Background
Gastric cancer (GC) is one of the most lethal malignancies worldwide [
1,
2]. Recently, the Cancer Genome Atlas (TCGA) suggested four types of GC [
3].The key driver genes in GC are still challenging to discern because the cancer has a high level of heterogeneity. Hence, there is an urgent need to better understand the detailed mechanisms that underline GC tumorigenesis and progression.
Receptor tyrosine kinases (RTKs) have emerged as key regulators in carcinogenesis among several solid tumors [
4,
5]. EphA2 belongs to the family of RTKs and functions in bi-directional signal transduction via direct contact with adjacent cells expressing its specific ligand, EphrinA1. Usually, EphA2 is expressed at low levels in normal epithelial cells [
6], whereas high levels of EphA2 have been observed in many solid tumors. Growing evidence indicate that EphA2 plays an important role in cellular transformation, primary tumor initiation, progression, and angiogenesis, and tumor invasion [
7‐
9].
Recently, much attention has been focused on targeting EphA2 in the treatment of pediatric malignant glioma [
10,
11]. We have previously demonstrated that EphA2 over-expression accelerated proliferative and metastatic properties in GC cells [
12,
13], and promoted the epithelial-mesenchymal transition (EMT) through activating Wnt/β-catenin signaling [
14]. Therefore, EphA2 is a proper candidate for developing targeted GC therapy that could inhibit metastasis and induce cytotoxicity in tumor cells while sparing normal cells. However, the regulation of EphA2 and cause of its overexpression in GC are still largely unknown.
miRNA usually negatively regulate gene expression primarily through interaction with the 3′-untranslated region (3’UTR) of target mRNAs [
15]. miRNAs are well known to contribute to tumorigenesis in multiple ways in several cancers, including GC. Recent studies have identified regulatory activities of several miRNAs in GC cell growth, invasion, and migration [
16‐
18], which supports that regulation of these miRNAs could potentially be developed into novel GC therapies.
In this study, we identified miR-302b as one of the most significantly downregulated miRNAs in GC cells. We show that miR-302b is a critical suppressor of GC cell growth and metastasis both in vitro and in vivo, and it inhibits downstream pathways (EMT and Wnt/β-catenin signaling) by directly targeting EphA2. Moreover, our results provide a potential epigenetic target for potential gastric cancer therapies that intervene with EphA2.
Methods
Antibodies and reagents
Primary antibodies for EphA2 (#6997, diluted1:1000), Snai1 (#3879, diluted1:1000), β-catenin (#8480, diluted1:1000), E-cadherin (#3195, diluted1:1000), N-cadherin (#13116, diluted1:1000), c-Myc (#13987, diluted1:1000), CyclinD1 (#2978, diluted1:1000), and GAPD- H (#2118, diluted1:1000) were purchased from Cell Signaling technology MA, USA.
Cell culture
The human gastric adenocarcinoma cell line SGC-7901 was purchased from the Cell Resource Center of Xiangya Central Experiment Laboratory, Central South University (Changsha, Hunan, China) and cultured in RPMI 1640 medium (Hyclone, Waltham, USA). The human gastric adenocarcinoma cell line AGS was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in F-12 K medium. All the cell lines were maintained with 10% fetal bovine serum (FBS) in a humidified incubator (37 °C, 5% CO2).
Cell infection, transfection, and conditioned media preparation
Cell transfection with 2 μM of miRNA mimics or miRNA inhibitors and their negative controls (designed and synthesized by Genepharma [Shanghai,China]) was conducted using Lipofectamine 2000 (Invitrogen,Grand Island, NY, USA). The sequences for these miRNAs are listed in Additional file
1: Table S1.
The 3’UTR of EphA2 sequence was amplified and sub-cloned into the pMIR-REPORT luciferase vector (Ambion, Austin, TX, USA). Mutations in the seed region of the putative miRNA-binding sites of EphA2 mRNA were generated by point mutation PCR. All primers used for this purpose are described in Additional file
2: Table S2.
MTT assay
Cells were seeded in 96-well plates at a density of 1 × 104 cells per well and incubated for 24 h. After varying lengths of time, 10 μl of MTT dye (5 mg/ml, Sigma-Aldrich) was added to each well, and cells were incubated for another 4 h at 37 °C. Afterward, DMSO (150 μl) was added to each well and mixed for 10 min. Spectrometric absorbance at 490 nm was determined using a microplate reader (Bio-Rad, Hercules, USA). Each sample had three replicates.
