Hsa-miR-506-3p Targeting ERK-2/ETS-1 Inhibits Angiogenesis And Cell Migration And Invation In Gastric Cancer

BackgroundAlthough the incidence of gastric cancer has declined by more than 80% in developed countries over the past 50 years, it remains one of the most significant health problems in developing countries. In China, gastric cancer is the fourth leading cause of cancer-related death gastric cancer patients in China and most other developing countries are usually diagnosed during the advanced stages of the disease. Due to the very high likelihood of metastasis in advanced gastric cancer, the characteristically late diagnosis contributes to the high rate of gastric cancer mortality. A better understanding of the molecular mechanisms underlying gastric cancer development and progression could help the development of novel approaches for the early detection and treatment of this type of cancer.In recent years, posttranscriptional regulation has been intensively studied as a phenotype-level control mechanism of gene expression regulation. Among the posttranscriptional regulators with metastatic activity, one of the most studied classes are small noncoding RNAs. MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression by binding to the 3’-untranslated region (3’-UTR) of target genes. miRNA regulation results in messenger RNA (mRNA) degradation and the inhibition of protein translation. Although an increasing number of studies examining different miRNAs in various pathophysiological settings have been reported, only a small fraction of these interactions have been validated experimentally.In this study, we demonstrate that hsa-miR-506-3p is deregulated in metastatic gastric cancer cell lines and that hsa-miR-506-3p deficiency is associated with poor overall gastric cancer patient survival. Additionally, we report that hsa-miR-506-3p overexpression in gastric cancer inhibits endothelial cell angiogenesis and metastatic invasion by targeting the extracellular signal regulated kinase 2 and the proto-oncogene ETS-1, which play several important roles in the angiopoietic and developmental processes of cancer cells.The necessity of ETS-1 for endothelial cells to adopt an angiogenic, blood vessel forming phenotype has been well documented. Accordingly, ETS-1 is abundant in regenerating adult tissues and in areas of the developing embryo that require the formation of new blood vessels. Since the acquisition of invasive behavior is part of the endothelial activation program, ETS-1 may be responsible for stimulating proteases necessary for these processes. Likewise, ETS-1 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. Thus, high ETS-1 levels in tumors often correlates with a poorer prognosis.ETS-1 was reported to be an MMP9 transcription factor. MMP9 plays a critical role in promoting tumor progression by degrading the extracellular matrix and altering cell adhesion.In this study, immunohistochemistry of clinical gastric cancer samples showed that ETS-1 expression was associated with MMP9 expression, indicating that ETS-1 and MMP9 synergistically regulate gastric cancer metastasis. Thus, it is possible that anhsa-miR-506-3p-ETS-1 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 hsa-miR-506-3p with respect to angiogenesis and metastasis, suggesting that hsa-miR-506-3p acts as a tumor suppressor in gastric cancer. Additional studies will be needed to explore the potential clinical utility of hsa-miR-506-3p as a potential biomarker for gastric cancer prognosis and as a new potential therapeutic target.Research Objectives1. To observe whether hsa-miR-506-3p expression levels affected the clinical outcomes of gastric cancer patients, we examined the possible role of hsa-miR-506-3p in gastric cancer, investigated its associations with the clinical outcomes of gastric cancer patients.2. Because angiogenesis and epithelial-to-mesenchymal transition (EMT) are two important processes that enables cancer cells to invade their surroundings and metastasize, we examined the potential role of hsa-miR-506-3p in angiogenesis and the metastasis of gastric cancer cells.3. To analyze and validate the direct targets of hsa-miR-506-3p, and explore the mechanism of miR-506-3p in gastric cancer invasion and metastasisMethods and ResultsPart I:Low expression of hsa-miR-506-3p was associated with shorter survival times of gastric cancer patientsMethods1. For hsa-miR-506-3p detection, gastric cancer specimens were obtained from 109 patients who underwent surgery without any radiotherapy, chemotherapy or biotherapy from The Department of Pathology, from January 2008 through November 2013. After collection, samples were