Background
Pancreatic cancer is a high-mortality tumour with a five-year overall survival rate of approximately 7% [
1,
2]. Among the causes of cancer-related death, this malignant tumour ranks fourth in the United States and sixth in China [
1,
3]. Approximately 80% of patients with pancreatic cancer have dissemination at the time of diagnosis [
1,
4]. These patients have lost the chance for radical treatment of pancreatic cancer. In the past decade, despite advancements in anti-metabolism therapy and targeted therapy, the overall survival rate of patients has not significantly improved due to the late pathological stage, high invasive phenotype and chemotherapy resistance.
Insulin-like growth factor 2-mRNA binding proteins (IGF2BPs), also known as IGF-II mRNA binding proteins (IMPs), are encoded by different genes that belong to the regulatory RNA binding protein family and are involved in the localization of their target RNA, stability and translation control [
5]. As the names of these proteins indicate, they are recognized members of the IGF axis that can be linked to IGF2 transcripts [
6,
7]. To date, insulin-like growth factor 2 mRNA binding proteins, including IGF2BP1 (IMP1), IGF2BP2 (IMP2), and IGF2BP3 (IMP3), are a unique family of m6A readers that target the common m6A sequence by recognizing thousands of mRNA transcripts [
8]. In mammals, the protein domains of the three members of the IGF2BP protein family are strikingly similar. All three members of the protein family contain two N-terminal RRMs and four C-terminal hnRNPK homology (KH) domains. The latter are arranged in two dual domains (KH1 + 2 and KH3 + 4) [
9]. Consistent with the conservation of six potential RNA binding domains, all three IGF2BPs bind to single-stranded RNA in vitro and in vivo [
9‐
11]. However, the role of the entire IGF2BP family in pancreatic cancer remains controversial. Therefore, it was necessary to probe the role of the IGF2BP family in pancreatic cancer.
The Cancer Genome Atlas (TCGA) is considered to be the largest cancer database, containing more than 20,000 primary cancer samples and normal matched samples for multiple cancer types. Therefore, we can use bioinformatics methods to study tumour data more deeply. To evaluate the relationship between the IGF2BP family and pancreatic cancer progression, we analysed mRNA expression in pancreatic cancer samples from the TCGA with R software and verified it in patients.
Methods
GEPIA dataset
Gene Expression Profiling Interactive Analysis (GEPIA) is a new web-based tool for gene expression analysis between tumour and normal data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project, applying a standard processing pipeline. It provides customizable functions such as tumour and normal differential expression analysis, and we can demonstrate the expression of IGF2BP1–3 in pancreatic cancer and normal tissues. GEPIA possesses key variable and interactive functions, including profile plotting, differential expression analysis, patient survival analysis, similar gene detection and dimensionality reduction analysis.
LinkedOmics dataset
LinkedOmics is a new and unique tool in the software ecosystem for disseminating data from all 32 TCGA cancer types. It can be used to access, analyse, and compare multiomics data within and across tumour types. We performed a prognostic analysis for the IGF2BP gene family using the LinkedOmics pancreatic cancer dataset.
TCGA data acquisition and differentially expressed IGF2BP gene analysis
The pancreatic cancer data in the TCGA contains 178 pancreatic cancer samples with important information, including pathological grade and clinical stage. All mRNA expression data, along with clinical data, were downloaded and further analysed with R software.
We utilized the “limma” package in R software to normalize the original expression levels of mRNAs downloaded from the TCGA. The “limma” package was used to analyse the expression of each IGF2BP gene between every grade and stage of cancer tissues. Last, a P-value < 0.05 was set as the filter condition for differentially expressed IGF2BP.
Gene set enrichment analysis of pancreatic cancer
Performed gene enrichment analysis (version 3.0, the broad Institute of MIT and Harvard,
http://software.broadinstitute.org/gsea/downloads.jsp) between pancreatic cancer and normal tissues to study the biological pathways of pancreatic cancer. Specifically, set “collapse data set to gene symbols” to false, set the number of marks to 1000, set the “permutation type” to phenotype, set the “enrichment statistic” to weighted, and utilized the Signal2Noise metric to rank genes. The high expression group was taken as the experimental group, and the low expression group was taken as the reference group. The “c2.cp.kegg.v7.0.symbols.gmt” gene set database was utilized for enrichment analysis. Cut-off criteria including gene set size > 500 and < 15, FDR < 0.25, and nominal
P-value < 0.05.
