Background
Gastric cancer (GC), one of the most common cancers, is the major cause of global cancer mortality, especially in Eastern Asia [
1]. Although several improvements have been made in diagnosis and therapy of patients with early stages of GC, it is extremely poor to treat GC at an advanced stage. Proliferation and metastasis are two important factors which are closely linked with poor prognosis and death in GC [
2,
3]. In recent years, molecules closely related to cell proliferation and metastasis have been studied as targeted agents for GC therapy [
4]. However, the molecular mechanism of GC cell growth and metastasis remains unclear. Therefore, it is urgent to make clear the key molecule involved in growth, invasion and migration of GC cells.
The DEAD-box RNA helicase 5 (DDX5), an ATP-dependent DEAD-box RNA helicase, acts as a transcriptional co-activator of several cancer-associated transcription factors and plays an important role in transcription initiation [
5]. As an oncogene, DDX5 is overexpressed in a variety of tumors and contributes to promoting cancer cell proliferation and metastasis [
6,
7]. In GC, high DDX5 expression was associated with advanced clinical stage [
8]. Furthermore, overexpression of DDX5 promoted GC cell growth in vitro and in vivo, while down-regulation of DDX5 expression could inhibit GC cell growth [
8].
Long non-coding RNA MIAT (Myocardial infarction associated transcript) is mainly expressed in the nucleus and highly conserved among mammalian species [
9]. In recent years, MIAT has been confirmed to be involved in development and progression of several cancers. MIAT had high expression in malignant mature B cells, which contributed the progression of chronic lymphocytic leukemias [
10]. Besides, MIAT was associated with the development of lung adenocarcinoma [
11]. Furthermore, MIAT exerted oncogenic effect on neuroendocrine prostate cancer [
12]. However, the exact role and mechanism of MIAT on GC cell proliferation and metastasis remains unclear.
MicroRNA-141 (miR-141) has been shown to be down-regulated in GC tissues and its low level was obviously associated with poor differentiation, metastasis, and advanced TNM stage of patients with GC [
13]. Further study demonstrated that over-expression of miR-141 could inhibit GC proliferation and metastasis, while knockdown of miR-141 could promote GC growth in vitro and in vivo [
14]. Therefore, miR-141 might serve as a prognostic factor and therapeutic candidate in GC patients.
In the present study, we found that MIAT expression was significantly increased in GC tissues and cells. Knockdown of MIAT led to impaired proliferation and metastasis of GC cells. We also observed that MIAT could interact with miR-141. The down-regulation of MIAT resulted in the increase of miR-141 expression and the decrease of DDX5 expression. Taken together, our data revealed an oncogenic role of the long noncoding RNA MIAT in GC and its role in regulating DDX5 expression.
Methods
Patients and tissue samples
Paired gastric cancer and normal gastric tissue were obtained between 2014 and 2016 from 120 patients (age: 28-85 years, median 60.4 years) who underwent primary surgical resection of gastric cancer in Department of Gastroenterology of Taizhou people’s Hospital affiliated of Nantong University of medicine (Taizhou, China). Follow-up information was obtained by reviewing patients’ medical records. None of the patients received radiotherapy or chemotherapy before surgical resection. All these tissue samples were immediately frozen in liquid nitrogen and stored at − 80 °C until total RNA was extracted.
Cell culture
Human gastric adenocarcinoma cell line BGC-823, gastric mucinous adenocarcinoma cell line MGC-803 were purchased from the Shanghai Institute of Cytobiology in Chinese Academy of Sciences. Human normal gastric epithelial cell line GES-1 was obtained from the Institute of Oncology in Beijing University. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL; Rockville, MD, USA) with 10% Fetal bovine serum (FBS; PAA Laboratories; GmbH, Linz, Austria) at 37 °C in a humidified incubator containing 5% CO2.
Real-time PCR assay
Total RNA of frozen tissues and cells were extracted using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s protocol. MIAT expression and mRNA level of DDX5 and PLK1 were quantified by real-time PCR using a LightCycler480 II Sequence Detection System (Roche, Basel, Switzerland). Sequences of the primers were available in Additional file
1: Table S1. GAPDH and U6 were used as internal standard using 2
-∆∆Ct method.
Western blotting
Total protein from tissue and cells were extracted in lysis buffer (Vazyme Biotech, Nanjing, China). Protein concentrations were determined using the Bio-Rad Protein Assay. Western blot analysis was performed as described [
15]. Individual immunoblots were probed with primary antibodies, anti-DDX5 antibody (Cell Signaling Technology; Beverly, MA, USA; diluted 1:1000) and anti-β-Actin antibody (Santa Cruz Biotechnologies; Santa Cruz, CA, USA; diluted 1:3000).
