Although the incidence of gastric cancer has declined by >80% in developed countries during the past 50 years, it remains one of the most significant health problems in the developing countries.
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In China, gastric cancer is the third leading cause of cancer-related death.
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In recent years, post-transcriptional regulation has been intensively studied as a phenotype-level control mechanism of gene expression regulation.
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7MicroRNAs: small RNAs with a big role in gene regulation.
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In this study, we demonstrate that miR-506 is deregulated in metastatic gastric cancer cell lines and that miR-506 deficiency is associated with poor overall gastric cancer patient survival. In addition, we report that miR-506 overexpression in gastric cancer inhibits endothelial cell angiogenesis and metastatic invasion by targeting the proto-oncogene
ETS1, which plays several important roles in the angiopoietic and developmental processes of cancer cells.
13- Lin Z.
- Liu Y.
- Sun Y.
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Expression of Ets-1, Ang-2 and maspin in ovarian cancer and their role in tumor angiogenesis.
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- Norman J.C.
ERK2 drives tumour cell migration in three-dimensional microenvironments by suppressing expression of Rab17 and liprin-beta2.
Therefore, we hypothesized that miR-506 targeting of
ETS1 influences the angiogenic activity of endothelial cells and gastric cancer metastasis.
Materials and Methods
Patients and Tissue Specimens
For miR-506 detection, gastric cancer specimens were obtained from 109 patients who underwent surgery without any radiotherapy, chemotherapy, or biotherapy from The Department of Pathology, The Third Affiliated Hospital, Kunming Medical University (Kunming, China), from January 2008 through November 2013. After collection, samples were immediately frozen and kept in liquid nitrogen until further use. Follow-up data were collected from 84 of the 109 patients. For immunohistochemistry, specimens from 173 gastric cancer patients were collected from The Department of Pathology, The Third Affiliated Hospital, Kunming Medical University, from January 2003 through November 2008. For all patients, detailed clinicopathological data were collected, including sex, age, TNM staging, and clinical staging, as defined according to the criteria of the American Joint Commission on Cancer (seventh edition).
157th Edition of the AJCC cancer staging manual: stomach.
Eight normal gastric tissue samples were obtained from macroscopically uninvolved areas 2 to 3 cm away from the benign nodules of patients with stomach leiomyoma (
n = 5) or adenomyoepithelioma (
n = 3) who underwent surgical resection. All normal gastric tissues were histopathologically assessed and were morphologically normal. This study was approved by the Ethics Committee of The Third Affiliated Hospital, Kunming Medical University, and all patients provided written informed consent.
Real-Time PCR
For miRNA expression analysis, frozen tissue specimens were ground into powder in liquid nitrogen. Total RNA was extracted from 50 to 100 mg of the powdered tissue using TRIzol reagent (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen), according to the manufacturer's protocols. Real-time PCR for miR-506 was performed in a reaction mixture containing SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan). Quantitation of miRNAs was performed using an ABI 7500 system (Life Technologies, Grand Island, NY), according to the manufacturer's protocol. All PCRs were performed in triplicate, and a relative quantitative method was applied using the averaged ΔCT from the normal tissues or untreated cells. The endogenous control was U6.
Cell Lines and Culture
Primary cultures of normal gastric epithelial cells were established from fresh specimens of the adjacent noncancerous gastric tissue taken from an area over 10 cm from the cancerous tissue. The human gastric cancer cell lines AGS, BGC-823, HGC-27, Kato-III, SGC-7901, MKN45, and MGC-803 were supplied and authenticated by the Cell Bank of Shanghai Institute of Cell Biology and the Chinese Academy of Sciences (Shanghai, China). Cell lines were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma, St. Louis, MO) in a humidified 5% CO2 atmosphere at 37°C.
Construction and Transfection of Expression Vectors Containing miR-506 Inhibitors and Mimics
The miR-506 mimics, inhibitors, and negative control were purchased from RiboBio Co Ltd (Guangzhou, China). For gene transfection, gastric cancer cells were grown overnight in the logarithmic growth phase and transiently transfected with miR-506 mimic, inhibitor, or negative control vectors using Lipofectamine 2000 (Invitrogen) for 48 hours, according to the manufacturer's protocol, before being subjected to analyses.
