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. 2023 Aug 27;37:101764. doi: 10.1016/j.tranon.2023.101764

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

Jian Qin a,1, Zheng Cui b,1, Jingyi Zhou a, Bosen Zhang c, Ruiqi Lu c, Youcheng Ding c, Hai Hu c, Jingli Cai c,⁎
PMCID: PMC10472310  PMID: 37643553

Highlights

  • •

    IGF2BP3 overexpression promote gallbladder cancer growth and indicate poor prognosis.

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    IGF2BP3 activates NF-kB pathway by upregulating CLDN4 to promote gallbladder cancer.

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    Gallbladder cancer-secreted IGF2BP3 induces macrophage M2 polarization via STAT3.

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

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.

Introduction

Gallbladder cancer has problems of late diagnosis and poor prognosis [1,2]. Because of the poor prognosis, the 5-year survival rate suggests that gallbladder cancer needs more effective treatment strategies [3]. The possible treatment for gallbladder cancer is complete surgical resection. Nevertheless, less than 10% of patients are considered candidates for surgery because most patients are in an advanced stage, resulting in poor clinical outcomes [4]. In recent years, with booming molecular biology and genetic sequencing, the molecular mechanism of gallbladder cancer are gradually being understood [5]. However, the main obstacle of gallbladder cancer is the lack of early biomarkers and effective chemotherapy with clear targets.

N6-methyladenosine (m6A) is a modification that a methylation of adenylate (A) at the sixth position. The preferential typical consensus sequence of m6A is DRACH (D = G, A, or U; R = G or A; H = A, C, or U) and the consensus sequence is highly dynamic with varying levels during development and in response to cellular stress, indicating that m6A have functional roles that influence mRNA fate [6]. m6A is an most abundant modification throughout the transcriptome with a wide range of functions in both protein-coding and non-coding RNAs, which has an important impact on cancer development [7,8]. m6A plays a regulatory role in RNA regulation, mainly though the m6A regulators writers (methyltransferase), erasers (demethylase), and readers [9]. Among them, m6A is catalyzed by writers and removed by erasers, and RNA reader protein recognizes m6A, binds the RNA and implements corresponding functions [10]. Recently, m6A regulators have been demonstrated to affect the growth and invasive of tumors [11]. For instance, insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), an m6A reader, is identified as a poor prognostic biomarker of glioma [12]. IGF2BP3-mediated JAK/STAT signalling pathway stimulates the malignant activities of bladder cancer [13]. Furthermore, high IGF2BP3 expression is associated with the aggressive carcinogenic behaviour of gallbladder adenocarcinoma [14]. However, the effects of IGF2BP3 served as m6A reader in the molecular, cellular, and tumor biology of gallbladder cancer are still largely unknown.

Tumor-associated macrophages (TAMs) are one of the most important tumor-infiltrating immune cell types in the tumor microenvironment. TAMs are specifically differentiated in different physiopathological environments and usually have two distinct fate endpoints: the M1-polarized macrophage and the M2-polarized macrophage [15]. Among them, CD206high M2 macrophages are considered to be a tumor growth-promoting factor, but CD86high M1 macrophages exerts anti-tumor functions. For example, Wnt5a-induced M2 TAMs promotes colorectal cancer development [16]. Lactate-induced TAMs promotes the invasion of pituitary adenoma [17]. In addition, more and more studies have shown that reprogramming TAMs towards the M1 phenotype is more beneficial to the survival of cancer patients. For instance, reprogramming M2 TAMs into M1-like macrophages by targeting IL-4 could inhibit tumor growth [18], by exoASO-STAT6 monotherapy could potent antitumor activity [19], and by STING agonism could overcome drug resistance [20]. Importantly, emerging studies have shown that m6A modifications are involved in TAM polarisation. Liu et al. proved that lncRNA-PACERR promotes polarization of M2 macrophage through binding to IGF2BP3 to enhance the stability of KLF12 in an m6A-dependent manner in pancreatic ductal adenocarcinoma [21]. However, whether IGF2BP3 can affect TAM polarization in an m6A-dependent manner to involve in gallbladder cancer is not fully understood.

In the present study, we aimed to excavate the key m6A regulator in gallbladder cancer and to investigate their functions and molecular mechanisms in gallbladder cancer progression. Hence, we profiled the expression profile of m6A-related genes in gallbladder cancer from the NCBI Gene Expression Omnibus (GEO) repository. Then, we selected the m6A gene with the most significant difference to study its effect on gallbladder cancer cells and the underlying mechanism. Our study may supply a feasible strategy for molecular diagnosis and targeted therapy in gallbladder cancer.

Materials and methods

Collection and preprocessing raw RNA-Seq data

The expression information of m6A-related genes was downloaded from the NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/), among which raw RNA-Seq data included GSE76633, GSE74048, and GSE139682. Using |log2FC| > 1 and p < 0.05 as a threshold, differential expression analysis between the tumor and normal groups was performed to acquire differential expression of m6A-related genes.