Small interfering RNA
Small interfering RNA specific for MIAT (si-MIAT) and control siRNA (si-control) was synthesized (Ribobio, Guangzhou, China) and transfected using Lipofectamine 2000 in gastric cells. The sequences of siRNA were on Additional file
1: Table S1.
Plasmid
The plasmid expressing DDX5 (pcDNA-DDX5) was constructed as previous description [
16]. BGC-823 cells were transfected with si-MIAT-1 and pcDNA-DDX5 for 48 h using Lipofectamine 2000 following the manufacturer’s protocol.
CCK-8 assay
Cell viability was measured by CCK-8 assay. Briefly, cells were plated in 96-well plates at a density of 3000 cells / well. Then, 10 μl of CCK-8 solution (Sigma, MO, USA) was added to each well and incubated at 37 °C for 2 h, and then the absorbance was measured at 450 nm. The experiment was performed independently in triplicate.
Flow cytometry analysis
Cell cycle was analyzed by flow cytometry analysis. Before transfection, serum was deprived for 24 h to synchronize cell cycle. Then, serum was added back, and si-MIAT or si-control transfected gastric cells were cultured in six-well plates. After transfection for 24 h, the cells were fixed with 80% cooled ethanol, and incubated with 0.5% Triton X-100 solution containing 1 mg / mL RNase A at 37 °C for 30 min. Next, PI (Sigma, MO, USA) was added into the wells at a final concentration of 50 μg / mL. Cellular DNA content was analyzed by a FACS (Becton Dickin-son). Data were processed using Cell-Quest software (Becton Dickinson).
Apoptosis was also analyzed by flow cytometry analysis. The si-MIAT or si-control transfected gastric cells were cultured in six-well plates for 48 h. The cells were harvested by trypsinization. Following double staining with FITC-annexin V and propidium iodide (PI), the cells were analyzed using flow cytometry (FACScan; BD Biosciences, San Jose, CA).
Lentivirus infection
BGC-823 cells were stably transduced with MIAT-set small interfering RNA (si-MIAT) Lentivector as well as control vector (GENECHEM, Shanghai, China). Transfection of BGC-823 cells was done at a 10-fold MOI (multiplicity of infection) virus particle concentration. MIAT expression was determined by Real-time PCR assay.
Invasion and migration assay
For the migration assays, 1 × 105 gastric cancer cells in serum-free media were placed into the upper chamber of a Transwell insert. For the invasion assays, gastric cancer cells in serum-free medium were placed into the upper chamber of an insert coated with Matrigel. Medium containing 10% FBS was added to the lower chamber. After incubation for 16 h, the cells remaining on the upper membrane were removed with cotton wool. Gastric cancer cells that had migrated or invaded through the membrane were fixed in methanol, stained with crystal violet (0.04% in water; 100 μl), counted using an inverted microscope and photographed.
Pull-down assay with biotinylated lncRNA-MIAT DNA probe
MIAT was transcribed from vector pSPT19-MIAT and biotin-labeled with the Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN) and SP6 RNA polymerase (Roche Applied Science, Basel, Switzerland), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). The biotinylated MIAT DNA probe was dissolved in binding and washing buffer, and incubated with Dynabeads M-280 Streptavidin (Invitrogen, CA, USA) at room temperature for 10 min to generate probe-coated beads according to the manufacturer’s protocol. Then, BGC-823 cell lysates were incubated with the probe-coated beads, and the RNA complexes bound to these beads were eluted and extracted for Real-time PCR analysis.
Pull-down assay with biotinylated miR-141
BGC-823 cells were transiently transfected with biotinylated miR-141, miR-141-Mut (mutation) and negative control (Ribobio, Guangzhou, China), harvested and lysed 48 h after transfection. 50 μL of the samples were aliquoted for input. The remaining lysates were incubated with Dynabeads M-280 Streptavidin (Invitrogen, CA, USA) according to the manufacturer’s protocol. In brief, the washed beads were treated in RNase-free solutions and incubated with equal volume of biotinylated miR-141 for 10 min at room temperature in binding and washing buffer on a rotator. Then, the beads with the immobilized miR-141 fragment were incubated with 10 mM EDTA (pH = 8.2) with 95% formamide at 65 °C for 5 min. The bound RNAs were purified using Trizol for the Real-time PCR analysis.
RNA immunoprecipitation (RIP)
RIP experiments were performed using the Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. The antibody for RIP assays of AGO2 (Cell Signaling Technology; Beverly, MA, USA) was diluted 1:50. Co-precipitated RNAs were detected by quantitative RT-PCR.
