Background
Gastric cancer (GC) is a common malignant disease worldwide [
1,
2]. Cancer cells invade locally and metastasize to distant sites, which leads to death [
3]. A better understanding of factors that contribute to GC cell behaviour could potentially improve GC therapies. Two decades ago, microRNA (miRNA) was discovered to be a non-coding RNA and identified as a regulatory gene [
4,
5]. Currently, miRNAs are reported to regulate the development of many diseases, especially malignant tumours, by acting as either oncogenes or tumour suppressors [
6‐
8]. miRNA dysregulation is involved in cancer progression and may provide targets for novel therapeutic approaches [
9‐
12]. Several miRNA-targeted therapeutic strategies have reached clinical or preclinical development [
13]. Currently, a miRNA-associated signature provides predictive power to classify and stratify EGC patients for endoscopic treatment [
14]. A mimic of the tumour suppressor miR-34 has reached phase I clinical trials for cancer treatment. Anti-miR-122 therapy has reached phase II trials for hepatitis treatment [
15]. Silencing of miR-632 inhibits EMT (epithelial-mesenchymal transition) and eliminates invasive ability in breast cancer cells. In addition, miR-632 is related to nasopharyngeal carcinoma, laryngeal cancer and myelodysplastic syndrome [
16‐
18]. Tuberculosis risk may be influenced by miR-632-mediated regulation [
19]. However, the relationship between miR-632 and GC therapeutics still needs to be clarified.
The trefoil factor family is a group of small-molecule polypeptides secreted by the mammalian gastrointestinal tract [
20]. Trefoil factor 1 (TFF1), a member of the trefoil peptide family, has been reported to inhibit gastrointestinal tumourigenesis. TFF1 is highly expressed in the human stomach and maintains gastric epithelial structure and function [
21]. However, this tissue-specific distribution is disrupted in pathological states. In metastatic GC, TFF1 is upregulated compared with its expression in primary cancer [
22]. After GC resection, secreted TFF1 in serum acts as a recurrence biomarker [
23]. We previously explored the effect of TFF1 in the maintenance of gastric mucosa integrity and continuity and found that TFF1 is closely associated with GC progression [
24,
25]. According to a computer-based set of predictions, target sequences within the TFF gene cluster demonstrate that multiple miRNAs can potentially bind the 3′-untranslated region and DNA coding sequence [
26]. We previously showed that miR-423-5p and miR-218-5p regulate GC proliferation and invasion by targeting TFF1, respectively [
27,
28].
Aberrant miRNA expression contributes to malignant cell behaviour, and in preclinical research, miRNA targeting has shown potential for improving GC therapy. Here, we demonstrate that miR-632 promotes tumour angiogenesis and endothelial recruitment in a TFF1-dependent manner.
Methods
Ethics statement
This study was approved by the Ethics Committee of The First Affiliated Hospital, Jinan University, China. Written consent was obtained from all participants.
Cell culture and transfection
AGS cell lines were purchased from ATCC (Manassas, VA, USA) and cultured in Ham’s F-12 K (Kaighn’s) medium (Life Technologies) supplemented with 10% foetal bovine serum (FBS, Life Technologies) and 1% penicillin G/streptomycin (Life Technologies). BGC823, MGC803, MKN45 and EAhy926 cell lines were purchased from Cell Bank, Shanghai Institutes for Biological Sciences (Cell Bank, CAS, Shanghai, China) and cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS and 1% penicillin G/streptomycin. All cell lines were incubated at 37 °C in an atmosphere of 95% air and 5% CO
2. The cell lines were checked free of mycoplasma contamination by PCR and culture, and authenticated with STR profiling (FBI, CODIS,
http://cellresource.cn).
miR-632-mimic (25 nM) and miR-632-inhibitor (50 nM) were purchased from Qiagen. The cell transfection method was described previously [
28].
RNA expression analysis
A miRNeasy Kit (Qiagen; cat. no. 217004) was used for total RNA extraction from human GC cells or tissues, and a miRNeasy Serum/Plasma Kit (Qiagen; cat. no. 217184) was used for miRNA extraction from GC patient serum following the manufacturer’s protocol. miRNA first-strand cDNA synthesis and real-time PCR were performed as previously described [
28]. miR-632 and control primers were purchased from Qiagen. TFF1 primers were described previously [
27].
In situ hybridization and immunohistochemical staining
Digoxin-labelled hsa-miR-632 probe (miRCURY LNA™ Detection probe; 250 pmol; 5’-DIG and 3’-DIG labelled; Exiqon) was used at a concentration of 1.5 pM to detect miR-632 expression. Rabbit anti-human TFF1 polyclonal antibody (ab92377) was purchased from Abcam and diluted 1:250 in IHC Antibody Diluent (ABD-0030; Maixin Biotech, Fuzhou, China). Anti-MMP9 (MAB-0245) and anti-CD34 (MAB-0034) antibodies were purchased from Maixin Biotech, Fuzhou, China. In situ hybridization and immunohistochemical staining were performed in serial paraffin sections of human GC tissue using previously described procedures [
28].
Western blot analysis
GC cells were transfected with miR-632 mimic or inhibitor or with corresponding controls for 48 h. Then, the cells were collected and lysed for Western blot analysis. Primary antibodies against TFF1 were purchased from Santa Cruz Biotechnology, and antibodies targeting MMP9, CD34, p-NFκB, and NFκB were purchased from Cell Signalling Technology. Primary antibodies against GAPDH and anti-rabbit/mouse secondary antibodies were purchased from Proteintech.