Protein Extraction and Western Blot Analysis
Total cellular protein was extracted from gastric cancer cells with or without miRNA transfection using a lysis buffer containing protease and phosphatase inhibitors. After protein quantification using a BCA Protein Assay Kit (Pierce 23,227; Thermo Fisher Scientific Inc., Rockford, IL), equal amounts of protein samples (50 g) were separated using SDS-PAGE and then transferred onto a 0.22-μm polyvinylidene difluoride membrane (ISEQ00010; Millipore, Billerica, MA). For Western blot analysis, membranes were first incubated with 8% skimmed milk/Tris-buffered saline/Tween 20 at room temperature for 4 hours and then with a primary antibody overnight at 4°C. The following primary antibodies were used: mouse monoclonal anti–E-cadherin (sc-59778; Santa Cruz Biotechnology Inc., Dallas, TX), mouse monoclonal anti-vimentin (sc-6260; Santa Cruz Biotechnology Inc.), and rabbit polyclonal anti–β-actin (ab8227; Abcam, Cambridge, UK). The following day, membranes were washed three times with Tris-buffered saline/Tween 20 and incubated for 1 hour with a secondary antibody (1:10,000 dilution for anti-mouse horseradish peroxidase–linked antibody 7074; 1:20,000 dilution for anti-rabbit horseradish peroxidase–linked antibody 7074; Cell Signaling Technology, Danvers, MA) and then detected using an ECL kit (RPN2235; Amersham ECL Select; GE Healthcare Life Sciences, Buckinghamshire, UK). Data were quantified using ImageJ software version 1.48u (NIH, Bethesda, MD;
http://imagej.nih.gov/ij) and β-actin or glyceraldehyde-3-phosphate dehydrogenase levels.
Wound-Healing Assay
SGC-7901 or BGC-823 cells were seeded at 2 × 106 cells per well in 6-well plates, grown to 100% confluency, and then kept in serum-free RPMI 1640 medium for 24 hours. Next, a wound was generated across the cell monolayer using a 100-μL plastic pipette tip. Cell migration into the wound area was inspected under an inverted microscope for up to 24 hours after scarification. Quantitative analysis of the wound closure was calculated by measuring the initial width of the wound and the final width of the wound and calculating the distance of wound closure.
Matrigel Invasion Assay
SGC-7901 or BGC-823 cells were seeded at 5 × 104 cells per well with serum-free medium into the upper Matrigel invasion chamber (Corning Inc., Corning, NY), the filter of which was precoated with Matrigel (Sigma-Aldrich Shanghai Trading Co. Ltd., Shanghai, China) and the lower chamber of which was filled with a medium containing 10% fetal bovine serum as a chemoattractant. The cells were cultured for 20 hours and, at the end of the experiment, the cells remaining in the upper chamber were carefully removed and the cells that had invaded the bottom of the membrane were fixed and stained with hematoxylin. Quantification was performed by counting the stained cells.
Three-Dimensional Cell Culture
Matrigel was dissolved at 4°C overnight and then used to coat 24-well plates (100 μL per well). After a 30-minute incubation at 37°C, BGC-823 or SGC-7901 cells (2 × 104) suspended in 2% liquid Matrigel were inoculated into the Matrigel-coated wells. Cells were then grown in a cell culture box for 10 to 14 days, and fresh culture medium was added every 3 to 4 days.
Tube Formation Assay
Cells (5 × 104 viable cells per well) were seeded onto a 48-well polystyrene plate coated with Matrigel (120 μL per well), which had been incubated at 37°C and 5% CO2 for 30 minutes. Six hours after seeding, representative phase-contrast images were obtained at ×4 magnification.
CAM Assay
Fertilized white leghorn chicken eggs were incubated in an incubator at 37°C with 60% humidity. A small window was made in the shell on day 7 of chick embryo development under aseptic conditions. The window was resealed with sterile adhesive tape, and the eggs were returned to the incubator until day 11 of chick embryo development. On day 11, 10-μL cell suspensions of BGC-823 or SGC-7901 cells were placed on top of the chick chorioallantoic membrane (CAM), and the eggs were resealed and returned to the incubator for 72 hours until day 14 (n = 6 chicken embryos per cell line). Fixation was performed using formaldehyde and acetone (1:1), and prefixation was performed for 15 minutes through the window. The transplanted tumors and surrounding chorioallantoic membrane were integrally unloaded for observation and imaging.