Tissue samples and tissue microarray analysis (TMA)

A TMA of gallbladder cancer tissue samples (GALCAR − 1, GAC1801) was purchased from a cohort of 165 gallbladder cancer patients from ZhuoHao Medicine (Shanghai, China). The TMA was constructed using a core diameter of 5 μm and stored at 4°C until they were ready for analysis. The expression level of IGF2BP3 was detected by immunohistochemistry (IHC). Briefly, TMA slices were dewaxed and treated with hydrogen peroxide [22]. Subsequently, the slices were soaked with IGF2BP3-antibodies at 4°C for 12 h. Next, the relationship between the expression of IGF2BP3 and the clinical features of patients was analysed. Prognostic values of IGF2BP3 expression in gallbladder cancer were evaluated by the Kaplan Meier plotter (http://kmplot.com/analysis/), displaying the overall survival (OS). Finally, univariate and multivariate regression analyses were performed on IGF2BP3 expression. The clinical factors of gallbladder cancer was used to determine whether IG2BP3 could be an independent prognostic predictor of gallbladder cancer.

Cell culture

The gallbladder cancer cell (GBC-SD and NOZ) lines and THP-1 cell line were provided by the Chinese Academy of Sciences (Shanghai, China). GBC-SD cells were conducted in RPMI 1640 medium (Corning, United States) with 10% fetal bovine serum (FBS, Gibco, United States). NOZ cells were conducted in DMEM medium (Corning, United States) with 10% FBS. The cells were cultured in 37°C, 5%CO2 incubator.

Plasmids and shRNAs

The mRNA of IGF2BP3 and CLDN4 were cloned into pcDNA3.1 (+) plasmid to construct the overexpression vectors, and then transfected into GBC-SD and NOZ cells using Lipofectamine 2000 (Invitrogen, USA). Additionally, to construct stable IGF2BP3 knockdown NOZ cells, cells were infected with the lentivirus-coated sh-IGF2BP3 or corresponding negative control.

Quantitative real-time PCR (q-PCR)

The total RNA was isolated by TRIzol reagent (Invitrogen, United States). Then, RNA was reverse transcribed into cDNA by Reverse Transcription Kit (Takara, China). The qPCR was conducted on ABI Q6 Real-Time PCR system (Applied Biosystems Inc., USA) using 2 × Master Mix kit (Roche). GAPDH was applied to normalize the expression of all genes. All primer sequences used in the experiment are listed in Table S1.

Western blot

Proteins were fractionated on a 10% SDS-PAGE gel and transferred to the PVDF membrane. The membranes were blocked with TBST solution containing 5% skim milk for 3 h, then probed with primary antibodies at 4°C 12 h, including anti-IGF2BP3 (1:5000, Proteintech, #14642-1-AP), anti-CLDN4 (1:200, Santa cruz, #sc-376643), anti-IKK beta (phospho Y188, 1:500, Abcam, #ab194519), anti-IKK beta (1:1000, Abcam, #ab124957), anti-NF-kB p65 (1:1000, Abcam, ab32536), anti-NF-kB p65 (phospho S536, 1:1000, Abcam, #ab76302), anti-Arg-1 (1:5000, Proteintech, #66129-1-Ig), anti-IL-10 (1:5000, Abcam, #ab133575), anti-STAT3 (1:2000, Proteintech, #10253-2-AP), and anti-GAPDH (1:10000, Proteintech, #60004-1-Lg). Next, the secondary antibody goat anti-rabbit IgG H&L (1:1000, Beyotime, #A0208) and goat anti-mouse IgG H&L (1:1000, Beyotime, #A0216) were incubated at 25 ± 5°C for 2 h. Protein visualization using enhanced chemiluminescence detection substrates, and protein expression was quantified by Image J software densitometer.

Cell proliferation assay

The infected cells were plated in 96-well plates (1000 cells/well) and incubation at 37°C for 2 h at 0, 24, 48, 72, and 96 h. Then, 10% CCK-8 reagent was added to each well and cultivated at 37°C for another 2 h. Finally, the absorbance was measured at the wavelength of 450 nm.

Transwell assays

Migration and invasion measurements were analysed by 24-well transwell inserts (Corning, USA). Cells were inoculated into the upper chambers with 0.4 mL serum-free culture media and added 0.6 mL of medium containing 10% FBS to the lower chambers. After incubation for one day, the cells were fixed with paraformal-dehyde and stained with crystal violet. After rinsing with PBS, a microscope (XSP-37XB, Shanghai Optical Instrument Factory) was used to count migrated or invaded cells. For macrophage migration, macrophages were added into the upper chambers, GBC or NOZ cells were added into the lower chambers, the remaining methods were consistent with those described above.

Apoptosis and cell cycle assay

The cells were digested with trypsin and then measured by FITC-Annexin V Apoptosis Detection Kit (BD Biosciences, USA). Cells were seeded in six well plates for the cell cycle assay and fixed with precooled 70% ethanol and propidium iodide staining. The DNA content was performed by flow cytometry.

