IGF2BP3 functions as a potential oncogene and is a crucial target of miR-34a in gastric carcinogenesis
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Gastric cancer (GC) is one of the frequent causes of cancer-related death in eastern Asian population. IGF2BP2 lists in the top rank up-regulated genes in GC, but its functional role is unclear.
The expression of IGF2BP3 in GC cell lines and primary samples was examined by qRT-PCR and Western blot. The biological role of IGF2BP3 was revealed by a series of functional in vitro studies. Its regulation by microRNAs (miRNAs) was predicted by TargetScan and confirmed by luciferase assays and rescue experiments.
IGF2BP3 ranked the No.1 of the up-regulated genes by expression microarray analysis in GC cell lines. The expression level of IGF2BP3 was observed in GC tissues comparing with non-tumorous gastric epitheliums. The up-regulated IGF2BP3 expression was associated with poor disease specific survival. IGF2BP3 knockdown significantly inhibited cell proliferation and invasion. Apart from copy number gain, IGF2BP3 has been confirmed to be negatively regulated by tumor-suppressive miRNA, namely miR-34a. The expression of miR-34a showed negative correlation with IGF2BP3 mRNA expression in primary GC samples and more importantly, re-overexpression of IGF2BP3 rescued the inhibitory effect of miR-34a.
We compressively revealed the oncogenic role of IGF2BP3 in gastric tumorigenesis and confirmed its activation is partly due to the silence of miR-34a. Our findings identified useful prognostic biomarker and provided clinical translational potential.
KeywordsGastric cancer IGF2BP3 miR-34a
Breast cancer resistance protein
Gene Set Enrichment Analysis
High-mobility group AT-hook 2.
Hepatocyte nuclear factor 4α
Heterogeneous Nuclear Ribonucleoprotein M
Insulin-like growth factor-2 mRNA-binding protein 3
- KH domains
K homology domains
- Per 1
RNA recognition motifs
The Cancer Genome Atlas
Transforming growth factor-β-induced factor 2
World Health Organization
Gastric cancer (GC) is one of the most prevalent malignancies worldwide. Accordingly, its incidence ranks the 4th in men and 5th in women while it causes the 3rd cancer-related death for men and 5th for women . Although the incidence appears to decrease in recent years, the mortality still remains high, which may due to the delayed diagnosis and the lack of effective treatment. Its occurrence and development often arise from the interaction between internal genetic heterogeneity and multiple external risk factors, such as Helicobacter Pylori infection and high-salt diet . For decades, this severe disease has attracted public attention. In 1965, Lauren P identified two types of GC, diffuse and intestinal, based on different histological features , which has been widely used in clinical pathology since then. Until 2010, another histological classification was suggested by World Health Organization (WHO): tubular, papillary, mucinous and poorly cohesive (including signet ring cell carcinoma), plus uncommon histologic variants . In 2014, The Cancer Genome Atlas (TCGA) proposed a novel characterization which was built upon the molecular mechanisms, and this classification leads to a brand new insight and an in-depth understanding of GC .
Many new emerging technologies are employed in GC research including expression microarray. By screening the putative dysregulated genes achieved by expression microarray analysis in nine GC cell lines, we found IGF2BP3 (Insulin-like growth factor-2 mRNA-binding protein 3) listing in the No.1 rank of the up-regulated genes.
IGF2BP3, also known as IMP3, belongs to a conserved IGF2 mRNA-binding protein family. IGF2BP3 was first identified due to its high abundance in pancreatic carcinoma . After its initial identification, IGF2BP3 has soon been explicated to be a mainly over-expressed member among the family in various tumor types, such as squamous cell carcinoma , lung cancer , melanoma , colon cancer , liver cancer . And the aberrant upregulation implicated its potential oncogenic role in tumorigenesis. Furthermore, accumulating evidences demonstrated that IGF2BP3 represented a promising biomarker in different cancers, such as colon cancer  and GC . However, knowledge of its function and regulation in GC is still quite limited.
High expression of IGF2BP3 mRNA but with low copy number gain rate, suggested that post-transcriptional regulation might play an important role for the IGF2BP3 upregulation in GC. By bioinformatic analysis, we found IGF2BP3 might be regulated by miR-34a (www.microrna.org), which was listed in the relative top rank. microRNAs (miRNAs) has been thought to be new regulators of gene expression through binding to the 3' untranslated regions (UTRs) of the targeted mRNAs , and then degrade or translationally inhibit those targeted mRNAs. Deviant expressions of miRNAs were detected in various human malignancies , and the aberrant expression is always correlated with oncogenesis [16, 17].
