Background
Gastric cancer is the second leading cause of cancer related death worldwide, and gastric cancer remains one of the most common malignancies, especially in East Asia [
1]. In most patients, gastric cancer is diagnosed at an advanced stage and is accompanied by malignant proliferation, extensive invasion and lymphatic metastasis. Although the clinical outcome of gastric cancer has gradually improved, the prognosis of patients with advanced disease is still disappointing and the 5-year survival rate of patients with gastric cancer still remains relatively low [
2]. Recently, multiple lines of evidence have revealed the contribution of long non-coding RNAs (lncRNAs) in tumorigenesis [
3]. Therefore, it is important to identify gastric cancer related lncRNAs and investigate their roles in gastric carcinogenesis.
In the past decade, fast development of sequencing technique and completion of ENCODE (encyclopedia of DNA elements) project have led to the discovery of a new group of RNAs, known as lncRNAs. lncRNAs are more than 200 nt in length with limited or no protein-coding capacity, which is often expressed in a disease-, tissue- or developmental stage-specific manner [
4‐
6]. Although lncRNAs are less well characterized compared with microRNAs, increasing evidence suggest that lncRNAs could play critical roles in regulation of diverse cellular processes such as stem cell pluripotency, cell differentiation, cell growth, cell apoptosis and cancer metastasis [
7‐
13]. Additionally, lncRNAs may function as
cis- or
trans-regulators of gene expression via playing as scaffolds for chromatin modifying complexes, decoys to transcription factors or serving as ‘sponge’ to microRNAs [
14‐
17].
SPRY4-IT1 (
SPRY4 intronic transcript 1), a lncRNA derived from an intron within
SPRY4 gene, has been recently revealed as oncogenic regulatory hubs or tumor suppressors in different cancers. SPRY4-IT1 was firstly reported to be over-expressed in melanoma cells, and knockdown of its expression inhibited cell growth, invasion and induced cell apoptosis [
11,
18]. Moreover, elevated expression of SPRY4-IT1 was associated with poor prognosis of clear cell renal cell carcinoma and esophageal squamous cell carcinoma [
19,
20]. SPRY4-IT1 also involved in trophoblast cells proliferation, migration and apoptosis [
21]. In previous study, we found that SPRY4-IT1 is down-regulated in non small cell lung cancer, and SPRY4-IT1 could function as a tumor suppressor via regulating cell growth and invasion [
22]. However, the expression pattern and biological roles of SPRY4-IT1 in gastric cancer is not well documented. The purpose of this study is to investigate the expression pattern and clinical significance of SPRY4-IT1 in gastric cancer, and identify its key role in gastric cancer cell proliferation and metastasis. This study may advance our understanding of the role of SPRY4-IT1 as a regulator of pathogenesis of gastric cancer and facilitate the development of lncRNA-directed diagnostics and therapeutics.
Methods
Tissue collection
61 Paired gastric cancer tissues and normal tissues were obtained from patients who had underwent surgery at Jiangsu province hospital between 2009 and 2011, and were diagnosed with gastric cancer (stages I, II, III, and IV; seventh edition of the AJCC Cancer Staging Manual) based on histopathological evaluation. No local or systemic treatment was conducted in these patients before the operation. All specimens were immediately frozen in liquid nitrogen, and stored at −80 °C until RNA extraction. This study was approved by the Research Ethics Committee of Nanjing Medical University, China. Informed consents were obtained from all patients.
Cell lines and culture conditions
Six gastric cancer cell lines (SGC7901, BGC823, MGC803, AGS, MKN45, MKN28, HCG-27), and a normal gastric epithelium cell line (GES-1) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 or DMEM (GIBCO-BRL) medium supplemented with 10% fetal bovine serum (10% FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin in humidified air at 37°C with 5% CO2.
RNA extraction and qRT-PCR analysis
Total RNA was extracted from tissues or cultured cells using TRIZOL reagent (Invitrogen, Carlsbad, CA). For qRT-PCR, 1 µg RNA was reverse transcribed to cDNA by using a Reverse Transcription Kit (Takara, Dalian, China). Real-time PCR analyses were performed with SYBR Premix ExTaq II kit (Takara, Dalian China). Results were normalized to the expression of GAPDH. The PCR primers were shown in Additional file
1: Table S1. The qRT-PCR assays and data collection were performed on ABI 7500, and results were analyzed and expressed relative to threshold cycle values (ΔCt), then converted to fold changes using the 2
−ΔΔCt method. GAPDH was used as an internal control.