Animal studies

We purchased BALB/c nude mice (male, 4–6 weeks old) from the Cloud-Clone Animal Inc. NOZ cells (2 × 106 cells) transfected with sh-IGF2BP3 or sh-NC were injected into the flanks of mice (n = 5) according to previously described criteria [23]. After 2 weeks of injection, the tumor volume was measured every 3 days. The tumor volume was calculated by volume = 1 / 2 × long × width2. On day 26, we weighed the tumor.

H&E staining and IHC staining

The tumor was fixed with paraformal-dehyde, dehydrated with ethanol, embedded in paraffin, sliced with slicer (3 μm), and finally stained with hematoxylin eosin (H&E). For IHC staining, the tissue sections were blocked with PBS supplemented with 5% BSA and then infiltrated by a specific antibody at 4°C for 12 h. After that, the secondary antibody infiltrated the slices at 25 ± 5°C for 1 h. Finally, the sections were examined under an optical microscope. We used the following antibodies: anti-IGF2BP3 (1:50, Proteintech, #14642-1-AP), anti-CLDN4 (1:100, Santa cruz, #sc-376643), goat anti-rabbit IgG H&L (1:50, Beyotime, #A0208) and goat anti-mouse IgG H&L (1:50, Beyotime, #A0216).

Immunofluorescence

For tissue immunofluorescence, the tissue sections were successively dewaxed, antigen repaired, and sealed (5% BSA, 30min). For cellular immunofluorescence, NOZ cells were cultured in 24-well plates, fixed with 4% paraformaldehyde for 10 min, and washed with PBS before antigen repair and sealed (1% BSA, 30min). Next, tissue sections were incubated with antibodies against Ki67 (1:100, Abcam, #ab16667), and cells were incubated with IGF2BP3 (1:50, Proteintech, #14642-1-AP), CLDN4 (1:100, Santa cruz, #sc-376643), and CD206 (1:250, Abcam, #ab64693) overnight. Thereafter, fluorescence-labelled secondary antibody Cy3 conjugated goat anti-rabbit IgG H&L (1:300, Servicebio, #GB21303) were used for tissue sections. The alexa Fluor-488 labeled goat anti-rabbit IgG H&L (1:200, Abcam, #ab150077) and Cy3 conjugated goat anti-mouse IgG H&L (1:500, Beyotime, #A0521) were added to cells. Nuclear counterstaining was assayed with DAPI (Servicebio, #G1012) and the images were taken with the fluorescence microscope.

RNA sequencing

The total RNA from normal control (NC) and sh-IGF2BP3 groups of NOZ cells were isolated by the RNeasy Mini Kit (Qiagen, Hilden, Germany), then the RNA-seq libraries were prepared on Agilent 2200 (Agilent, USA). The Illumina HI-SEQ platform was used for deep sequencing analysis of the library. The gene expression level of each transcript was counted as RPKM. In addition, the R Bioconductor DESeq2 package was used to detect differential gene expression.

Gene function enrichment analysis

The Gene Ontology (GO) terms for IGF2BP3 target genes based on homologies were extracted (http://www.geneontology.org). Kyoto Encyclopedia of Genes and Genome (KEGG) (http://www.genome.jp/kegg) was used to analyse the pathway. Gene set enrichment analysis (GSEA) was performed for estimating the enrichment of various pathways in sample.

RNA immunoprecipitation (RIP) assay

According to the procedure specification, RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, United States) was used for the RIP assay. Briefly, cell samples were lysed with a complete RIP lysis buffer (Millipore) containing protease inhibitors and RNase. In addition, antibody against IgG or IGF2BP3 were used to pre-coat magnetic beads. The magnetic beads were then incubated with cell lysate to capture antibodies. Finally, RNA was isolated and evaluated by qPCR.

MeRIP-qPCR

Total RNA was separated from pre-processed cells and fragmented into 100 nucleotides at random. Then, RNA samples were immunoprecipitated with magnetic beads pre-coated with anti-m6A antibody (Millipore, Germany) or anti-mouse IgG (Millipore). Next, m6A modified RNA fragments were eluted with N6-methyladenosine 5′- sodium monophosphate. Finally, qPCR detection was performed.

RNA stability assay

Actinomycin D (5 μg/mL) was incubated cells for 0 h, 1h, 2 h, and 4 h, respectively. Then the cells were harvested and RNA was extracted for q-PCR as described above. The formula for RNA degradation rate (k) is e-kt = N0/Nt, where k means the degradation rate, t represents the time after transcription inhibition, and Nt and N0 are the RNA quantities at time t and time 0. RNA half-life was calculated according to degradation rate t1/2 = ln2/k.

Correlation between IGF2BP3 and CLDN4

The mRNA expression data of IGF2BP3 and CLDN4 in patients with gallbladder cancer were downloaded from the NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/) (GSE76633 and GSE139682). The mRNA expression profile and the correlation between IGF2BP3 and CLDN4 were analysed.

