Background
Gastric cancer (GC) is the fourth most common cancer in the world and the second leading cause of cancer-related deaths. It is estimated that there are approximately 750,000 new cases diagnosed annually around the world, and 5-year overall survival rates are less than 25% [
1]. In spite of the progress in chemotherapy, radiotherapy and surgical techniques for GC in recent years, the survival rate of GC patients remains unsatisfactory [
2]. One of the main reasons for the low overall survival is the lack of appropriate molecular biomarkers, which results in most of GC patients being diagnosed at advanced stage, missing the best opportunity for curative surgery.
Exosomes are small vesicles with several unique properties such as classic dish or cup morphology, 50–100 nm in diameter, double lipid layer, and a density of 1.12–1.19 g/mL [
3,
4]. Exosomes are generated from the internal vesicles of multivesicular bodies (MVBs), which release their contents into biological fluids [
5,
6]. Exosomes in the body fluids such as blood and urine could serve as diagnostic markers for various diseases including cancer because they may reflect the pathological state of their derived cells [
7‐
9]. Exosomes contain proteins, nucleic acids, and lipids, which usually vary with the cellular and tissue origins of the exosomes and are adapted to their functions [
10,
11]. The identification and isolation of cancer cells derived exosomes in body fluids could enable the specific detection of DNA, RNA, and proteins, which will aid in the diagnosis and treatment of cancer.
Tripartite motif (TRIM) proteins, one of the subfamilies of the RING-type E3 ubiquitin ligases, are regarded as critical regulators of neoplastic processes [
12,
13]. These proteins play important roles in a variety of biological processes, such as cell proliferation, cell differentiation, DNA repair, transcriptional regulation, and apoptosis [
12,
13]. Tripartite motif-containing 3 (TRIM3) is a member of the TRIM protein family, which maps to chromosome 11p15.5 [
14,
15]. It has been reported that the loss of TRIM3 gene promotes the development and progression of glioblastomas, while overexpression reduces the tumorigenicity of GBM. The tumor suppressive function of TRIM3 in GBM may be linked to its regulation of p21 [
16,
17]. TRIM3 has also been reported to be lowly expressed in glioma cells [
18]. However, the roles of TRIM3 in gastric cancer have not been well characterized.
In the present study, we isolated exosomes from the serum of gastric cancer patients and identified the proteomic profile of serum exosomes. We found that TRIM3 protein level was decreased in the serum exosomes of gastric cancer patients compared to that in healthy controls. TRIM3 exerted a tumor suppressive role in gastric cancer. TRIM3 overexpression inhibited while TRIM3 knockdown promotedthe growth and metastasis of gastric cancer through the regulation of stem cell factors and EMT regulators. Moreover, we demonstrated that exosomes-mediated delivery of overexpressed TRIM3 could suppress gastric cancer growth and metastasis in vitro and in vivo.
Methods
Cell lines and cell culture
Human gastric cancer cell lines SGC-7901 and MGC-803, normal gastric epithelial cell line GES-1, and HEK-293 cell line were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). The cells were propagated in RPMI-1640 medium (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). The cells were cultured at 37 °C in humidified air with 5% CO2.
Human gastric cancer samples
Peripheral blood samples were collected after obtaining informed consent from 80 gastric cancer patients (63 male and 17 female), and 80 healthy volunteers (50 male and 30 female) used as age and sex matched control groups, which were collected from the Affiliated Peoples’ Hospital of Jiangsu University, China. Blood samples were centrifuged and serum was isolated and kept at − 80 °C until analysis. The serum samples of 20 gastric cancer patients (14 male and 6 female) and age matched 20 healthy volunteers (13 male and 7 female) were collected from the Affiliated Peoples’ Hospital of Jiangsu University. Paraffin sections of 20 (13 male and 7 female) gastric cancer and adjacent noncancerous tissues were collected from the Affiliated Peoples’ Hospital of Jiangsu University, China. The primary gastric cancer tissues and their matched, adjacent noncancerous tissues (located more than 5 cm away from the primary site) were collected from 60 gastric cancer patients (46 male and 14 female) undergoing surgery at the Affiliated Peoples’ Hospital of Jiangsu University, China. Informed consent was given in all patients examined. All samples were confirmed by pathological examination. Histological grade was defined according to the World Health Organization classification. Documented informed consent was obtained from all subjects and the Ethics Committee of Jiangsu University approved all aspects of the study (2012258).