Luciferase reporter assays
The 3’-UTR of human DDX5 or lncRNA-MIAT was amplified from human genomic DNA and individually inserted into the pmiR-RB-REPORT™ (Ribobio, Guangzhou, China) using the XhoI and NotI sites. Similarly, the fragment of DDX5 or lncRNA-MIAT 3’-UTR mutant was inserted into the pmiR-RB-REPORT™ control vector at the same sites. For reporter assays, BGC-823 cells were co-transfected with wild-type (mutant) reporter plasmid and miR-141-Ribo™ mimic (miR-Ribo™ negative control). Luciferase activity was measured 48 h post-transfection as described previously [
17].
Animal tumor model
Male athymic nude mice (5-6 weeks) were purchased from Shanghai Laboratory Animal Centre (Chinese Academy of Sciences, Shanghai, China). Cultured BGC-823 and MGC-803 cells were transfected with si-MIAT or control vector lentivirus. To generate the orthotopic model, stable infected cells (1 × 107) were injected subcutaneously into the flank region of the nude mice. Tumor diameters were measured every three days, and volumes calculated using the estimation: width2 × length× 0.5. Animals were sacrificed on day 24 and tumor weights were measured.
To generate the lung metastasis model, stable infected BGC-823 cells (1 × 107) were injected into tail vein of the nude mice. The mice were sacrificed 5 weeks after the injection. The size and weight of the lungs were assessed, and visible tumors on the lung surface were counted.
Immunofluorescence
Tumor tissues from mice (5 μm thick) were immunolabeled with anti-DDX5 monoclonal antibody (Cell Signaling Technology; Beverly, MA, USA. 1:100 dilution). Then, dishes were washed and incubated with Alexa Fluor 488-conjugated secondary antibodies (1:50 dilution) for 1 h at room temperature. Nuclei were stained with Hoechst (10 mg/ml) for 2 min. Samples were examined with fluorescence microscope (Zeiss, Oberkochen, German).
Statistical analysis
Statistical analysis was performed with statistical analysis software SPSS 19.0 software. Statistical analyses were performed using either an analysis of variance (ANOVA) or Student’s t-test. Data were expressed as mean ± standard deviation. P < 0.05 was considered to be significant.
Discussion
Long noncoding RNAs (lncRNAs) have been demonstrated to play important roles in carcinogenesis and development of human GC. LncRNA MIAT has been confirmed to be involved in development and progression of several cancers, such as chronic lymphocytic leukemias, lung adenocarcinoma and prostate cancer [
10‐
12]. However, the role of MIAT in GC has not been investigated. In the present study, we found that MIAT expression was upregulated in GC tissues compared with adjacent normal tissues. In particular, patients with advanced clinical stages, poor differentiation, or metastasis expressed higher levels of MIAT.
To explore the biological function of MIAT in GC progression, we performed in vitro and in vivo assays. Our data showed that MIAT deletion resulted in significant inhibition of cell migration and invasion, while induced cell cycle arrest at S phase and apoptosis in GC cells. Furthermore, our data demonstrated that MIAT knockdown inhibited tumor growth in nude mice xenografts. These data indicated that high expression of MIAT promoted the progression of GC and MIAT may play oncogenic role in human GC progression.
LncRNA can act as competing endogenous RNAs (ceRNAs) to absorb miRNA by sequence complementarity and in turn affecting biological functions of miRNA [
19,
20]. Li et al. revealed that MIAT functioned as ceRNAs to activate MAPK signaling pathways by sponging miR-106 in lung adenocarcinoma cells [
11]. Initially, we found that MIAT knockdown significantly increased miR-141 level but had no effect on expression of miR-503, miR-133, miR-139, miR-204, miR-338 and miR-128 in GC cells. Then, we used luciferase reporter assays to demonstrate that MIAT contained the miR-141 binding site. Moreover, we verified that MIAT could directly bind to miR-141 and serve as miR-141 sponges in GC cells. Since miR-141 could inhibit proliferation and metastasis of GC [
21‐
23], MIAT exerted oncogenic effect on human GC by negatively regulating miR-141 expression.
DDX5, a transcriptional co-activator of several cancer-associated transcription factors, promoted cancer cell proliferation and metastasis [
24]. Previous study demonstrated overexpression of DDX5 promoted GC cell growth, whereas, DDX5 knockdown inhibited cell growth [
8]. In the current study, we identified that DDX5 is a novel target for miR-141 in GC cells. Moreover, MIAT and miR-141 appeared to positively and negatively regulate DDX5 expression, respectively. Most importantly, knockdown of MIAT impaired GC cell proliferation and metastasis by inhibiting DDX5 expression. Thus, MIAT/miR-141/DDX5 will provide a novel insight into the mechanism of GC growth and metastasis.