IGF2BP3 drives gallbladder cancer progression by m6A-modified CLDN4 and inducing macrophage immunosuppressive polarization

Abstract

Introduction: N6-methyladenosine (m6A) is an emerging epigenetic modification, which plays a crucial role in the development of cancer. Nevertheless, the underlying mechanism of m6A-associated proteins and m6A modification in gallbladder cancer remains largely unknown.

Materials and methods: The Gene Expression Omnibus database and tissue microarray were used to identify the key m6A-related gene in gallbladder cancer. The function and mechanism of IGF2BP3 were further investigated by knockdown and overexpression techniques in vitro and in vivo.

Results: We found that IGF2BP3 was elevated and correlated with poor prognosis in gallbladder cancer, which can be used as an independent prognostic factor for gallbladder cancer. IGF2BP3 accelerated the proliferation, invasion and migration of gallbladder cancer cells in vitro and in vivo. Mechanistically, IGF2BP3 interacted with and augmented the stability of CLDN4 mRNA by m6A modification. Enhancement of CLDN4 reversed the inhibitory effect of IGF2BP3 deficiency on gallbladder cancer. Furthermore, we demonstrated that IGF2BP3 promotes the activation of NF-κB signaling pathway by up-regulation of CLDN4. Overexpression of IGF2BP3 in gallbladder cancer cells obviously promoted the polarization of immunosuppressive phenotype in macrophages. Besides, Gallbladder cancer cells-derived IGF2BP3 up-regulated the levels of STAT3 in M2 macrophages, and promoted M2 polarization.

Conclusions: We manifested IGF2BP3 promotes the aggressive phenotype of gallbladder cancer by stabilizing CLDN4 mRNA in an m6A-dependent manner and induces macrophage immunosuppressive polarization, which might offer a new theoretical basis for against gallbladder cancer.

Keywords: Claudin-4; Gallbladder cancer; M2 macrophages; N6-methyladenosine; Prognosis.

Fig. 1

The expression levels and prognostic meaning of IGF2BP3 in gallbladder cancer. (A) The relative expression of IGF2BP3 in gallbladder cancer and normal tissues from the GEO database. (B-C) Representative images of tumor stage and lymph node metastasis under IGF2BP3 staining by using gallbladder cancer TMA. A total of 165 patients. Scale bars = 500 μm or 100 μm. (D) Score of tumor stage and lymph node metastasis. (E) Kaplan–Meier curves. (F-G) Univariate and multivariate regression analysis. *p < 0.05, ***p < 0.001.

Fig. 2

A xenograft model revealed that knockdown IGF2BP3 inhibits the progression of gallbladder cancer. (A) Typical diagrams of xenograft tumors (n = 5). (B) The tumor volume (n = 5). (C) The tumor weight (n = 5). (D) H&E staining of tumor tissues. (E) Typical IHC diagrams of IGF2BP3 staining. (F) Representative immunofluorescence diagrams of Ki67 staining. Scale bar = 100 μm. *p < 0.05, **p < 0.01.

Fig. 3

IGF2BP3 enhances the mRNA stability of CLDN4 in an m 6 A-dependent manner. (A) Venn diagrams. (B-C) The effects of knockdown and overexpression of IGF2BP3 on target genes were explored by q-PCR (n = 3). (D-E) CLDN4 expression were performed by western blot in NOZ cells after infected with IGF2BP3 shRNA or overexpression IGF2BP3, respectively (n = 3). (F) RIP assay was performed using IgG or IGF2BP3 antibody in NOZ cells (n = 3). (G) Co-expression of IGF2BP3 and CLDN4 in NOZ cells was determined by immunofluorescence assay (n=3). (H) CLDN4 enrichment was detected by MeRIP-qPCR (n = 3). (I) q-PCR was used to detect the effect of IGF2BP3 interference on mRNA stability of CLDN4 (n = 3). (J) Schematic representation of CLDN4-WT plasmids and CLDN4-MUT plasmids containing m6A motif mutations in the CDS region. (K) The relative luciferase activity of the WT or MUT CLDN4 reporter (n=3). *p < 0.05, **p < 0.01, ***p < 0.001. NS: no significant.

Fig. 4

IGF2BP3 regulates malignant activities of gallbladder cancer cells by up-regulating CLDN4. (A) The CCK8 assay (n = 6). (B) Cell migration and invasion were quantified. (C-D) Representative images of cell migration and invasion (n = 3). Scale bar = 100 μm. (E) Apoptosis rate was analyzed by flow cytometry (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5

Overexpression of IGF2BP3 in gallbladder cancer induces M2-like polarization of macrophages. (A) Schematic representation of the co-culture system. (B) Knockdown or overexpressing IGF2BP3 in cancer cells was co-cultured with macrophages, and the migration of macrophages was then assessed by transwell assays (n = 3). Scale bar = 100 μm. Macrophages in the upper chamber, NOZ/GBC cells in the lower chamber. (C) q-PCR was used to detect the effect of GBC cells-treated with IGF2BP3 overexpression on M2 macrophage activation markers (n = 3). (D) Western blot showed the expression of IL-10 and Arg-1 (n = 3). The flow cytometry (E) and immunofluorescence (F) were used to detect CD206 distribution in macrophages co-cultured with GBC cells-treated with IGF2BP3 overexpression (n = 3). Scale bar = 50 μm. *p < 0.05, **p < 0.01.

Fig. 6

Overexpression of IGF2BP3 in gallbladder cancer drives M2-like polarization by activating STAT3 signalling pathway. (A) q-PCR was used to detect the level of STAT3 in M2 macrophage-treated with sh/OE-IGF2BP3-induced gallbladder cancer cells (n = 3). (B) Western blot was used to detect the level of STAT3 in M2 macrophage-treated with IGF2BP3 overexpression-induced GBC cells or sh-IGF2BP3-induced NOZ cells (n = 3). (C) q-PCR was used to detect the c-myc in M2 macrophage-treated with sh/OE-IGF2BP3-induced gallbladder cancer cells (n = 3). (D) Diagram of the molecular mechanism by which IGF2BP3 regulates gallbladder cancer progression.*p < 0.05, **p < 0.01.