Luciferase reporter assay

The sequence of CLDN4 containing the predicted m6A binding site was ligated into pGL3-Basic vector to construct wild-type (WT) CLDN4 reporter. We also generated a recombinant reporter of the mutant type (MUT) CLDN4 covering the mutant binding site of m6A. The recombinant plasmid was co-transfected with pcDNA3.1-IGFBP3 or blank vector into 293T cells. Next, the dual-luciferase activity was measured by Dual-Glo Luciferase Assay system. Briefly, after 48 h transfection, the cells were lysed by passive lysis buffer. Finally, the relative ratio of firefly luciferase activity to Renni luciferase activity was calculated.

Macrophages M2 polarization

The THP-1 cells were conducted in RPMI 1640 medium with 10% FBS and 0.05 mM β-mercaptoethanol. THP-1 cells were treated with 100 ng/mL PMA for 24 h and then stimulated with 20 ng/mL IL-4 for 24 h to induce M2 macrophages. Subsequently, cells were co-cultured with NOZ or GBS cells for 24 h. The M2 macrophages was determined by CD206 positive cells using flow cytometry.

Statistical analysis

All statistics were conducted by Graphpad Prism 9.0. The outcomes were represented as the mean ± standard deviation (SD). The t test was accustomed to comparing two groups of data. One-way analysis of variance followed by Tukey test was accustomed to examining the difference among three or more groups. Differences were deemed statistically significant at a p < 0.05.

Results

IGF2BP3 is up-regulated in gallbladder cancer and correlated with poor prognosis

To identify the differential expression of m6A-related genes in gallbladder cancer, we obtained the sequencing data of gallbladder cancer through the GEO database. Differential expression analysis revealed that only IGF2BP3 was up-regulated in gallbladder cancer tissues relative to the normal group in all databases (Figs. 1A and S1A). We also analysed IGF2BP3 expression in the GEPIA database, based on cholangio carcinoma data within TCGA, and found that IGF2BP3 was upregulated in cancer tissue compared with the normal tissues (Fig. S1B). Hence, we used TMA to further verify the relationship between IGF2BP3 expression and gallbladder cancer. According to the clinical analysis data, there was a prominent association between IGF2BP3 expression and TNM stage and lymph node metastasis (Table S2, Fig. 1B–D). Subsequently, we evaluated the prognostic influence of IGF2BP3 in gallbladder cancer by Kaplan-Meier methods, which revealed the OS of patients with high IGF2BP3 expression was sharply shorter than those with low expression (Fig. 1E). In addition, univariate Cox regression analysis and Multivariate Cox regression analysis indicated that IGF2BP3 was regard as an independent predictor of the prognosis of patients with gallbladder cancer (Fig. 1F, G). Collectively, these results indicated that m6A reader IGF2BP3 is up-regulated in gallbladder cancer and its expression is an independent prognostic factor for gallbladder cancer.

Fig. 1.

Fig. 1

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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.

IGF2BP3 promotes malignant activities of gallbladder cancer cells

Considering the high expression of IGF2BP3 in gallbladder cancer and its relationship with poor prognosis, we speculated that IGF2BP3 might regulate the pernicious activities of gallbladder cancer cells. To verify our supposition, we detected the expression of IGF2BP3 in NOZ and GBC-SD cells, and found that the expression of IGF2BP3 in NOZ was higher than that in GBC-SD (Figs. S2A and 2B). Subsequently, IGF2BP3 knockdown and IGF2BP3 overexpression assays were performed in NOZ cells (Fig. S2C and 2D) and GBC-SD cells (Fig. S3A and S3B), respectively. The results of proliferation assays represented that inhibition of IGF2BP3 remarkably prevented the proliferation of NOZ cells (Fig. S2E), whereas overexpression of IGF2BP3 had the opposite effect (Fig. S3C). Further, we found that blocking the endogenous IGF2BP3 expression reduced cell migration and invasion (Fig. S2F and 2G), whereas IGF2BP3 overexpression significantly accelerated cellular behaviours in vitro (Fig. S3D and S3E). To further understand the role of IGF2BP3 in gallbladder cancer cell progression, we assessed the apoptosis and cell cycle by flow cytometry. The cell cycle result revealed that inhibition of IGF2BP3 markedly enhanced the proportion of cells in the G1 peak while lessening the proportion of S phase (Fig. S2H). However, overexpression of IGF2BP3 did not influence the cell cycle of GBC-SD cells (Fig. S3F). Furthermore, as shown in Fig. S2I, knockdown of IGF2BP3 markedly facilitated cell apoptosis, while overexpression of IGF2BP3 elicited the opposite effects (Fig. S3G). In summary, the above results demonstrated that IGF2BP3 had a tumor stimulative function in gallbladder cancer.

Knockdown of IGF2BP3 inhibits the tumor growth of gallbladder cancer in vivo

To further validate the critical role of IGF2BP3 in vivo, we inoculated NOZ cells stably transfected with sh-IGF2BP3 or control into nude mice. Compared with the control, IGF2BP3 knockdown notably decreased tumorigenesis and suppressed tumor growth (Fig. 2A–C). Additionally, H&E staining of tissue sections showed that IGF2BP3-knockdown exhibited a weak nuclear heterogeneity in tumors compared with sh-NC (Fig. 2D). Correspondingly, immunohistochemical staining of IGF2BP3 was remarkably reduced in the sh-IGF2BP3 group (Fig. 2E). Finally, knockdown of IGF2BP3 decreased Ki67 staining (a marker of cell proliferation), compared with sh-NC xenografts (Fig. 2F). Collectively, these results suggested that IGF2BP3 served as an oncogenic role in gallbladder cancer in vivo.