Dual-luciferase reporter assay
AGS cells were seeded in 24-well plates at a density of 6 × 104 cells per well immediately prior to transfection. pmirGLO dual-luciferase miRNA target expression vector was purchased from Promega (cat. E1330). The full-length or a mutated 3’UTR region of TFF1 was inserted into a luciferase reporter vector. AGS cells were co-transfected with miR-632-mimic along with the vectors. After 48 h, the cells were assessed for both firefly and Renilla luciferase activity using a dual-luciferase reporter assay system (Promega; E1910).
Enzyme-linked immunosorbent assay (ELISA)
After 24 h of miR-632 mimic or inhibitor treatment, cell supernatants were collected and examined using a TFF1 ELISA kit (USCN Life Science, Houston, TX, USA) following the manufacturer’s protocol. The absorbance at 450 nm was measured using a microplate reader.
Endothelial recruitment experiment
EAHY926 cells were grown to 100% confluency in 60-mm dishes. Three coverslips seeded with GC cells transfected with hsa-miR-632 mimic, hsa-miR-632 inhibitor or corresponding controls were transferred onto the EAHY926 monolayer and a scratch was made across the EAHY926 monolayer using a 200-μl pipet tip. At least three images were collected along each scratch and analysed for the area covered by EAHY926 cells.
Gastric cells were treated with hsa-miR-632-mimic, hsa-miR-632-inhibitor or corresponding controls and incubated at 37 °C for 24 h. The supernatant was collected and added to EAHY926 cells. A total of 100 μl Matrigel (BD Bioscience) was added to a 96-well plate and allowed to polymerize. Then, 3 × 104 EAHY926 cells in serum-free medium were added to each Matrigel-coated well. Cells were incubated for 6 h at 37 °C and then imaged via microscopy. Each group was evaluated in triplicate.
Cell migration and wound healing assays
Cell migration was analysed using Transwell and wound healing assays. The Transwell assay was performed using 24-well Transwell plates (Costar, USA) polycarbonate filters containing 8-μm-poros. Briefly, 200 μl of cell suspension containing 1 × 105 cells in the absence of FBS was added into each upper Transwell chamber, and 500 μl of medium containing 10% FBS was added to the lower chamber. The cells were incubated for 20 h and then fixed in 4% paraformaldehyde for 10 min. Cells were stained with 1% crystal violet for 30 min and counted.
Wound healing assays were performed in 6-well plates. Cells were cultured in 6-well plates at a density of 5 × 106 cells per well for 24 h, and then, a scratch was made across the cell monolayer with a 200-μl pipet tip. At least three images were collected along each scratch and analysed for the area covered by cells.
Statistical analysis
Statistical analysis was performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Student’s t-test (means ± standard deviation) and chi-square test were used for data analysis according to different data types. All of the values are expressed as the mean ± SD of at least three independent experiments performed in triplicate, and P < 0.05 was considered to be statistically significant. Graphs were plotted using GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA).
Discussion
In our current study, we demonstrated that miR-632 promotes GC progression by accelerating angiogenesis in a TFF1-dependent manner. Our results showed that miR-632 is highly expressed in GC tissue and serum and negatively associated with TFF1 in GC. Here, we detected miR-632 in serum to provide an experimental basis for future non-invasive rapid diagnosis of GC using peripheral blood samples. miR-632 improves tube formation and endothelial cell recruitment by negatively regulating TFF1 in GC cells. Recombinant TFF1 reversed angiogenesis mediated by miR-632. TFF1 is a target gene of miR-632.
As important regulators of gene expression, miRNAs have not only been implicated in various signalling pathways but also in embryonic development, tissue homeostasis, stem cell transition, anticancer therapy, and other biological processes [
29‐
31]. A miRNA-associated diagnosis provides predictive power to inform early GC patients and has the potential to be applied in endoscopic treatment [
14]. We previously found that miR-218-5p and miR-423-5p regulate GC proliferation and invasion, respectively [
27,
28]. miR-218-5p regulates GC cell the proliferation by targeting TFF1 in an Erk1/2-dependent manner. miR-423-5p regulates cell proliferation and invasion by targeting TFF1 in GC cells. We previously suggested that TFF1 improves gastric mucosal protection and epithelial integrity [
25]. Trefoil peptides are also used to protect against mucosal injury via oral administration or other approaches [
32,
33]. Additionally, increased TFF1 expression in para-carcinoma tissue suggests that TFF1 is associated with tumour suppression and differentiation. TFF1 is involved in inhibition of tumour invasion and migration, and it may be used as a target to enhance the chemotherapy sensitivity by regulating apoptosis resistance [
34]. In addition, miRNAs can regulate TFF1 expression and secretion. In our present study, we found that miR-632 may inhibit TFF1 expression and secretion in GC cells.
Conclusions
Therefore, our research demonstrated that miR-632 is upregulated in GC tissue and serum and negatively associated with TFF1. miR-632 improves tube formation and endothelial cell recruitment by negatively regulating TFF1 in GC cells. We conclude that miR-632 promotes GC progression by accelerating angiogenesis in a TFF1-dependent manner.
Acknowledgements
We appreciate our colleagues Yesen Li and Zhide Guo (State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Xiamen University, P. R. China) for their participation in the molecular and cell imaging in this work.
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