Cell cycle analysis
Cells were harvested 48 h after seeding, and single-cell suspensions containing 1 × 10
6 cells were fixed with 75% alcohol ethanol. The cell cycle was monitored using propidium iodide (PI) staining of the nuclei. The fluorescence of DNA-bound PI in the cells was measured with a FACScan Flow Cytometer (BD Biosciences, San Diego, CA, USA) [
14].
Scratch wound-healing assay
Cells were plated and grown overnight to confluence in a 6-well plate. Monolayers of cells were wounded by dragging a pipette tip across the surface of the monolayer. Cells were washed to remove cellular debris and allowed to migrate for 24 h. Images were taken at 0 h and 24 h after wounding using an inverted microscope (Olympus, Japan) [
14].
Cell invasion assay
Transwell invasion assays were performed in 24-well, 8-μm pore size, transwell plates according to the manufacturer’s instructions (Corning, New York, NY, USA). The bottom of transwell chamber was coated with BD Matrigel Basement Membrane Matrix. The upper chamber was filled with 1 × 105 cells in RPMI 1640 containing 5% FBS. The lower chamber was filled with RPMI 1640 containing 25% FBS as a chemo-attractant. After the chambers were incubated for 24 or 48 h at 37 °C, non-invading cells on the upper side of the chamber were removed from the surface of the membrane by scrubbing, and invading cells on the lower surface of the membrane were fixed with methanol, mounted, and dried. The number of cells invading through the matrigel was counted by a technician blinded to the experimental settings in four randomly selected microscopic fields of each filter. The test was conducted in three biological replicates.
Western blot
Whole cell extracts were prepared using 0.14 M NaCl, 0.2 M triethanolamine, 0.2% sodium deoxycholate, 0.5% Nonidet P-40 and supplemented with a protease inhibitor (Sigma-Aldrich). Fifty micrograms of protein lysate was loaded into each well lysates were resolved by SDS-PAGE on 10% polyacrylamide gels, and then they were transferred to nitrocellulose or PVDF membranes. After blocking with 5% milk, the transferred membranes were subsequently incubated overnight at 4 °C with primary antibody, followed by secondary antibody for 1 h at routine temperature. Bands were visualized using an ECL Advance Detection System (Amersham Biosciences, Piscataway, NJ, USA).
In vivo tumorigenesis
For in vivo studies, 1 × 10
6 SGC-7901 cells stably expressing miR-302b, miR-NC, or no vector (wild-type) were injected subcutaneously into the flanks of male BALB/c nude mice at 5 weeks of age as previously described [
19]. After 30 days, the mice were sacrificed, and tumor masses were measured. GC lung metastases were formalin-fixed, paraffin-embedded, and assessed by hematoxylin and eosin (H&E) staining. The experiments were performed using three mice per group, and all animal experiments were performed in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Central South University.
Luciferase assay
The 3’-UTR sequence of EphA2 was amplified from normal human genomic DNA (NM_004431) and sub-cloned into the pmirGLO luciferase reporter vector (Promega). SGC-7901 or AGS cells at 70–80% were co-transfected with wild-type (WT) or mutant (Mut) 3’-UTR vectors and miR-302b-3p mimics or inhibitors using Lipofectamine 2000. At 48 h post-transfection, the cells were assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Firefly luciferase activity in each sample was normalized to Renilla luciferase activity. The firefly luciferase activity of the cells that were transfected with miRNA mimics or inhibitors is represented as the percentage of activity relative to that of cells that were transfected with negative controls. All experiments were performed in triplicate.
Statistical analysis
Results are expressed as mean ± SEM from at least three independent experiments. Using the GraphPad Prism statistical program, data were analyzed using Student’s t-test, unless otherwise specified (Mann–Whitney test, ROC, Pearson correlation, etc.). Statistical analyses were performed using IBM SPSS22.0 software (SPSS). Two-tailed P < 0.05 was considered to be statistically significant.
