK1 mRNA was determined by RT-qPCR. (E-F) BxPC3 cells were treated with 3-Denitroadenosine (DAA) (1 μM) or transfected with si-METTL3. The level of MEKK1 mRNA was detected by RT-qPCR; MEKK1 protein was determined by immunoblotting with β-actin as loading control. (G) The predicated m6A-modified sites on MEKK1 mRNA. (H) Methylation-RNA-immunoprecipitation (with anti-m6A) and RT-qPCR were performed to detect the enrichment of m6A modification on predicted sites. (I-J) BxPC3 cells were transfected with plasmid expressing hsa_circ_0058495 or si-0058495 for 48 hours (I) or transfected over-IGF2BP2 with or without si-0058495 for 48 hours (J), IGF2BP2, MEKK1, p-ERK1/2, ERK1/2, p-SAPK/JNK, SAPK/JNK, p-p38 and p38 proteins were determined by immunoblotting with β-actin as loading controls. ns, no significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 7

Single-cell transcriptomic characterization of PDAC TME stratified by hsa_circ_0058495 expression. (A) UMAP visualization of annotated cell clusters in PDAC TME. (B) UMAP projection of cellular distribution stratified by hsa_circ_0058495 expression status. (C) Cellular composition analysis comparing hsa_circ_0058495 high- and low-expression cohorts. (D) Differential expression heatmap of cluster-specific markers across cell populations. (E) Cell type-specific marker expression profile across TME compartments. (F) UMAP representation of macrophage subtypes (M1/M2) in PDAC TME. (G) Macrophage subtype distribution in hsa_circ_0058495 expression cohorts. (H) Polarization-specific marker expression profile in macrophage subtypes. (I) Pseudotemporal trajectory analysis of macrophage differentiation dynamics. (J) Expression dynamics of key regulatory genes during macrophage polarization. Color gradient represents normalized expression levels (blue: low; red: high).