immediately frozen and kept in liquid nitrogen until further use. Follow up data was collected from 84 of the 109 patients. Eight normal gastric tissue samples were obtained from macroscopically uninvolved areas 2 to 3 cm away from the benign nodules of patients of leiomyoma and adenomyoepithelioma of stomach 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 (Kunming, Yunnan, China) and all patients involved in the study provided written informed consent.2. To observe whether hsa-miR-506-3p expression levels affected the clinical outcomes of gastric cancer patients, we used quantitative real time PCR in a blinded manner to detect hsa-miR-506-3p expression levels in a cohort of human gastric cancer samples.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, USA) and reverse transcribed into complementary DNA using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s protocols. Real-time PCR for hsa-miR-506-3p was performed in a reaction mixture containing SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan). Quantitation of microRNAs was performed using an ABI 7500 system (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s protocol. All PCR reactions 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 U6.3. 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 Chinese Academy of Sciences (Shanghai, China). Cell lines were grown in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen),100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St Louis, MO, USA) in a humidified 5% CO2 atmosphere at 37℃.Results1. To observe whether hsa-miR-506-3p expression levels affected the clinical outcomes of gastric cancer patients, we used quantitative real time PCR in a blinded manner to detect hsa-miR-506-3p expression levels in a cohort of human gastric cancer samples. Strikingly, hsa-miR-506-3p expression levels robustly stratified advanced clinical stage and early stage patients.2. High expression of hsa-miR-506-3p was associated with longer survival times of gastric cancer patients. Patients whose primary gastric cancer lesions expressed high hsa-miR-506-3p levels (hsa-miR-506-3p levels greater than the mean) exhibited significantly longer overall survival times than those whose primary lesions expressed low hsa-miR-506-3p levels (hsa-miR-506-3p levels less than the mean).3. Ubiquitous basal hsa-miR-506-3p expression was lower in gastric cancer cells than in normal gastric tissues. Additionally, hsa-miR-506-3p expression was closely associated with the invasion properties of gastric cancer cells. Of the cell lines examined, hsa-miR-506-3p expression was lowest in the SGC-7901 cell line, and this cell line had the highest invasive ability. Conversely, hsa-miR-506-3p expression was highest in the BGC-823 cell line, and this cell line had the lowest invasive ability of all the gastric cancer cell lines examined. Because of these divergent results, the SGC-7901 and BGC-823 cell lines were chosen for further hsa-miR-506-3p knock-in and knockdown experiments.Part II:miR-506 inhibits angiogenesis and the epithelial to mesenchymal transition of gastric cancer cellsMethods1. Construction and transfection of expression vectors containing hsa-miR-506-3p inhibitors and mimics. For gene transfection, gastric cancer cells were grown overnight in the logarithmic growth phase and transiently transfected with hsa-miR-506-3p mimic, inhibitor or negative control vectors using Lipofectamine 2000 (Invitrogen) for 48 h according to the manufacturer’s protocol before being subjected to analyses.2. Chick Chorioallantoic Membrane (CAM) Assay:Fertilized white leghorn chicken eggs were incubated in an incubator at 37℃ with 60% humidity. A small window was made in the shell on day seven 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 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 done using formaldehyde and acetone (1:1) and pre-fixation was performed for 15 min through the window. The transplanted tumors and surrounding chorioallantoic membrane were integrally unloaded for observation and photography.3. Tube formation assay:Cells (5×104 viable cells/well) were seeded on a 48-well polystyrene plate coated with matrigel (120 μl/well) which had been incubated at 37℃ and 5% CO2 for 30 min. Six h post-seeding, representative phase-contrast images were photographed at 4 x magnification.4. 