Zhou et al. Molecular Cancer (2017) 16:77 DOI 10.1186/s12943-017-0647-2 RESEARCH Open Access IGF2BP3 functions as a potential oncogene and is a crucial target of miR-34a in gastric carcinogenesis Yuhang Zhou1,2,3†, Tingting Huang1,2,3,4†, Ho Lam Siu1†, Chi Chun Wong2, Yujuan Dong2, Feng Wu1, Bin Zhang5, William K. K. Wu2,6, Alfred S. L. Cheng4,7, Jun Yu2,4,8, Ka Fai To1,2,3,4* and Wei Kang1,2,3,4* Abstract Background: 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. Method: 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. Results: 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. Conclusion: 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. Keywords: Gastric cancer, IGF2BP3, miR-34a Background infection and high-salt diet [2]. For decades, this severe Gastric cancer (GC) is one of the most prevalent malig- disease has attracted public attention. In 1965, Lauren P nancies worldwide. Accordingly, its incidence ranks the identified two types of GC, diffuse and intestinal, based 4th in men and 5th in women while it causes the 3rd on different histological features [3], which has been cancer-related death for men and 5th for women [1]. widely used in clinical pathology since then. Until 2010, Although the incidence appears to decrease in recent another histological classification was suggested by years, the mortality still remains high, which may due to World Health Organization (WHO): tubular, papillary, the delayed diagnosis and the lack of effective treatment. mucinous and poorly cohesive (including signet ring cell Its occurrence and development often arise from the carcinoma), plus uncommon histologic variants [4]. In interaction between internal genetic heterogeneity and 2014, The Cancer Genome Atlas (TCGA) proposed a multiple external risk factors, such as Helicobacter Pylori novel characterization which was built upon the molecu- lar mechanisms, and this classification leads to a brand * Correspondence: [email protected]; [email protected] † new insight and an in-depth understanding of GC [5]. Equal contributors Many new emerging technologies are employed in GC 1Department of Anatomical and Cellular Pathology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The Chinese University of research including expression microarray. By screening Hong Kong, Hong Kong, SAR, People’s Republic of China the putative dysregulated genes achieved by expression Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. Molecular Cancer (2017) 16:77 Page 2 of 14 microarray analysis in nine GC cell lines, we found 1999 and 2006 in the Prince of Wales Hospital, Hong IGF2BP3 (Insulin-like growth factor-2 mRNA-binding Kong was retrieved. Ethical approval was obtained from protein 3) listing in the No.1 rank of the up-regulated the Joint Chinese University of Hong Kong-New Terri- genes. tories East Cluster Clinical Research Ethics Committee. 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 RNA extraction and quantitative real-time polymerase carcinoma [6]. After its initial identification, IGF2BP3 chain reaction (qRT-PCR) has soon been explicated to be a mainly over-expressed Cultured cells were harvested for extracting total RNA member among the family in various tumor types, such with TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA as squamous cell carcinoma [7], lung cancer [8], melan- synthesis was performed with a High-Capacity cDNA oma [9], colon cancer [10], liver cancer [11]. And the Reverse Transcription Kits (Applied Biosystems, Carls- aberrant upregulation implicated its potential oncogenic bad, CA). The variations of mRNA expression of related role in tumorigenesis. Furthermore, accumulating genes were quantified by qRT-PCR and primers were evidences demonstrated that IGF2BP3 represented a listed as following: IGF2BP3 (sense: AGT TGT TGT promising biomarker in different cancers, such as colon CCC TCG TGA CC; anti-sense: GTC CAC TTT GCA cancer [12] and GC [13]. However, knowledge of its GAG CCT TC); B2M (sense: ACT CTC TCT TTC function and regulation in GC is still quite limited. TGG CCT GG; anti-sense: ATG TCG GAT GGA TGA High expression