Thus in current study, we will firstly investigate the basic expression and functional role of IGF2BP3 in GC. Secondly, we will comprehensively reveal the expression regulation of IGF2BP3 by miR-34a in GC and detect their expression correlation in primary samples. Finally, the clinical correlation and survival-prediction significance of these potential biomarkers will be revealed. Collectively, we aim to deeply explore the molecular mechanism of up-regulated IGF2BP3 in gastric tumorigenesis and offer a translational potential for clinical intervention of GC.
GC cell lines and primary gastric tumor samples
Human GC cell lines (MKN1, MKN7, MKN28, MKN45, SNU1, SNU16, AGS, KatoIII, NCI-N87, MGC-803, SGC-7901, TMK-1) and immortalized gastric epithelial cells (GES-1 and HFE-145) were described previously . Cells were cultured at 37 °C in humidified air atmosphere containing 5% CO2 in RPMI 1640 (GIBCO, Grand Island, NY) medium supplemented with 10% fetal bovine serum (GIBCO). A cohort of 247 formalin-fixed paraffin embedded tissues of GCs diagnosed between 1999 and 2006 in the Prince of Wales Hospital, Hong Kong was retrieved. Ethical approval was obtained from the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Cultured cells were harvested for extracting total RNA with TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA synthesis was performed with a High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Carlsbad, CA). The variations of mRNA expression of related genes were quantified by qRT-PCR and primers were listed as following: IGF2BP3 (sense: AGT TGT TGT CCC TCG TGA CC; anti-sense: GTC CAC TTT GCA GAG CCT TC); B2M (sense: ACT CTC TCT TTC TGG CCT GG; anti-sense: ATG TCG GAT GGA TGA AAC CC). The protocol of qRT-PCR was described in a previous study . For microRNA expression detection, miR-34a expression in GC was detected by Taqman miRNA assays, and they were used to quantify the levels of mature miR-34a (Assay ID: #4427975, Life Technologies). The relative expression level of miR-34a was normalized by RNU6B expression (Assay ID: #001093). 7500 Fast Real-Time System (Applied Biosystems) was used for the qPCR reaction. And the reaction system was incubated at 95 °C for 30 s, followed by 40 cycles of 95 °C for 8 s and 60 °C for 30 s.
Protein extraction and western blot analysis
The protein extraction and western blot analysis protocol were described in our previous study . The primary antibodies detected IGF2BP3 (#07-104) was from Merck Millipore. Other primary antibodies were from Cell Signaling (Danvers, MA) including p21 (#2946), p27 (#2552), p-Rb (Ser807/811) (#9308), cleaved-PARP (Asp214) (#9541), Cyclin D3 (#2936), CDK4 (# 12790), CDK6 (#3136) and GAPDH (#2118). The secondary antibodies were anti-Mouse IgG-HRP (Dako, 00049039, 1:30000) and anti-Rabbit IgG-HRP (Dako, 00028856, 1:10000).
Immunohistochemistry was to conduct tissue microarray within a 4 μm-thick section of each clinical sample using Ventana NexES automated Stainer (Ventana Corporation). All sections were performed microwaving in EDTA antigen retrieval buffer after de-waxing in xylene and graded ethanol. The IGF2BP3 primary antibody (1:100, 07–104) was from Merck Millipore. The cytoplasmic expression of IGF2BP3 was evaluated according to the proportion of tumor cells with intensity of cytoplasmic staining .
miRNA and siRNA transfection for functional assays
The miRNA precursor miR-34a (PM11030) and scramble control (AM17110) were commercially available from Life Technologies. siIGF2BP3-1 (SI03230759) and siIGF2BP3-2 (SI04234167) were obtained from Qiagen (Valencia, CA). Lipofectamine 2000 Transfection Reagent (Invitrogen) was used for all transfection assays. The cell proliferation experiments, colony formation assays in monolayer, cell invasion assays, flow cytometry analysis for cell cycle distribution have been described in our previous work . The experiments were repeated in triplicate to obtain standard deviations.