Fig. 2.

Fig. 2

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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.

Silencing of IGF2BP3 alters the gene expression profile of gallbladder cancer cells

To elucidate the regulatory mechanism of IGF2BP3 in gallbladder cancer, the target genes were screened by transcriptome sequencing in gallbladder cancer cells after IGF2BP3 silencing. The heat map revealed distinct mRNA expression profiles between sh-IGF2BP3 and NC groups (Fig. S4A). Table S3 exhibited the top 10 up-regulated and top 10 down-regulated mRNAs. The GO analysis demonstrated that these target genes were observably enriched in proliferation-related GO terms, such as mitotic cell cycle, cell cycle, cell division, DNA replication, and some other cell cycle processes (Fig. S4B). For KEGG pathway analysis, differentially expressed genes participated in the pathways including Wnt signalling pathway, NF-κB signalling pathway, and Hippo signalling pathway (Fig. S4C).

IGF2BP3 promotes CLDN4 mRNA stability in an m6A-dependent manner

To explore the target gene of IGF2BP3, we first identified the up-regulated genes in gallbladder carcinoma compared to the normal tissues using GSE139682 and GSE90639 datasets. Then, the IGF2BP3 binding genes were obtained from IGF2BP3 RIP-Seq data from GSE76633. Next, sh-IGF2BP3 induced down-regulated genes in our transcriptome sequencing also requisitioned. Finally, these 4 datasets were intersected and 14 target genes were obtained (Fig. 3A). Then, 5 tumor-related genes (LEMD1, B4GALNT3, SLC29A2, TMC5, and CLDN4), according to the literature [24], [25], [26], [27], were screened for q-PCR verification. The interference samples verified that these five genes were significantly down-regulated, which consistent with the sequencing results (Fig. 3B). However, IGF2BP3 overexpression only significantly elevated CLDN4 expression while the changes of the remaining four genes did not meet expectations (Fig. 3C). Therefore, we selected CLDN4 as the target gene of IGF2BP3.

Fig. 3.

Fig. 3

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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.

Further analysis exhibited that IGF2BP3 expression was positively related to CLDN4 levels in the GSE7663 and GSE139682 datasets (Fig. S5A). Moreover, sh-IGF2BP3 significantly inhibited the protein expression of CLDN4, while IGF2BP3 overexpression exhibited the opposite effects (Fig. 3D and 3E). IHC staining showed that silencing of IGF2BP3 remarkably decreased the CLDN4 protein level in gallbladder carcinoma tissues of the xenograft mice (Fig. S5B). Conclusively, the aforementioned data indicated that IGF2BP3 regulates CLDN4 expression.

Next, we sought to investigate how IGF2BP3 enhances the expression of CLDN4. Consistent with the eCLIP-seq data from ENCODE database, our RIP-qPCR assays demonstrated IGF2BP3 could combine with the mRNA of CLDN4 in NOZ cells (Figs. 3F and S5C). Immunofluorescence staining showed that IGF2BP3 was co-localized with CLDN4 in the cytoplasm of NOZ cells (Fig. 3G). Next, we examined whether IGF2BP3 binds CLDN4 via an m6A-dependent manner. MeRIP-qPCR verified that CLDN4 had an m6A RNA methylation site (Fig. 3H). It has been reported that IGF2BP3 affects the mRNA stability of target gene after binding to the target gene through the m6A site [28]. Therefore, we further explore whether IGF2BP3 affects the stability of CLDN4. By actinomycin D treatment, IGF2BP3 knockdown dramatically decreased the mRNA stability of the target gene CLDN4 (Fig. 3I). Based on the above information, we speculated that the interaction between IGF2BP3 and CLDN4 mRNA depends on m6A site. To verify this hypothesis, we constructed the WT and MUT CLDN4 luciferase reporters containing the m6A site or mutation site, respectively (Fig. 3J). The mutation was performed at the m6A site with the highest m6A score predicted by the SRAMP database (Fig. S5D). Luciferase reporter gene experiment demonstrated that overexpression of IGF2BP3 enhanced the luciferase activity of WT-CLDN4 reporter, but did not affect the luciferase activity of MUT CLDN4 (Fig. 3K). Therefore, these results revealed that IGF2BP3 promotes CLDN4 expression by elevation mRNA stability in an m6A-dependent manner.

Overexpression of CLDN4 triggers malignant activity of gallbladder cancer cells

To determine whether CLDN4 regulates the malignant activity of gallbladder cancer, we overexpressed CLDN4 in the NOZ cell line (Fig. S6A and 6B). It was observed that overexpression of CLDN4 boosted the proliferation, migration, and invasion of NOZ cells, while suppressed apoptosis (Fig. S6C–G). Therefore, overexpression of CLDN4 accelerated gallbladder cancer cell development.