of IGF2BP3 mRNA but with low copy AAC CC). The protocol of qRT-PCR was described in a number gain rate, suggested that post-transcriptional previous study [19]. For microRNA expression detection, regulation might play an important role for the IGF2BP3 miR-34a expression in GC was detected by Taqman upregulation in GC. By bioinformatic analysis, we found miRNA assays, and they were used to quantify the levels IGF2BP3 might be regulated by miR-34a (www.micror- of mature miR-34a (Assay ID: #4427975, Life Technolo- na.org), which was listed in the relative top rank. micro- gies). The relative expression level of miR-34a was nor- RNAs (miRNAs) has been thought to be new regulators malized by RNU6B expression (Assay ID: #001093). of gene expression through binding to the 3' untranslated 7500 Fast Real-Time System (Applied Biosystems) was regions (UTRs) of the targeted mRNAs [14], and then used for the qPCR reaction. And the reaction system degrade or translationally inhibit those targeted mRNAs. was incubated at 95 °C for 30 s, followed by 40 cycles of Deviant expressions of miRNAs were detected in various 95 °C for 8 s and 60 °C for 30 s. human malignancies [15], and the aberrant expression is always correlated with oncogenesis [16, 17]. Protein extraction and western blot analysis Thus in current study, we will firstly investigate the The protein extraction and western blot analysis protocol basic expression and functional role of IGF2BP3 in GC. were described in our previous study [20]. The primary Secondly, we will comprehensively reveal the expression antibodies detected IGF2BP3 (#07-104) was from Merck regulation of IGF2BP3 by miR-34a in GC and detect Millipore. Other primary antibodies were from Cell Sig- their expression correlation in primary samples. Finally, naling (Danvers, MA) including p21 (#2946), p27 (#2552), the clinical correlation and survival-prediction signifi- p-Rb (Ser807/811) (#9308), cleaved-PARP (Asp214) cance of these potential biomarkers will be revealed. (#9541), Cyclin D3 (#2936), CDK4 (# 12790), CDK6 Collectively, we aim to deeply explore the molecular (#3136) and GAPDH (#2118). The secondary antibodies mechanism of up-regulated IGF2BP3 in gastric tumori- were anti-Mouse IgG-HRP (Dako, 00049039, 1:30000) genesis and offer a translational potential for clinical and anti-Rabbit IgG-HRP (Dako, 00028856, 1:10000). intervention of GC. Methods Immunohistochemistry GC cell lines and primary gastric tumor samples Immunohistochemistry was to conduct tissue microarray Human GC cell lines (MKN1, MKN7, MKN28, MKN45, within a 4 μm-thick section of each clinical sample using SNU1, SNU16, AGS, KatoIII, NCI-N87, MGC-803, Ventana NexES automated Stainer (Ventana Corpor- SGC-7901, TMK-1) and immortalized gastric epithelial ation). All sections were performed microwaving in EDTA cells (GES-1 and HFE-145) were described previously antigen retrieval buffer after de-waxing in xylene and [18]. Cells were cultured at 37 °C in humidified air at- graded ethanol. The IGF2BP3 primary antibody (1:100, mosphere containing 5% CO2 in RPMI 1640 (GIBCO, 07–104) was from Merck Millipore. The cytoplasmic Grand Island, NY) medium supplemented with 10% fetal expression of IGF2BP3 was evaluated according to the bovine serum (GIBCO). A cohort of 247 formalin-fixed proportion of tumor cells with intensity of cytoplasmic paraffin embedded tissues of GCs diagnosed between staining [20]. Zhou et al. Molecular Cancer (2017) 16:77 Page 3 of 14 miRNA and siRNA transfection for functional assays Rescue experiments The miRNA precursor miR-34a (PM11030) and scram- For the rescue experiments, AGS and MKN28 cells were ble control (AM17110) were commercially available first transfected with miR-34a precursor or negative con- from Life Technologies. siIGF2BP3-1 (SI03230759) and trol respectively. After 24 h incubation, IGF2BP3 expres- siIGF2BP3-2 (SI04234167) were obtained from Qiagen sion plasmid (#19879, Addgene) together with empty (Valencia, CA). Lipofectamine 2000 Transfection vector (pcDNA3.1, Life Technologies) were transfected Reagent (Invitrogen) was used for all transfection assays.