Luciferase activity assays
The putative miR-34a binding site in IGF2BP3 3'UTR of was subcloned into pMIR-REPORT Vector (Ambion). The oligonucleotides that encompass the miR-34a recognition site and the oligonucleotides which contain the mutated binding site were listed in Additional file 1: Table S1. Prior to digestion and subcloning, oligonucleotides were annealed in 30 mmol/L HEPES buffer that contains 100 nmol/L potassium acetate and 2 mmol/L magnesium acetate . The firefly luciferase constructs were co-transfected with Renilla luciferase vector control into MGC-803 cells. Dual luciferase reporter assays (Promega, Madison, WI) were performed 24 h after transfection.
Treatment of cell lines with 5-Aza and TSA
AGS, MKN1, NCI-N87 and MGC-803 were treated with demethyltransferase inhibitor (5-Aza, Sigma, St Louis, MO) and histone-deacetylase inhibitor trichostatin A (TSA, Sigma) . Cells were incubated with 10 μM 5-Aza for 72 h in 5-Aza group, while in TSA group, cells were treated with 100 nM TSA for 24 h. As for the combination treatment group, cells were treated with 5-Aza for 96 h and 100 nM TSA was added into the culture medium in the last 24 h. Equal amount of vehicle DMSO (Sigma) was used in negative control groups.
In vivo tumorigenicity model
MGC-803 cells (107 cells suspended in 0.1 ml PBS) were transfected with miR-34a or scramble control then were injected subcutaneously into the left and right dorsal flank of 4-week old Balb/c nude mice respectively. Diameters of tumors were measured and documented each 5 days with a total of 25 days. Tumor volume (mm3) was accessed by measuring the longest and shortest diameter of the tumor and calculating as follows: volume = (shortest diameter) 2 × (longest diameter) × 0.5. All animal handling and experimental procedures were approved by Department of Health in Hong Kong.
For the rescue experiments, AGS and MKN28 cells were first transfected with miR-34a precursor or negative control respectively. After 24 h incubation, IGF2BP3 expression plasmid (#19879, Addgene) together with empty vector (pcDNA3.1, Life Technologies) were transfected to the cells using FuGENE HD Transfection Reagent (Roche, Nutley, NJ). After another 24 h, cells were harvested for functional study (MTT proliferation, monolayer colony formation, and cell invasion assays). The in vivo rescue experiments were performed using MGC-803 cells .
The Student T test was used to compare the differences in functional differences between siIGF2BP3 and siScramble control transfected cells. It is also used to compare the biological behavior between miR-34a transfected cells and scramble miRNA transfectant counterparts. Nonparametric Pearson Chi-Square test was used to evaluate the correlation between IGF2BP3 expression and selected clinicopathologic parameters. Kaplan-Meier method was used to estimate the survival rate for each parameter and the equivalences of the survival curve were examined by log-rank statistics. For those parameters were found statistically significant in the univariate survival analysis (P < 0.05), the Cox proportional hazards model was employed to further evaluate them for multivariate survival analysis. All statistical analysis was performed by SPSS software (version 22.0; SPSS Inc). A two-tailed P-value of less than 0.05 was considered statistically significant and the P-value less than 0.001 was considered highly significant.
IGF2BP3 is highly expressed in GC
Overexpression of IGF2BP3 correlates with poor survival in GC
Silence of IGF2BP3 exerts tumor-suppressive effects both in vitro and in vivo
IGF2BP3 is a direct target of miR-34a in GC
miR-34a functions as potential tumor suppressor in GC cells
Downregulation of miR-34a correlates with poor survival in GC
IGF2BP3 re-expression rescues the tumor-suppressive function of miR-34a
IGF2BP3, contains a structure of two N-terminal RNA recognition motifs (RRM) and four C-terminal messenger ribonucleoprotein K homology (KH) domains . The C-terminal KH domains are required for RNA-binding, and this attribute decides the cytoplasmic localization and granular distribution of IGF2BP3 [38, 39].
IGF2BP3 has been reported to participate in tumorigenicity in numerous kinds of cancers and its overexpression in tumorous tissues makes it a promising biomarker for diagnosis or prognosis accordingly . The enrichment of IGF2PB3 in human malignancies might promote tumor growth via raising the quantity of IGF2 . Moreover, IGF2BP3 enhanced cell proliferation through synergizing with hnRNPM in the nucleus, leading to an elevated level of cyclins . Recently, IGF2BP3 was believed to, mainly through the let-7 family, boost the expression level of High-mobility group AT-hook 2 (HMGA2) by preventing miRNA binding . In breast cancer, apart from the capability of elevating the invasive potential , IGF2BP3 was also believed to be involved in chemo-resistance by stabilizing the mRNA of breast cancer resistance protein (ABCG2) .