Enhancement of CLDN4 reverses the inhibitory effects of IGF2BP3 knockdown on gallbladder cancer cells

To indicate whether IGF2BP3 regulates the malignant phenotype of gallbladder cancer cells by targeting CLDN4, we performed a rescue experiment. Knockout of IGF2BP3 inhibited cells proliferation, migration, and invasion (Fig. 4A–D), as well as supported cell apoptosis (Fig. 4E). However, CLDN4 overexpression could attenuate the effects of sh-IGF2BP3 on NOZ cells. These data revealed that IGF2BP3 enhanced the malignant activities of gallbladder cancer cells via CLDN4.

Fig. 4.

Fig. 4

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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.

IGF2BP3 activates NF-kB pathway by upregulating CLDN4 in gallbladder cancer

To explore the underlying signalling pathway by which IGF2BP3 regulates the gallbladder cancer, we performed GSEA analysis using our transcriptome data from the sh-NC and sh-IGF2BP3 groups. GSEA confirmed that NF-κB signalling pathway was enriched by the two different datasets (Fig. S7A). String database also showed that CLDN4 interacts with IGF2BP3-mediated genes involved in the NF-κB pathways (Fig. S7B). Western blot analysis showed that knockdown of IGF2BP3 inhibited the phosphorylation of IKKβ and NF-κB, but overexpression of CLDN4 dramatically reversed this effect (Fig. S7C), indicating that IGF2BP3 activates NF-κB signalling pathway by CLDN4. Given that NF-κB signalling pathway is an important regulatory pathway in gallbladder cancer [29,30], we hypothesized that IGF2BP3 motivated NF-κB pathway by enhancing CLDN4 to regulate gallbladder cancer development.

Gallbladder cancer cells-derived IGF2BP3 induces M2 polarization of macrophages

In TME, immunosuppressive macrophage differentiation is the main cause of reduced anticancer immunity and poor overall survival in patients with gallbladder cancer [31]. Therefore, we further explored the relationship between IGF2BP3 expression and macrophages M2 polarization. We transfected sh-IGF2BP3/NC and pcDNA3.1-IGF2BP3/vector into NOZ and GBC cell lines, respectively, and then co-cultured them with M2 macrophages in transwell co-culture system (Fig. 5A). As shown in Fig. 5B, M2 macrophages showed higher migration ability when co-cultured with OE-IGF2BP3 GBC cells compared to OE-NC GBC cells. However, M2 macrophages showed the opposite trend when co-cultured with sh-IGF2BP3 NOZ cells. Moreover, compared with NC, macrophages incubated with IGF2BP3-overexoressed GBC cells exhibited a significant up-regulation in the level of IL-10, TGF-β1 and Arg-1 (Fig. 5C and D). Meanwhile, the percentage of M2 macrophage with CD206+ phenotype was significantly increased when treated with OE-IGF2BP3 GBC cells (Fig. 5E and F). Hence, IGF2BP3 mediated by gallbladder cancer cells promoted M2 polarization of macrophages in vitro.

Fig. 5.

Fig. 5

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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.

Gallbladder cancer cells-overexpressed IGF2BP3 drives macrophages M2 polarization by activating STAT3 signalling pathway

We further explored the potential regulatory mechanism of IGF2BP3 in promoting M2 macrophage polarization in gallbladder cancer. STAT3 signalling pathway has been reported to induce M2 macrophage polarization of gallbladder cancer [32]. As indicated in Fig. 6A and B, overexpression of IGF2BP3 in GBC cells promoted the mRNA and protein expression of STAT3 in M2 macrophages, while inhibition of IGF2BP3 had the opposite effect. Meanwhile, IGF2BP3 overexpression in GBC cells promoted the expression of c-myc (a marker of activation of STAT3 signalling pathway) in M2 macrophages; however, this phenomenon was opposite when M2 macrophages co-incubated by sh-IGF2BP3-transfected GBC cells (Fig. 6C). Collectively, IGF2BP3 overexpression in gallbladder cancer cells promoted M2 polarization of macrophages via activating STAT3 signalling pathway.

Fig. 6.

Fig. 6

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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.

Discussion

Gallbladder cancer is very easy to metastasize and has a high degree of malignancy [33]. Moreover, gallbladder cancer is not sensitive to radiotherapy and chemotherapy [34,35]. However, the primary research on gallbladder cancer is relatively weak for the moment, so it is imperative to find the treatment target of gallbladder cancer. In recent years, the important role of RNA m6A methylation modification and related regulatory factors in various cancers has attracted extensive attention from researchers [36]. With the progress of genetic sequencing, the development of some cancers had been identified to be associated with m6A methylation dysregulation [37]. In this project, our research was the first to demonstrate that m6A reader IGF2BP3 was highly expressed in gallbladder cancer with high diagnostic value, and could be recognized as an independent prognostic factor for gallbladder cancer. Additionally, we found that overexpressed IGF2BP3 notably enhanced tumor growth ability. Mechanistically, IGF2BP3 targets CLDN4 depending on m6A modification to activities NF-kB pathway, thus promoting the progression of gallbladder cancer. Furthermore, overexpression of IGF2BP3 in gallbladder cancer promoted macrophages recruitment and induced M2 macrophage polarization by activating STAT3 signalling pathway (Fig. 6D).