Functional enrichment analyses of pancreatic cancer
Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) functional enrichment analyses were performed to analyse IGF2BP2 and IGF2BP3. The Database for Annotation, Visualization, and Integrated Discovery (DAVID,
https://david.ncifcrf.gov/) was applied to identify enriched KEGG and GO pathways and terms.
Cell lines and reagents
The human pancreatic cancer cell lines, including ASPC-1, SW1990, PANC-1, MIA Paca-2 and HPDE6-C7, were purchased from the University of Colorado Cancer Center Cell Bank. ASPC-1, PANC-1, MIA Paca-2 and HPDE6-C7 cells were cultured in DMEM medium with 10% FBS (Invitrogen, Carlsbad, CA, USA) at 37 °C in a 5% CO2 atmosphere. SW1990 cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) at 37 °C in a 5% CO2 atmosphere. Cells were digested and passaged when cell confluence reached 80–90%.
Quantitative reverse transcription polymerase chain reaction
Total RNA was extracted from the ASPC-1, SW1990, PANC-1, MIA Paca-2 and HPDE6-C7 cell lines using TRIzol reagent (Life Technologies) according to the instructions provided by the manufacturer. Total RNA (1 μg) was used as a template to synthesize complementary DNA (cDNA) using a PrimeScript RT Reagent Kit with cDNA Eraser (Takara Biotechnology). Subsequently, qRT-PCR was performed using SYBR Premix Ex Taq (Takara Bio Inc.). The primer sequences used for real-time PCR are listed in Table
S1. All qRT-PCR assays were performed on an ABI 7900 system (Applied Biosystems).
Cell proliferation assay
The Cell Counting Kit-8 (CCK-8) assay (MedChemExpress) was used according to the protocol provided by the manufacturer to assess cell proliferation. ASPC-1 and SW1990 cells were seeded into 96-well plates (5 × 103 cells/plate) and cultured at 37 °C. Ten microlitres of CCK-8 solution was added to each well of the plate at the following times: 0 h, 24 h, 48 h, 72 h and 96 h. Optical density (OD) was measured at 1–4 days at a wavelength of 450 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific, Inc.).
Protein extraction and Western blot analysis
Utilized RIPA lysis buffer with 1% phenylmethanesulfonyl fluoride (PMSF) and DL-dithiothreitol (DTT) to extracted total protein. The BCA protein assay kit (Beyotime Biotechnology) was used to determine the concentration of the protein lysate. Equivalent (30 μg) protein was isolated by 10% SDS-PAGE. Then, the proteins were transferred to PVDF membranes (0.45 mm; Beijing Solarbio Science & Technology Co., China). Before incubated the membranes with IGF2BP2 and IGF2BP3 antibodies (1:1000, R&D Systems, MN, USA) at 4 °C for 12 h, the membranes were blocked at room temperature with 5% BSA for 1 h. Then GAPDH rabbit polyclonal antibody (1:4000, Proteintech, USA) was utilized as a loading control for normalization. HRP-conjugated secondary anti-rabbit antibody (1:4000; ProteinTech Group) was incubated at room temperature about 1 h. Finally, the bands were placed on an Omega Lum G machine (Aplegen, USA) and visualized using ECL reagents (Thermo Fisher Scientific).
SW1990 cells were seeded into 6-well plates (1 × 103 cells/plate) and cultured for 14 days. Then, cells were fixed with 10% formaldehyde for 5 min and stained with 1% crystal violet for 30 s prior to counting the number of colonies.
Cell invasion assay
Cell invasion was performed with transwell plates (24-well insert, 8 μm pore size; BD Biosciences, Bedford, MA, USA). The filters (Corning Inc., USA) were covered with 55 μL of Matrigel (1:8 dilution; BD Biosciences). Then, 5 × 104 SW1990 cells were distributed in 100 μl of serum-free RPMI-1640 medium and inoculated into the upper chamber. Next, 600 μl of 90% RPMI-1640 medium supplemented with 10% FBS was added to the bottom chamber. After 24 h of incubation, the chamber was fixed with 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet for 30 min. Finally, magnification microscope to count the number of invading cells in the bottom chamber.