Gelatin Zymography Assay
For the gelatin zymography assay, proteins were extracted from the cultured supernatant of the BGC-823 or SGC-7901 gastric cancer cells, with or without miRNA transfection. Equal amounts of protein samples (30 μg) were separated by electrophoresis under denaturing conditions (SDS-PAGE). After electrophoresis, the gel was washed four times in elutriant. After shock rinsing twice, the gel was incubated for 48 hours at 37°C. Next, after the gel was rinsed three times, it was stained using SimplyBlue Safestain (Invitrogen) and incubated for 3 hours at room temperature under gentle agitation. Next, SimplyBlue SafeStain was removed, and the gel was destained according to the manufacturer's instructions. The gel was scanned with an image analysis system (Quantity One; Bio-Rad, Hercules, CA).
Luciferase Assay
TargetScan (
http://www.targetscan.org, last accessed May 20, 2015) was used to predict whether miR-506 targeted the 3′-UTRs of
ETS1. Mutations in the miR-506 target sites were generated. For the luciferase assay, cells (2 × 10
5) were seeded in triplicate in 6-well plates and allowed to settle for 24 hours. Different concentrations of miR-506 mutant, inhibitor, mimics, or the control-luciferase plasmid were synthesized at Kunming Medical University and RiboBio Co Ltd. Plasmids were transfected into gastric cancer cells using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer's recommendations. Luciferase signals were measured 48 hours after transfection using the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI), according to the manufacturer's protocol. Three independent experiments were performed, and the data are presented as the means ± SD.
Immunohistochemistry
An EnVision detection kit (GK500705; Dako, Glostrup, Denmark) was used for immunohistochemical analysis of ETS1 and matrix metalloproteinase (MMP)-9 proteins in gastric cancer tissues, according to the manufacturer's protocol. Briefly, primary antibodies were diluted in phosphate-buffered saline, according to the manufacturers' recommendations: 1:50 for ETS1 (NCL-ETS-1; Novocastra, Newcastle upon Tyne, UK) and 1:400 for MMP-9 (sc-21733; Santa Cruz Biotechnology Inc.). A total volume of 50 μL diluted antibody solution was added to each section and incubated overnight at 4°C. The next day, after washing with phosphate-buffered saline, sections were incubated with Dako REAL EnVision/HRP (Dako Corp., Carpinteria, CA) for 30 minutes each. The color of the complexes was visualized by incubating tissue sections with a chromogenic substrate, 3,3′-diaminobenzidine (Dako Corp.) for 3 minutes, after which slides were rinsed in water, counterstained with hematoxylin, and mounted with Eukitt (O. Kindler GmbH & Co, Freiburg, Germany). ETS1 and MMP-9 protein expression in gastric cancer tissue specimens, as detected by antibodies, was reviewed and scored under a light microscope by two independent pathologists (Y.W. and S.D.) who were not aware of the clinicopathological data.
Statistical Analysis
All statistical analyses were performed using SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL). The t-test was used to evaluate whether there was a significant difference between the two groups of data in all of the pertinent experiments. P < 0.05 (using a two-tailed paired t-test) was considered statistically significant.
Discussion
EMT and angiogenesis are two essential processes in cancer progression.
16Noncanonical FZD2 signaling induces EMT and tumor progression.
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Cessation of CCL2 inhibition accelerates breast cancer metastasis by promoting angiogenesis.
In this study, we report a significant role for miR-506 in gastric cancer EMT and angiogenesis suppression. In addition, we demonstrated that higher endogenous miR-506 expression was associated with longer survival times in gastric cancer patients. miR-506 is located at Xq27.3, a chromosomal region closely associated with fragile X syndrome.
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Fragile X syndrome and Friedreich's ataxia: two different paradigms for repeat induced transcript insufficiency.
Down-regulation of miR-506 has been detected in several solid tumors, including serous ovarian cancer and breast cancer,
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Integrated analyses identify a master microRNA regulatory network for the mesenchymal subtype in serous ovarian cancer.