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. Following shock rinsing twice, the gel was incubated for 48 h at 37℃. Next, after the gel was rinsed three times, it was stained using SimplyBlue Safestain (Invitrogen) and incubated for three 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, USA).5. Wound-healing assay:SGC-7901 or BGC-823 cells were seeded at 2 ×106 cells/well in 6-well plates, grown to 100% confluency and then kept in serum-free RPMI 1640 for 24 h. Next, a wound was created across the cell monolayer using a 100μl plastic pipette tip. Cell migration into the wound area was inspected under an inverted microscope for up to 24 h 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.6. Matrigel invasion assay:SGC-7901 or BGC-823 cells were seeded at 5 ×104 cells/well with serum-free medium into the upper matrigel invasion chamber (Corning, USA) the filter of which was pre-coated with matrigel (Sigma) and the lower chamber of which was filled with a medium containing 10% FBS as a chemoattractant. The cells were cultured for 20 h 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.7.3D cell culture:Matrigel was dissolved at 4℃ overnight and then used to coat 24-well plates (100 μl per well). After 30 min incubation at 37℃, 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.Results:1. For gene transfection, gastric cancer cells SGC-7901 with relative lower expression of hsa-miR-506-3p was transfected with hsa-miR-506-3p mimics to gain cell SGC-7901-miR506 OE. Gastric cancer cells BGC-823 with relative higher expression of hsa-miR-506-3pwas transfected with hsa-miR-506-3p inhibitor to gain cell BGC-823-miR506KD.2. Because angiogenesis is an important factor in cancer metastasis, we examined the angiogenic effects of hsa-miR-506-3p expression in gastric cancer cells. A matrigel-based tube formation assay using SGC-7901 cells showed that hsa-miR-506-3p overexpression suppressed vascular tubule development. Accordingly, as quantified by histological morphometric evaluation of chick chorioallantoic membrane (CAM) sections, vessel density was significantly reduced in SGC-7901 cells with hsa-miR-506-3p overexpression. Conversely, hsa-miR-506-3p inhibition in BGC-823 cells promoted the formation of a tubular polygonal network and induced vessel growth on the CAM.3. Consistently, gelatin zymography assays showed that the expression of matrix metalloproteinase (MMP) 9 and 2 were decreased by hsa-miR-506-3p overexpression. This suggests that hsa-miR-506-3p contributes to extracellular matrix maintenance in the gastric cancer microenvironment. Furthermore, gelatin zymography assays showed that hsa-miR-506-3p expression inhibition was associated with the expression of MMP9 and MMP2. These findings indicate that hsa-miR-506-3p is necessary and sufficient for angiogenesis suppression during gastric cancer progression.4. Because epithelial to mesenchymal transition (EMT) is an important process that enables cancer cells to invade their surroundings and metastasize, we examined whether hsa-miR-506-3p overexpression was correlated with EMT in human gastric cancer cell lines. We observed, as expected, that hsa-miR-506-3p overexpression reduced the ability of SGC-7901 cells to invade through matrigel. Additionally, wound healing assays revealed that the migration ability of SGC-7091 cells was diminished. These results indicated that hsa-miR-506-3p could be a potential suppressor of gastric cancer metastasis. Furthermore, the growth of gastric cancer cell pseudopodia is considered a pro-metastatic phenotype, and this was robustly decreased by hsa-miR-506-3p overexpression. Consistent with the hsa-miR-506-3p overexpression results, hsa-miR-506-3p inhibitors significantly increased the invasive and motive ability of BGC-823 cells. Furthermore, hsa-miR-506-3p suppression increased the growth of gastric cancer cell pseudopodia.5. Moreover, expression of E-cadherin and a-catenin was elevated, and expression of N-cadherin and vimentin was decreased by hsa-miR-506-3p overexpression, suggesting that hsa-miR-506-3p acts as an epithelial transition promoter in gastric cancer cells.Similarly, Western blot analysis showed that hsa-miR-506-3p inhibition caused decreased expression of E-cadherin and a-catenin, as well as increased expression of N-cadherin and vimentin. Collectively, these findings indicate that hsa-miR-506-3p plays a role in EMT suppression in gastric cancer cells.Part Ⅲ:Hsa-miR-506-3p targeting ERK-2/ETS-1 to regulate MMP9 expression supresses the metastasis and invasion of gastric cancerMethods1. Hsa-miR-506-3p target genes were predicted and selected using TargetScan software (http://www.targetscan.org, last accessed May 20,2015).2. Luciferase assay:TargetScan (http://www.targetscan.org/) was used to predict whether hsa-miR-506-3p targeted the 3’UTRs of ETS-1. Mutations in the hsa-miR-506-3p target sites were generated. For the luciferase assay, cells (2×105) were seeded in triplicate in 6-well plates and allowed to settle for 24 