Treatment cells with 5-aza-CdR
BGC823 and SGC7901 cells (2.5 × 105) were seeded into six-well culture plate on day 0 and exposed to 0, 5 µM 5-aza-CdR(Sigma-Aldrich, USA)for 3 days. The cells treated with 5-aza-CdR were harvested and used for detection of SPRY4-IT1 expression.
Chromatin immunoprecipitation assays
The ChIP assays were performed using EZ-Magna CHIP KIT according to the manufacturer’s instruction (Millipore, Billerica, MA, USA). SGC7901 and BGC823 cells were treated with formaldehyde and incubated for 10 min to generate DNA–protein cross-links. Cell lysates were then sonicated to generate chromatin fragments of 200–300 bp and immunoprecipitated with DNMT1 (Millipore) or the negative control IgG (Millipore). Anti-AcH3 (Millipore) was used as the positive control for the CHIP procedure. Precipitated chromatin DNA was recovered and analyzed by qRT-PCR (Additional file
1: Table S1).
Transfection of gastric cancer cells
SPRY4-IT1 over-expression plasmid and siRNA has been described in previous study [
22]. siRNAs for the human DNMT1 and the negative control olignucleotides were purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA) (Additional file
1: Table S1). All plasmid vectors for transfection (pCDNA-SPRY4-IT1 and empty vector) were extracted by DNA Midiprep or Midiprep kit (Qiagen, Hilden, Germany). Gastric cells cultured on six-well plate were transfected with the pCDNA-SPRY4-IT1, si-SPRY4-IT1 or si-DNMT1 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells were harvested after 48 h for qRT-PCR and western blot analyses.
Cell proliferation assays
Cell proliferation was monitored using Cell Proliferation Reagent Kit I (MTT) (Roche Applied Science). Si-SPRY4-IT1-transfected BGC823 cells (3,000/well), and pCDNA-SPRY4-IT1-transfected BGC823 or SGC7901 cells (3,000/well) were allowed to grow in 96-well plates. Cell proliferation was documented every 24 h following the manufacturer’s protocol. All experiments were performed in quadruplicate. For the colony formation assay, a total of 500 cells were placed in a fresh six-well plate and maintained in media containing 10% FBS, replacing the medium every 4 days. After 14 days, cells were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich). Visible colonies were manually counted. For each treatment group wells were assessed in triplicate.
Cell migration and invasion assays
For the transwell assays, at 48 h post-transfection, 5 × 104 (migration) or 1 × 105 (invasion) cells in serum-free media were placed into the upper chamber of an insert (8-μm pore size; Millipore, Billerica, MA, USA). Medium containing 10% FBS was added to the lower chamber. After incubation for 24 h, the cells remaining on the upper membrane were removed with cotton wool, whereas the cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet, imaged, and counted using an IX71 inverted microscope (Olympus, Tokyo, Japan). Experiments were independently repeated three times.
5 weeks female athymic BALB/c nude mice were maintained under pathogen-free conditions and manipulated according to protocols approved by the Committee on the Ethics of Animal Experiments of Nanjing medical University. BGC823 cells transfected with pCDNA-SPRY4-IT1 or empty vector were harvested from six-well cell culture plates and resuspended at a concentration of 1 × 108 cells/mL. A volume of 100 µL of suspended cells was subcutaneously injected into a single side of the posterior flank of each mouse. Tumor growth was examined every 3 days, and tumor volumes were calculated using the equation V = 0.5 × D × d2 (V, volume; D, longitudinal diameter; d, latitudinal diameter). At 15 days post injection, the mice were euthanized and tumor weights were measured and also used for further analysis.
Tail vein injection of cells for metastasis in athymic mice
5-weeks-old male athymic mice were purchased from the Animal Center of the Nanjing University (Nanjing, China) and maintained in laminar flow cabinets under specific pathogen-free conditions. BGC823 cells transfected with pCDNA-SPRY4-IT1 or the empty vector were harvested from 6-well plates and resuspended at 2 × 107 cells/mL. A volume of 0.1 mL of suspended cells was injected into the tail veins of mice, which were sacrificed 7 weeks after injection. The lungs were removed and photographed, and visible tumors on the lung surface were counted. This study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Our protocol was approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
Western blotting analysis
Cells were lysed with RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail (Roche, CA, USA). The concentration of protein was determined using the Bio-Rad protein assay kit. Protein extracts (40 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to nitrocellulose membranes (Sigma) and incubated with antibodies. ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad, CA, USA). GAPDH was used as a control. Antibodies against E-cadherin and Vimentin (1:1,000 dilution) was purchased from Cell Signaling Technology (CST, MA, USA).