, 20- Arora H.
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miR-506 regulates epithelial mesenchymal transition in breast cancer cell lines.
suggesting that miR-506 plays an important role in tumor suppression. Our study presents evidence that miR-506 is a potent inhibitor of EMT and that miR-506 overexpression is associated with decreased vimentin and increased E-cadherin expression in gastric cancer. We also explored the potential role of miR-506 in angiogenesis and found that miR-506 both suppresses angiogenesis in gastric cancer and is associated with decreased MMP-9 expression. These findings suggest that miR-506 acts as a suppressor of angiogenesis and metastasis in gastric cancer.
We also demonstrated that ETS1 is a direct target of miR-506. The necessity of ETS1 for endothelial cells to adopt an angiogenic, blood vessel–forming phenotype has been well documented.
21- Naito S.
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Ets-1 upregulates matrix metalloproteinase-1 expression through extracellular matrix adhesion in vascular endothelial cells.
, 22The biology of the Ets1 proto-oncogene.
, 23- Arderiu G.
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Angiogenic microvascular endothelial cells release microparticles rich in tissue factor that promotes postischemic collateral vessel formation.
Accordingly, ETS1 is abundant in regenerating adult tissues and in areas of the developing embryo that require the formation of new blood vessels.
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Mediator Med23 deficiency enhances neural differentiation of murine embryonic stem cells through modulating BMP signaling.
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Transcriptome analysis reveals novel players in the cranial neural crest gene regulatory network.
Because the acquisition of invasive behavior is a part of the endothelial activation program, ETS1 may be responsible for stimulating proteases necessary for these processes.
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Role of transcription factors in angiogenesis: Ets-1 promotes angiogenesis as well as endothelial apoptosis.
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miR-125b is methylated and functions as a tumor suppressor by regulating the ETS1 proto-oncogene in human invasive breast cancer.
Likewise, ETS1 may help cancer cells get nutrients and oxygen by inducing tumor vascularization, and may promote tumor metastasis and invasion by activating ECM-degrading proteases in tumor or mesenchymal cells.
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Inhibitory effects of the transcription factor Ets-1 on the expression of type I collagen in TGF-beta1-stimulated renal epithelial cells.
Thus, high ETS1 levels in tumors often correlate with a poorer prognosis.
22The biology of the Ets1 proto-oncogene.
ETS1 was reported to be an MMP-9 transcription factor.
29- Ghosh S.
- Basu M.
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ETS-1 protein regulates vascular endothelial growth factor-induced matrix metalloproteinase-9 and matrix metalloproteinase-13 expression in human ovarian carcinoma cell line SKOV-3.
MMP-9 plays a critical role in promoting tumor progression by degrading the extracellular matrix and altering cell adhesion.
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In this study, immunohistochemistry of clinical gastric cancer samples showed that ETS1 expression was associated with MMP-9 expression, indicating that ETS1 and MMP-9 synergistically regulate gastric cancer metastasis. Thus, it is possible that a miR-506–ETS1 axis plays an important role in the regulation of gastric cancer angiogenesis.
In summary, cancer is a complex disease and controlling cancer development and progression requires system-level and integrative approaches. Our study revealed the functional relevance of miR-506 with respect to angiogenesis and metastasis, suggesting that miR-506 acts as a tumor suppressor in gastric cancer. Additional studies will be needed to explore the potential clinical utility of miR-506 as a potential biomarker for gastric cancer prognosis and as a new potential therapeutic target.
Article info
Footnotes
Supported, in part, by National Natural Science Foundation of China grants 30671904, 81360322, 81060185, 81260307, and 81470005, Scientific Research Project of the Department of Education of Yunnan Province grant 2011J043, Leading Talent of Health Systems of Yunnan Province grant L-201213, and National Clinical Key Specialty Construction Projects of Oncology of the National Health and Family Planning Commission of China (awarded to the Tumor Hospital of Yunnan Province: 2013-2014).
Z. Li, Z. Liu, and S.D. contributed equally to this work.
Disclosures: None declared.
Copyright
© 2015 American Society for Investigative Pathology. Published by Elsevier Inc.