M6A is the most abundant modification in higher eukaryotic mRNAs, which is essential for mRNA metabolism and a variety of organisms. Currently, increasing studies have proved that m6A influences the development of tumor, such as proliferation, growth, invasion and metastasis [38,39]. For instance, IGF2BP3 was overexpressed in stomach cancer, promoting cancer cell migration and angiogenesis [28]. Similarly, IGF2BP3 regulated cell proliferation and apoptosis in esophageal squamous cancer [40]. Due to the huge role of m6A in cancer, m6A and its regulators are expected to become a diagnostic or therapeutic target for various cancers [41]. Thus, our study confirmed that IGF2BP3 was elevated in gallbladder cancer tissues, and heightened the proliferation, metastasis and invasion of gallbladder cancer cells and hindered apoptosis, accelerating G1/S transition and tumor growth, which indicated that IGF2BP3 was an oncogene and had good diagnostic value. In addition, as a deep understanding of the molecular mechanisms regulating cancer development are key to achieving personalized treatment [42,43], it is crucial to explore how IGF2BP3 regulates gallbladder cancer.

To dissect the mechanisms of IGF2BP3 in gallbladder cancer, we searched for potential m6A targets of IGF2BP3 based on gallbladder cancer database, IGF2BP3 RIP-Seq, and transcriptome sequencing by lentivirus interfered with IGF2BP3. The results manifested that CLDN4 was the target gene of IGF2BP3, and IGF2BP3 increase the expression of CLDN4 by m6A modification. CLDN4, an essential member of the claudins family, is extensively expressed in multiple cancers and participates in cell migration and invasion [44]. CLDN4 has been revealed to induce epithelial-mesenchymal transition, which reflects the metastasis of tumor [45]. Moreover, we proved that differentially downstream target genes with IGF2BP3 were enriched in cell division, DNA replication, and functions related to cell proliferation. As we all know, abnormal cell division and DNA replication are signatures of cancer progression [46]. Convincing evidence has shown that an abnormal expression of CLDN4 may contribute to cancer metastasis [47,48]. Taken together with our results that the overexpression of CLDN4 prominently enhanced malignant phenotype of gallbladder cancer cell, it was demonstrated that the increased expression of CLDN4 reflects the progression of gallbladder cancer.

As a reader of m6A, IGF2BP3 controls the destiny of target mRNA by identifying the m6A sequence of RNA. For example, IGF2BP3 accelerated the growth activity of cancer cells by up-regulating the expression of CDK4 in an m6A-dependent manner [49]. Thus, we demonstrated that IGF2BP3 promoted the malignant activity of gallbladder cancer cells by targeting CLDN4 in an m6A-dependent manner. In addition, our study found that interfering with IGF2BP3 inhibited the phosphorylation of NF-κB and IKKβ, and CLDN4 overexpression could deprive this effect. It has been reported that deregulation of NF-κB and IKKβ phosphorylations is a hallmark of cancer [50]. Meanwhile, other studies have reported that inhibition of NF-κB can enhance the antitumor activity of gemcitabine in gallbladder cancer, which is a key target for tumor therapy [51]. Therefore, we suggest that IGF2BP3 activates NF-kB pathway by upregulating CLDN4, which is a promising area for gallbladder cancer therapy.

Macrophages are the most abundant immune infiltrating cells in tumors, which can be differentiated into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages [52]. Our study found that IGF2BP3 promoted macrophages recruitment in gallbladder cancer and induced M2 macrophage polarization. M2-polarized macrophages, commonly considered TAMs, play a pro-tumor role in cancer through regulation [53]. Studies have shown that M2-polarized macrophages drives tumor immune escape, thereby promoting tumor progression [54]. Therefore, we hypothesized that high expression of IGF2BP3 leading to poor prognosis in patients with gallbladder cancer may closely associated with its induction of M2-polarized macrophages in the TME. In addition, our results indicated that high expression of IGF2BP3 in gallbladder cancer not only upregulated STAT3 mRNA and protein expression in THP-1 differentiated macrophages, but also induced M2 macrophage differentiation. STAT3 has been reported to be closely related to M2 macrophage polarization [55]. Thus, we hypothesized that gallbladder cancer cells-drived IGF2BP3 promoted macrophage M2 polarization via activating STAT3 signalling pathway. Therefore, it is necessary to further explore the mechanism by which IGF2BP3 regulates the STAT3 signalling pathway to induce M2 macrophage polarization in gallbladder cancer in the future.