h. Different concentrations of hsa-miR-506-3p mutant, inhibitor, mimics or the control-luciferase plasmid were synthesized at Kunming Medical University and RiboBio (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 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Three independent experiments were performed and the data are presented as the mean ±SD.3. Immunohistochemistry:For immunohistochemistry, specimens from 173 gastric cancer patients were collected from The Department of Pathology, from January 2003 through November 2008. For all patients, detailed clinicopathological data were collected, including gender, age, TNM staging and clinical staging as defined according to the criteria of the American Joint Commission on Cancer (AJCC,7th Edition). An EnVisionTM detection kit (GK500705, DAKO, Glostrup, Denmark) was used for immunohistochemical analysis of ETS-1 and MMP9 proteins in gastric cancer tissues according to the manufacturer’s protocol. Briefly, primary antibodies were diluted in phosphate buffered saline (PBS) according to the manufacturers’ recommendations:1:50 for ETS-1 (NCL-ETS-1, Novocastra, Newcastle upon Tyne, UK) and 1:400 for MMP9 (sc-21733, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and a total volume of 50μl diluted antibody solution was added to each section and incubated overnight at 4℃. The next day, after washing with PBS, sections were incubated with Dako REALTM EnVisionTm/HRP (Dako Corporation, Carpinteria, USA) for 30 min each. Color visualization of the complexes was achieved by incubating tissue sections with a chromogenic substrate 3,3’-diaminobenzidine (DAB; Dako Corporation) for 3 min, after which slides were rinsed in water, counterstained with hematoxylin and mounted with Eukitt (O. Kindler GmbH & Co., Freiburg, Germany). Levels of ETS-1 and MMP9 expression as detected by antibodies in gastric cancer tissue specimens were reviewed and scored under a light microscope by two independent pathologists who were not aware of the clinicopathological data.4. Statistical analysis:All statistical analyses were performed using SPSS for Windows (Version 13.0, SPSS Inc., Chicago, IL, USA). The Student’s t-test was used to evaluate whether there was a significant difference between two groups of data in all the pertinent experiments. A p value< 0.05 (using a two-tailed paired t test) was considered statistically significant.Results1. ERK-2 and ETS-1 are two direct targets of hsa-miR-506-3p regulation: hsa-miR-506-3p target genes were predicted and selected using TargetScan software.2. Based on our analysis, we found that hsa-miR-506-3p targets the 3’UTRs of ERK-2 and ETS-1. The expression levels of ERK-2 and ETS-1 were lower in hsa-miR-506-3p transfected SGC-7901 cells; however, their expression was augmented following hsa-miR-506-3p inhibition in BGC-823 cells. Moreover, the expression levels of VEGFR2 and MMP9 were also lower in hsa-miR-506-3p transfected SGC-7901 cells and augmented following hsa-miR-506-3p inhibition in BGC-823 cells.3. To confirm hsa-miR-506-3p interaction with the 3’UTR of ETS-1, wild type or mutant hsa-miR-506-3p-binding sequences from the 3’-UTR of ERK-2 and ETS-1 genes were cloned into the 3’UTR of luciferase reporter gene for luciferase assays. Overexpression of hsa-miR-506-3p with the wild type ERK-2 or ETS-13’UTR in SGC-7901 and BGC-823 cells resulted in a significant decrease in luciferase activity; however, luciferase activity was not decreased by overexpression of hsa-miR-506-3p with mutant 3’UTR hsa-miR-506-3p-binding sequences. Collectively, these results indicate that ERK-2 and ETS-1 may be two potential targets mediating hsa-miR-506-3p-dependent regulation in gastric cancer cells.4. ETS-1 expression is associated with MMP9 in gastric cancer tissues:Because MMP9 is known to participate in gastric cancer metastasis and ETS-1 was reported to be MMP9 transcription factor, we examined the expression of both proteins in gastric cancer tissues. Protein expression levels were examined in a cohort of human gastric cancer samples in a blinded manner. Nuclear accumulation of ETS-1 was observed in 71.10% of the gastric cancer tissues examined, while 75.14%(130/173) of the tissue samples examined stained positively for MMP9. ETS-1 expression was associated with MMP9 expression (r= 0.459, p≤0.001), indicating that ETS-1 and MMP9 synergistically regulate gastric cancer metastasis.Conclusions1. Additional studies will be needed to explore the potential clinical utility of hsa-miR-506-3p as a potential biomarker for gastric cancer prognosis.2. Hsa-miR-506-3p targeting ERK-2/ETS-1 inhibits angiogenesis and cell migration and invation in gastric cancer...