Statistical analysis
In this study, the experiments were carried out in triplicate, and the data were expressed as the mean ± standard deviation. The t-test was utilized for the statistical analysis of the data. Comparisons between multiple groups were performed with one-way ANOVA followed by an LSD-t test. P < 0.05 was considered significant.
Discussion
In the past few years, despite tremendous efforts in pancreatic cancer research, the 5-year patient survival rate has not improved significantly. Patients with early pancreatic cancer have a good prognosis and can be cured by surgery combined with adjuvant therapy. However, most patients with advanced pancreatic cancer cannot undergo surgical resection alone. For patients with advanced pancreatic cancer, it is essential to explore more effective prognostic markers and therapeutic targets. Therefore, we screened the IGF2BP protein family through bioinformatics and conducted a differential analysis. IGF2BP2 and IGF2BP3, which are related to pancreatic cancer progression and survival, were further analysed, and their functions were verified in vitro.
In the preliminary analysis, three members of the IGF2BP protein family were identified to have differential expression between pancreatic cancer and adjacent tissues. Further analysis confirmed that only IGF2BP2 and IGF2BP3 were associated with pancreatic cancer progression. Therefore, only IGF2BP2 and IGF2BP3 were subjected to gene enrichment analysis to assess their cell compositions, molecular functions and biological characteristics.
In the gene set enrichment analysis, the base excision repair (BER) pathway was determined to be the most relevant pathway for IGF2BP2. Notably, the BER pathway plays a significant role in maintaining genome integrity, and many human health issues occur when any part of the BER pathway is aberrant [
17]. This pathway begins with glycosylation enzymes and recognizes and excises lesions through the cleavage of glycosidic bonds [
17]. Dianov et al. verified that aberrant P53 signalling could lead to failure of the BER coordination mechanism, APE1 overexpression and genome instability [
18]. In our enrichment analysis, P53 was also upregulated, consistent with the conclusion of Dianov et al. Although the relationship between abnormalities in the BER pathway and the development and prognosis of cancer has been studied [
19‐
21], in pancreatic cancer, whether IGF2BP2 is associated with this process has not yet been elucidated. The positive correlation between pathogenic
Escherichia coli (
E. coli) infection and colon cancer has been confirmed in multiple studies [
22,
23]. The infection of pathogenic
E. coli destroys the microenvironment of the microflora in the intestinal tract, thereby inducing colon cancer [
22‐
24]. In studies of pathogenic
Escherichia coli infection-induced pathways for pancreatic cancer, there is a lack of clear evidence that this pathway is associated with pancreatic cancer. The upregulation of IGF2BP3 expression in pancreatic cancer tissues supports research on this pathway. IGF2BP3 imbalance-induced pancreatic cancer may be related to pathogenic
E. coli infection.
The autoimmune response to IGF2BP2 observed in hepatocellular carcinoma and colorectal, ovarian, and breast cancers supports the potential of autoantibodies against IGF2BP2 as biomarkers for cancer screening, diagnosis, and prognosis [
5]. Consistent with the results of our Cox regression model in pancreatic cancer, the overexpression of IGF2BP2 in basal-like breast cancer and oesophageal adenocarcinoma predicts short-term survival for patients. At the cellular level, IGF2BP2 enhances genome instability and stimulates cancer cell proliferation and migration. Cao et al. believed that the dysregulation of IGF2BP2 was related to insulin resistance, diabetes and carcinogenesis and may potentially become a powerful biomarker and candidate target for related diseases [
24]. In fact, IGF2BP3 might differentiate normal tissues from cancerous tissues and serve as a prognostic marker for colorectal, hepatocellular, and ovarian clear-cell carcinomas [
25‐
27]. Previous research has confirmed that IGF2BP3 is involved in cell growth and migration in early embryonic development [
28]. Similarly, both of our results confirmed the role of IGF2BP2 and IGF2BP3 in inhibiting tumour progression.
Conclusion
In summary, we successfully revealed that members of the IGF2BP protein family can be used for the diagnosis and prognosis of advanced pancreatic cancer. Both IGF2BP2 and IGF2BP3 have great potential to become biomarkers for pancreatic cancer, as verified in patients. Although we explored the mutation types and possible carcinogenic mechanisms of IGF2BP2 and IGF2BP3 in pancreatic cancer, the mechanisms that promote the progression of pancreatic cancer need further study.
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