Conclusions

In summary, the m6A reader IGF2BP3 is up-regulated in patients with gallbladder cancer and can be served as an independent prognostic factor for gallbladder cancer. IGF2BP3 promotes malignant activities of gallbladder cancer in vitro and in vivo. Mechanistically, silencing of IGF2BP3 alters the gene expression profile of gallbladder cancer cells, specifically supressing CLDN4 expression. Overexpression of CLDN4 triggers malignant activity of gallbladder cancer cells. IGF2BP3 enhances the mRNA stability of CLDN4 depending on m6A modification to activate the NF-kB signalling pathway to boost the progression of gallbladder cancer. Overexpression of IGF2BP3 in gallbladder cancer promoted macrophage M2 polarization by activating STAT3 pathways. Taken together, our study provides new insights into the pathological mechanism and immunotherapy of gallbladder cancer.

Funding

This research was supported by The Featured Clinical Discipline Project of Shanghai Pudong (Grant Number PWYts2021-06).

Ethics approval and consent to participate

Laboratory mouse were handled instrict accordance with good animal practice as defined by the National Regulations for the Administration of Experimental Animals of China. All protocol was approved by the Ethics Committee of Tongji University School of Medicine and all patients were provided with informed consent.

Supplementary materials

Supplementary Fig. 1 The expression profile of m6A-related genes in GSE76633, GSE74048, and GSE139682. Red * indicates a difference.

Supplementary Fig. 2 Influences of IGF2BP3 knockdown on proliferation, migration, and invasion in vitro. (A-B) Expression of IGF2BP3 in NOZ and GBC-SD cell lines by q-PCR and western blot examination (n = 3). (C-D) IGF2BP3 mRNA and protein expression were performed by q-PCR and western blot in NOZ cells infected with IGF2BP3 shRNA (n = 3). (E) Cell proliferation assays (n = 6). (F-G) Cell migration and invasion were assessed by transwell assays (n = 3). Scale bar = 100 μm. (H-I) Cell cycle progression and apoptosis rate were analyzed by flow cytometry (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. NS: no significant.

Supplementary Fig. 3 Influences of IGF2BP3 overexpression on proliferation, migration, and invasion in vitro. (A-B) IGF2BP3 mRNA and protein expression were performed by q-PCR and western blot in GBC-SD cells infected with overexpression vector of IGF2BP3 (n = 3). (C) Cell proliferation assays (n = 6). (D-E) Cell migration and invasion were assessed by transwell assays (n = 3). Scale bar = 100 μm. (F-G) Cell cycle progression and apoptosis rate were analyzed by flow cytometry (n = 3). **p < 0.01, ***p < 0.001. NS: no significant.

Supplementary Fig. 4 Transcriptional analysis of downstream genes and related signaling pathways for IGF2BP3. (A) The heat map showed the differentially expressed genes in sh-IGF2BP3 and normal cells. (B) GO enrichment analysis. (C) KEGG-enriched terms of differential genes.

Supplementary Fig. 5 IGF2BP3 enhances CLDN4 expression by m6A modification. (A) Correlation between IGF2BP3 and CLDN4 from the GSE76623 and GSE139682 databases. (B) Typical IHC diagrams of CLDN4 staining in sh-NC and sh- IGF2BP3 xenograft tumors (n = 5). (C) eCLIP-seq data from ENCODE database shows IGF2BP3 binds to CLDN4 mRNA. (D) SRAMP database shows the m6A sites of CLDN4 mRNA.

Supplementary Fig. 6 The influence of CLDN4 on gallbladder cancer cells. (A-B) CLDN4 mRNA levels and protein expression were detected in NOZ cells infected with overexpression vector of CLDN4 (n = 3). (C) The proliferation ability was determined by CCK-8 assays (n = 6). (D-E) Representative images of cell migration and invasion (n = 3). Scale bar = 100 μm. (F) Cell migration and invasion were quantified. (G) Apoptosis rate was analyzed by flow cytometry (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Supplementary Fig. 7 IGF2BP3 induces the phosphorylation of NF-kB/IKKβ signaling pathway by upregulating CLDN4. (A) GSEA for samples with sh-IGF2BP3. (B) The network between CLDN4 and the IGF2BP3-mediated genes in NF-kB pathway. (C) Western blot showed the phosphorylation of NF-kB and IKKβ. *p < 0.05. NS: no significant.

CRediT authorship contribution statement

Jian Qin: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing – original draft. Zheng Cui: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing – original draft. Jingyi Zhou: Software, Validation, Formal analysis, Investigation, Visualization, Writing – original draft. Bosen Zhang: Validation, Formal analysis, Resources, Visualization, Supervision, Writing – original draft. Ruiqi Lu: Formal analysis, Resources, Visualization, Supervision, Writing – review & editing. Youcheng Ding: Resources, Writing – review & editing. Hai Hu: Project administration, Writing – review & editing. Jingli Cai: Conceptualization, Formal analysis, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

Not applicable.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2023.101764.

Appendix. Supplementary materials

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Data availability

  • Data available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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mmc8.docx (17KB, docx)
mmc9.docx (16.1KB, docx)
mmc10.docx (17.3KB, docx)

Data Availability Statement

  • Data available on request.

Articles from Translational Oncology are provided here courtesy of Neoplasia Press

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