Exosome isolation
Exosomes were obtained from the serum and the culture supernatants by using ExoQuick precipitation solution (System Biosciences, Mountain View, CA, USA) according to manufacturer’s instructions. Briefly, 250 μL of serum were mixed with 63 μL of ExoQuick solution and incubated overnight at 4 °C. After centrifugation at 1500 g for 30 min, the pellets were suspended in 50 μL PBS. The culture supernatants from cells that had grown to sub-confluence (70–80%) in serum-free medium were harvested. The appropriate volume of ExoQuick-TC Exosome Precipitation Solution was added into the cell supernatant (1:5 ratio) and incubated at 4 °C overnight. After centrifugation at 1500 g for 30 min, the pellets were resuspended in PBS and filtrated through a 0.22 μm filter (EMD Millipore, Billerica, MA, USA). The isolated exosomes were stored at − 70 °C until use.
Transmission electron microscopy
Twenty microliters of the prepared exosomes were pipetted onto formvar carbon-coated copper grids and allowed to adsorb for 10 min before excess fluid was drained. The adsorbed exosomes were then negatively stained with 2% (w/v) phospphotungstic acid (pH 6.8) for 5 min and was air-dried under an electric incandescent lamp, and analyzed with a transmission electron microscope (FEI Tecnai 12, Philips), bar = 50 nm.
Size and concentration analyses of exosomes
The isolated exosomes were diluted in PBS and were analyzed using a NanoSight LM 10-HSBFT 14 Instrument (NanoSight, Malvern, UK) according to the manufacturer’s protocol. The size and concentration of exosomes were then analyzed by using the Nanoparticle Tracking Analysis 2.0 (NTA 2.0) software.
LC-MS/MS analysis
Exosomes were obtained from the serum of gastric cancer patients and healthy controls (n = 3) with ExoQuick precipitation solution (System Biosciences, Mountain View, CA, USA) according to manufacturer’s instructions. The serum exosomes were re-suspended in 50 μl of PBS, 2 μl triton X-100, and 5 μl phenylmethylsulfonyl fluoride with vortexing to dissolve the vesicles. The insoluble fraction was pelleted by centrifugation 20,000 g. The insoluble fraction was acetone precipitated at − 20 °C and digested in-gel with 1μg/ul trypsin (sequencing grade, Promega) for 18 h at 37 °C. Resulting peptides were analyzed by LC-MS/MS on an Q-Exactive-Orbitrap mass spectrometer (Thermo Scientific, Waltham MA, USA). Fold change means the ratio of direct reporter group strength, this experiment selected 1.13 times as the difference threshold, and P value< 0.05.
ELISA for TRIM3 detection
Exosomes were homogenized and lysed in RIPA buffer supplemented with proteinase inhibitors. TRIM3 concentration in exosomes was measured by using a commercial ELISA Kit according to the manufacturer’s instruction (Sanco, Hong Kong, China). The absolute amount of TRIM3 protein was calculated based on standard curves using human recombinant TRIM3 as the standard material. The concentration of TRIM3 was expressed as pictograms per milliliter.
TRIM3 plasmid transfection
The TRIM3 expression vector and control vector were purchased from Genechem (Shanghai, China). The TRIM3 expression vector or control vector were transfected into MGC-803 and SGC-7901 cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The expression of TRIM3 was confirmed by using real-time quantitative RT-PCR and western blot at 48 h after transfection.
siRNA transfection
Chemically synthesized TRIM3 siRNA and the scramble control siRNA were purchased from Genepharma (Shanghai, China). The sequences of siRNAs are shown in Additional file
1: Table S1. The siRNAs were transiently transfected into MGC-803 and SGC-7901 cells by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells were plated in 6-well plates at a density of 1 × 10
5 cells/well.
Exosome treatment
MGC-803 and SGC-7901 cells were treated with various doses of control and TRIM3-overexpressing exosome (10 μg, 25 μg, 50 μg) and cultured for 48 h, MGC-803 cells were treated with TRIM3-overexpressing exosome derived from MGC-803 cells and SGC-7901 cells were treated with TRIM3-overexpressing exosome derived from SGC-7901 cells.
Cells were harvested and seeded into 35 mm plates (1000 cells/well) and incubated for 10 days under standard conditions. At the end of the incubation period, the colonies were fixed with 4% paraformaldehyde and stained with crystal violet.
Transwell migration assay
Cells (1 × 105/well) were plated into the top chamber and 10% FBS containing medium was placed into the bottom chamber. After incubation at 37 °C in 5% CO2 for 12 h, the cells remaining at the upper surface of the membrane were removed with a cotton swab. The cells that migrated through the 8 μm sized pores and adhered to the lower surface of the membrane were fixed with 4% paraformaldehyde, stained with crystal violet and photographed.