Discussion
Identification of crucial molecular determinant(s) is essential to find alternative strategies to overcome the current bottleneck in tumor therapies, including those for GC. As a member of RTK family, it is well known that EphA2 is an emerging target for cancer therapeutics, due to its increased expression in tumor tissues [
12‐
14]. Downregulation of EphA2 expression through various approaches inhibits malignant behavior in vitro and in vivo [
31‐
33]. To date, the knowledge of functional roles and regulatory mechanism of EphA2 in GC are remains unclear due to the lack of applicable and practicable method to target it.
Recent studies have indicated that dysregulation of miRNAs results in dysregulated EphA2 in several kinds of solid tumors, including prostate, breast, and colon cancers [
27,
28,
34]. Among these reported microRNA profiles and online predicted results, we found seven miRNAs (miR-124, miR-302b, miR-125a-5p, miR-143, miR-29b-1, miR-29b-2, miR-29c) that were predicted to target EphA2 in GC. Further experiments demonstrated that miR-302b was the most significant downregulating miRNA of EphA2 in GC cells. Thus, we focused our studies on miR-302b and its role in regulating EphA2 in GC. Our results establish EphA2 as a novel, direct, functional effector of miR-302b in GC. This finding is supported by the observation that the overexpression of miR-302b in GC cells markedly downregulated EphA2 expression through targeting with the 3’UTR of EphA2 mRNA, and vice versa.
MiR-302b, an embryonic stem cell (ESC)-specific microRNA, has been documented to regulate the EMT, which endows tumor cells with the ability to leave the primary tumor and invade the local tissue for metastatic spread [
29,
35]. Loss of epithelial-cell markers (e.g., E-cadherin) and gain of mesenchymal-cell markers (e.g., N-cadherin, Snail), are reliable markers of EMT, particularly at the leading edge or invasion front of human solid tumors [
36,
37]. It’s reported that miR-302b expression is lower in gastric cancer, and it can regulate cell proliferation and cell cycle through different pathways [
24,
38‐
40]. Recently several reports, including our research in GC, have suggested that EphA2 may function as an activator of EMT in carcinogenesis [
14,
41]. Here, we showed that miR-302b modulates EphA2-associated EMT changes in GC cells. This result is supported by the observation that miR-302b overexpression reduced the transformation of EphA2-mediated mesenchymal-like phenotype in GC cells. Our in vitro studies further showed that miR-302b also dramatically suppressed N-cadherin, Snail, and β-catenin expression levels, the markers of EMT, by regulating the expression of EphA2.
Because of the complexity of EMT induction in the tumor microenvironment, the EMT involves several related signaling pathways, such as tumor growth factor-β, nuclear factor-κB, Notch, and Wnt/β-catenin [
42]. Wnt/β-catenin signaling has a major impact on EMT [
43] during cancer progression. After Wnt receptor on the cell membrane is stimulated by the signal, promoting the translocation of β-catenin into the nucleus may result in the loss of E-cadherin and subsequent induction of EMT [
44‐
46]. Inhibition of Wnt/β-catenin signaling can block EMT transcription factors and promote epithelial differentiation. We previously demonstrated that EphA2 regulated the EMT through Wnt/β-catenin signaling. Consistently, we found that miR-302b affects downstream Wnt/β-catenin signaling via EphA2.This result was confirmed by that EphA2 downregulation by miR-302b also decreased expression of c-Myc and CyclinD1, specific target oncogenes of the Wnt/β-catenin signal pathway. This is the first study to report this mechanism of miR-302b involvement in the EMT.
miR-302b acts as a tumor suppressor in several types of tumors by inhibiting cell growth and migration, and by modulating the cell cycle [
47‐
49]. Several studies have demonstrated that reprogramming of the EMT promotes tumorigenesis and invasiveness, which favors uncontrolled tumor cell growth and metastasis [
50,
51]. Our in vitro data suggest that miR-302b overexpression can attenuate GC cell migration and invasiveness, suppress cell proliferation, and induce cell cycle arrest in G0–G1 phase. Further research showed that these effects of miR-302b on GC cells are due to phenocopied changes in EphA2. Moreover, our in vivo studies using subcutaneous xenograft and tail vein injection models in mice indicates that overexpression of miR-302b led to a pronounced decrease in tumor volume and lung metastases. Based on these results, we posit that miR-302b could reduce GC proliferation and progression both in vitro and in vivo by immediately targeting EphA2-induced EMT through Wnt/β-catenin signaling.