Fluorescence immunohistochemistry
BGC823 cells were fixed in 4% paraformaldehyde following a standard protocol. Rabbit anti-E-cadherin and Vimentin polyclonal antibodies (1:50; CST) were used as primary antibodies, with TRITC-labeled anti-Rabbit IgG (1:200; Sigma-Aldrich, St. Louis, MO, USA) used as a secondary antibody. Sections were mounted onto slides using Gel Mount Aqueous Mounting Medium (G0918, Sigma-Aldrich, St. Louis, MO, USA) and examined with an Olympus BX51 microscope (Olympus, Tokyo, Japan).
Statistical analysis
Statistical analysis was performed using the SPASS17.0 statistical software package. The expression levels of SPRY4-IT1 in tumor tissues were compared with normal adjacent mucosa by Wilcoxon test, while the associations between SPRY4-IT1 expression and clinical characteristics were evaluated by Chi square test. Survival curves were estimated by the Kaplan–Meier method. The log-rank test was used to estimate the statistical differences between survival curves. A two-tailed P < 0.05 was considered significantly.
Discussion
Over the past decade, miRNAs have moved to the forefront of ncRNA research in gastric cancer. However, lncRNAs in gastric cancer are still an emerging field. GAPLINC (gastric adenocarcinoma predictive long intergenic noncoding RNA), a 924-bp lncRNA, is highly expressed and displays considerable predictive effect in the diagnosis and prognosis of gastric cancer. Manipulating GAPLINC expression altered CD44 mRNA abundance and regulated cell migration and proliferation by suppressing CD44 expression as a molecular decoy for miR211-3p [
24]. Similarly, HOTAIR may function as an completing endogenous RNA to regulate the expression of human epithelial growth factor receptor 2 (HER2) through sponging miR-331-3p [
25]. FENDRR is down-regulated in gastric cancer, and decreased expression of FENDRR induces FN1 expression, which resulted in activation of MMP2/MMP9 [
26]. Thus, identification of dysregulated lncRNAs will enhance our knowledge of lncRNAs function in the progression and metastasis of gastric cancer and could be used as new diagnostic or therapeutic targets.
SPRY4-IT1 expression was significantly decreased in gastric cancer tissues and cell lines. In addition, the decreased expression of SPRY4-IT1 was associated with poor prognosis and shorter survival time. We also showed that DNMT1 could directly bind to SPRY4-IT1 promoter region and may contribute to the lost expression of SPRY4-IT1 in gastric cancer cells. Moreover, ectopic expression of SPRY4-IT1 led to the significant inhibited malignant phenotype of gastric cancer cells both in vitro and
vivo. However, SPRY4-IT1 has been found to be up-regulated in esophageal squamous cell carcinoma, breast cancer and bladder cancer, which suggests that SPRY4-IT1 has an tissue-specific expression pattern and may function as oncogene or tumor suppressor in different cancer [
20,
27,
28]. Taken together, these findings indicate that SPRY4-IT1 could function as a tumor suppressor and may be useful as a novel prognostic or progression marker for gastric cancer.
The invasion and metastasis of cancer cells are landmark events that involve many changes in cellular behavior, and lead to different steps of the metastatic cascade. Although SPRY4-IT1 can suppress migratory and invasive phenotype of gastric cancer cells, the underlying mechanism is still elusive. We previously showed that SPRY4-IT1 is decreased in NSCLC, and elevation of its expression impairs cell invasion and metastasis through the regulation of EMT process. EMT is a key step toward cancer metastasis, a biological process where epithelial cells lose their polarity and undergo transition into a mesenchymal phenotype. Loss of E-cadherin expression is a hallmark of EMT process and is likely required for enhanced tumor cell motility [
29‐
31]. Perl et al. reported that loss of E-cadherin expression coincides with the transition from well differentiated adenoma to invasive carcinoma in a transgenic mouse model of pancreatic beta-cell carcinogenesis [
32]. In this study, we determined the protein levels of these EMT-induced markers following SPRY4-IT1 over-expression or knockdown. Our results indicate that inhibitory effects of SPRY4-IT1 on cell migration and invasion were partly associated with EMT process.
Conclusions
In summary, we demonstrated that the decreased SPRY4-IT1 expression is a common event underlying the progression and metastasis of gastric cancer, indicating that SPRY4-IT1 may be an indicator of poor survival rate and a negative prognostic factor for gastric cancer patients. SPRY4-IT1 contributed to gastric cancer cells invasion and metastasis may partly via regulating epithelial–mesenchymal transition process. However, the underlying molecular mechanisms through which SPRY4-IT1 involved in EMT requires further investigation.
Authors’ contributions
MX, FN, MS carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. RX carried out the immunoassays. YL and PZ participated in the design of the study and performed the statistical analysis. WD, XL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.