There were several reports focusing on IGF2BP3 in GC in the past years. A group of Japanese scientists suggested IGF2BP3 to be an independent poor prognostic marker and an indicator for peritoneal dissemination in GC after surgery . Similarly, merely based on their own clinical cohort, Kim HJ et al. also indicated that IGF2BP3 predicted worse outcome and malignant effusion among GC patients . Recently, IGF2BP3 was again proposed to be associated with poor survival in Brazilian population . However, all the previous studies failed to investigate the functional role of IGF2BP3 in gastric carcinogenesis. In our study, we provided the first evidence to comprehensively reveal the oncogenic function of IGF2BP3 in GC. Upregulation of IGF2BP3 was detected in both GC cell lines and primary GC samples in our cohort, as well as other published databases. In addition, we described IGF2BP3 expression with molecular classification proposed by TCGA. The IGF2BP3 was uniformly high expressed in all the four subtypes in contrast with normal gastric tissues. This abundance was proved to promote cell growth and invasive ability in GC cell lines via functional assays, which was further confirmed by GSEA results. Moreover, data from flow cytometry demonstrated that IGF2BP3 might play a role in G1 to M phase transition. And a previous work done by Rivera Vargas T et al. explained that IGF2BP3 protein directly bound with the mRNAs of CCND1/3, together with hnRNPM, to control the expression of CCND1/3 in the nucleus .
To further investigate the mechanism of IGF2BP3 upregulation in gastric carcinogenesis, we firstly checked the copy number change and mRNA expression in TCGA cohort. However, more cases with mRNA expression than the cases with copy number gain were detected, we thereby focused on post-transcriptional regulation mechanism, such as dysregulated related miRNAs. After screening and a series validation assays, miR-34a was confirmed to be the main regulator of IGF2BP3 and their expression showed negative correlation in primary samples, especially in intestinal type GC. Rescue experiments demonstrated that IGF2BP3 is a crucial target of miR-34a in GC because IGF2BP3 re-overexpression partly relieved the tumor-suppressive effect of miR-34a. miR-34a has been identified as a classical tumor suppressive miRNA in variety of malignancies [48, 49]. Multiple putative targets have been revealed for miR-34a to exert its tumor-suppressive role , such as period 1 (Per1) in cholangiocarcinoma , NOTCH1 in colon cancer stem cells , Hepatocyte nuclear factor 4α (HNF4α) in hepatocellular carcinoma  and transforming growth factor-β-induced factor 2 (Tgif2) in bone metastasis . It has been also reported that miR-34a enhances the sensitivity of GC cells against cisplatin through PI3K/AKT/survivin pathway [50, 55]. On the other hand, our study suggested the activation of IGF2BP3 in GC is partly due to silence of miR-34a and enriched the target pool of miR-34a. Furthermore, a great number of papers have proposed that miR-34a mimics had therapeutic potential to inhibit cancer progression [56, 57, 58].
In summary, high expression of IGF2BP3 in GC was associated with poor prognosis. Knockdown of IGF2BP3 significantly suppressed its oncogenic role. The upregulation of IGF2BP3 was partly due to the silence of tumor-suppressive miR-34a in some GC samples. Our findings not only clarified the mechanism of IGF2BP3 upregulation in GC but also provided therapeutic target for clinical intervention.
We acknowledge the TCGA research Network (http://cancergenome.nih.gov/), The UCSC Cancer Genomics Browser (https://genome-cancer.ucsc.edu/), and NCI Center for Cancer Genomics Office (http://gdc.nci.nih.gov/) for providing the gastric cancer data set and analysis.
This study is supported by General Research Fund (RGC Reference No. CUHK14114414 and CUHK14110016) from The Research Grants Council of Hong Kong.
Availability of data and materials
KFT and WK designed the experiments, provided direction and guidance on the whole project. YZ, TH, HLS, CCW, YD, FW and BZ conducted the experiments, analyzed the results and performed bioinformatics analysis. YZ, TH and WK drafted the manuscript. WKKW, ASLC and JY reviewed the manuscript and made significant revisions on the drafts. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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