RNA extraction, RT-PCR and real-time RT-PCR
Total RNA was extracted from cells and tissues using TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA was extracted from serum exosomes using QIAGEN RNA extraction Kit and equal amount of RNA was used for RT-PCR and real-time RT-PCR analyses. β-actin was used as the internal control. The sequences of specific primers are listed in Additional file
2: Table S2.
Western blot
The cells and isolated exosomes were homogenized and lysed in RIPA buffer supplemented with proteinase inhibitors. Equal amount of proteins was loaded and separated on a 10% SDS-PAGE gel. Following electrophoresis, the proteins were transferred to a PVDF (polyvinylidene difluoride) membrane, blocked in 5% (w/v) non-fat milk and incubated with the primary antibodies. The sources of primary antibodies were: GAPDH and E-cadherin (Cell Signaling Technology, Beverly, MA, USA); N-cadherin (Abcam, USA); Sox2 (Millipore, USA); PCNA (Bioworld Technology, Louis Park, MN, USA); Oct4,Vimentin (Signalway Antibody, USA); GAPDH, Goat anti rabbit IgG (HRP), and Goat anti mouse IgG (HRP) (CWBIO, China).
Animal model
The animal studies were performed with approval of the University Committee on Use and Care of Animals of Jiangsu University. The male BALB/c nu/nu mice aged 4–6 weeks were purchased from the Laboratory Animal Center of Shanghai (Academy of Science, Shanghai, China) and were randomly divided into six groups (n = 6).
Tumor growth assay in vivo
Two groups received subcutaneous injections of either vector or TRIM3-overexpressing MGC-803 cells (2 × 106 cells in 200 μl PBS), and two groups received subcutaneous injections of either NC-siRNA or TRIM3-siRNA transfected MGC-803 cells (2 × 106 cells in 200 μl PBS). MGC-803 pre-treated with TRIM3-overexpressing or control exosomes were subcutaneously injected into the two-flank of BALB/c nude mice (2 × 106 cells in 200 μl PBS). Tumors were surgically removed 20 days after injection, photographed and weighted. Tumor volume was assessed by caliper measurement and calculated by the formula (L × W × W/2), where L represents length, and W represents width.
The mice were grouped as described above. MGC-803 cells were intraperitoneally injected into the mice (2 × 106 cells in 500 μl PBS). Mice were inspected every 2 days and killed at 1 month after injection. The metastatic tumor nodes were removed and examined.
Vector construction and luciferase activity assay
The 3′ untranslated regions (3’-UTR) of TRIM3 mRNA were obtained by PCR and ligated into the pmirGLO dual-luciferase miRNA target expression vector (Promega, Madison, WI, USA). The mutant constructs were generated using Quick Change II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). MiRNA-20a mimics and NC-mimics, miRNA-20a inhibitors and NC-inhibitor were purchased from Genepharma (Shanghai, China), and the sequences are shown in Additional file
3: Table S3. For the overexpression studies, HEK-293 cells were co-transfected with 500 ng reporter vector with miRNA-20a mimics or NC-mimics. For the knockdown studies, HEK-393 cells were co-transfected with 500 ng reporter vector with miRNA-20a inhibitors or NC-inhibitor. Firefly luciferase activity was examined after transfection by using a dual-luciferase reporter assay system (Promega, USA) and normalized to that of Renilla luciferase.
Immunohistochemistry
The protein levels of TRIM3 in formalin-fixed paraffin-embedded gastric cancer tissues and adjacent non tumor tissue were detected by immunohistochemistry. Briefly, the sections were incubated with primary antibody and secondary antibody, visualized with 3,3′-diaminobenzidine (DAB) and then counterstained with hematoxylin for examination by the microscope. Twenty gastric cancer tissues and the corresponding adjacent noncancerous normal tissues were chosen randomly. The sections were photographed under a microscope and at least six fields were examined.
Statistical analyses
All results were confirmed in at least three independent experiments, and data from one representative experiment were shown. Data are presented as the mean ± SD. A two-tailed Student’s t-test was used to test the differences in sample means for data with normally distributed means. Mann-Whitney U-test was used for non-parametric data. The statistical analysis was performed using SPSS Statistics software. Values of P < 0.05 were considered significant.
Discussion
Cancer cells release excessive quantities of exosomes. The plasma of patients with acute myeloid leukemia (AML) were found to contain a 60-fold greater quantity of exosomes than that of normal donors [
19]. Melanoma cells also release large quantities of exosomes, in contrast to normal melanocytes [
20,
21]. Patients with ovarian cancer exhibit significantly increased levels of serum exosomes compared to benign disease or healthy controls [
22]. Silva et al. have reported that high levels of exosomes in the plasma of colorectal cancer patients were significantly associated with poorly differentiated tumors and with decreased overall survival [
23]. These reports indicate that the level of exosomes is elevated in the circulation of cancer patients and is positively associated with disease progression. In this study, we found that the concentration of exosomes from the serum of gastric cancer patients was higher than that from the healthy controls, which is consistent with that reported in the other cancers, suggesting that the increased level of exosomes is an important indicator of cancer progression.
Exosomes perform a variety of extracellular functions in the processes of cancer development and progression [
24,
25]. Exosomes contain a wide range of functional proteins and nucleic acids that can be transferred to local and distant recipient cells [
26‐
29]. Tumor cells secrete large amounts of exosomes that promote tumor progression through communication between the tumor and surrounding stromal tissue, activation of proliferative and angiogenic pathways, and initiation of premetastatic sites [
30‐
34]. Therefore, exosomes are an important component of the tumor microenvironment and are currently considered to be one of the main contributors to tumor progression and metastasis [
24]. In this study, we initially purified exosomes from the serum of gastric cancer patients and explored the protein profiles of exosomes by using LC-MS/MS. We identified a total of 243 differentially expressed proteins between the exosomes from the serum of gastric cancer patients and that from the serum of healthy controls. The altered expression of proteins in exosomes may act as messengers that transport signals between cells to promote cancer progression.
At present, there are few studies about the role of TRIM3 in cancer. It has been reported that the mRNA and protein levels of TRIM3 are downregulated in hepatocellular carcinoma (HCC) and are correlated with an unfavorable prognosis in patients with HCC, suggesting that TRIM3 could be a prognostic marker and novel therapeutic target for HCC [
35]. Liu et al. have reported that TRIM3 is a tumor suppressor mapping to chromosome 11p15.5 and that it might block tumor growth by sequestering p21 and preventing it from facilitating the accumulation of cyclin D1-cdk4 [
17]. Chen et al. have shown that TRIM3 acts as a tumor suppressor in GBM by restoring asymmetric cell division [
16]. Boulay et al. have reported the homozygous deletions of TRIM3 in brain tumor [
36]. Piao et al. suggest that TRIM3 functions as a tumor suppressor in colorectal cancer (CRC), which is exerted partially through the regulation of p53 [
37]. We found that TRIM3 was significantly downregulated in gastric cancer tissues. We investigated the functional roles of TRIM3 in gastric cancer by using gain- and loss-of-function studies. We found that TRIM3 inhibited gastric cancer growth and metastasis through the regulation of stem cell factors and EMT regulators, indicating that TRIM3 also plays tumor suppressive roles in gastric cancer. However, more studies are warranted to clarify the molecular mechanism responsible for the roles of TRIM3 in gastric cancer. We further identified TRIM3 as a direct target of miR-20a, and negatively regulated by miR-20a. MiR-20a act as an oncogenic miRNA that has been previously reported to be overexpressed in most cancers, suggesting that the upregulated expression of miRNAs in cancer may be associated with the decreased expression of TRIM in gastric cancer.
Exosomes contain bioactive molecules that reflect the pathological state of the originated cells, thus providing an enriched source of biomarkers [
38‐
40]. Melo et al. have reported that the cell surface proteoglycan glypican-1 can be detected on exosomes harvested from the serum of patients with pancreatic cancer and breast cancer [
41]. EBV-positive nasopharyngeal carcinoma (NPC) cell-derived exosomes contain HIF-1α, which increases the migration and invasiveness of EBV-negative NPC cells [
42]. In this study, we demonstrated that the expression of TRIM3 was lowly expressed in exosomes from the serum of gastric cancer patients and from the culture supernatants of gastric cancer cells, suggesting that the decreased expression of TRIM3 in serum exosomes could be a diagnostic biomarker for gastric cancer.
The tumor suppressive role of TRIM3 in gastric cancer and the presence of TRIM3 in exosomes led us to explore the possibility of delivering TRIM3 by exosomes to treat gastric cancer. We overexpressed TRIM3 in gastric cancer cells and collected the TRIM3-overexpressing exosomes and incubated gastric cancer cells with the TRIM3-overexpressing exosomes. We found that the TRIM3-overexpressing exosomes could be internalized into the gastric cancer cells. Exosomal TRIM3 could inhibit gastric cancer growth and metastasis in vitro and in vivo, suggesting that exosomes-mediated transfer of TRIM3 might serve as a new strategy for gastric cancer therapy.
Acknowledgments
The funding body had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.