miR-124 represses the mesenchymal features and suppresses metastasis in Ewing sarcoma

Metastasis is the most powerful predictor of poor outcome of Ewing sarcoma (ES). Thus, identification of new molecules involved in tumor metastasis is of crucial importance to reduce morbidity and mortality of this devastating disease. In this study, we found that miR-124, a highly conserved miRNA, was suppressed in ES tissues and might be associated with tumor metastasis through suppressing its mesenchymal features. Overexpression of miR-124 suppressed the invasion of ES cells in vitro and tumor metastasis in vivo, which might be achieved through suppressing its mesenchymal features, as overexpression of miR-124 could repress the mesenchymal genes expression, and inhibit cell differentiation to mesenchymal lineages in ES cells. However, when SLUG was experimentally restored in these cells, mesenchymal features including suppressed expression of mesenchymal genes and decreased invasive ability were observed. We also found that cyclin D2 (CCND2) was a novel target gene of miR-124, and was directly involved in miR-124-mediated suppressive effects on cell growth. Lastly, we found that treatment with 5-Aza-CdR restored the expression of miR-124, accompanied with suppressed cell proliferation, invasion and mesenchymal features of ES cells, which demonstrated that hypermethylation might be involved in the regulation of miR-124 expression. Collectively, our data suggest that hypermethylation-mediated suppression of miR-124 might be involved in the tumor initiation and metastasis through suppressing the mesenchymal features of ES cells.


Cell culture and transfection
Human ES cell lines (A673, SK-ES-1, RD-ES, SK-N-MC) were obtained from the American Type Culture Collection. These cell lines were immediately expanded and frozen such that they could be restarted every 3 to 4 months from a frozen vial of the same batch of cells. Human mesenchymal stem cells (MSCs) were isolated from normal adult human bone marrow withdrawn from bilateral punctures of the posterior iliac crests of three normal volunteers. MSCs were maintained at low confluency in IMDM, 10% FBS, and PDGF-BB (10 ng/ml). All cell lines were authenticated (Hybribio Bioscience & Technology Inc., Guangzhou, China). A673 and RD-ES cells were maintained in RPMI 1640 medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria), streptomycin (100 μg/ml), and penicillin (100 U/ml). SK-ES-1 cells were propagated in McCoy's 5A medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% FBS. SK-N-MC cells were maintained in DMEM medium, supplemented with 10% FBS. All cells were incubated in a humidified atmosphere of 5% CO 2 at 37°C as previously reported (30). MiR-124/scramble mimics were purchased from Dharmacon (Austin, TX, USA). According to manufacturer's instructions, all oligonucleotides were transfected into ES cells to a final concentration of 50 nM by Dhamafect 1 (Dharmacon, Lafayette, CO, USA). Cells were collected for further experiments 48 h post-transfection.

Cell proliferation and cell cycle analyses
For cell proliferation analysis, cells were seeded into 24-well plates at 8 × 10 3 cells per well, and then incubated in 10% Cell Counting Kit-8 buffer (CCK-8, Dojindo, Japan) diluted in normal culture medium at 37°C until visible color conversion occurred. The proliferation rate was determined 0, 24, 48, and 72 h after transfection. The absorbance in each well was measured at 450 nM and 630 nM using a microplate reader. For cell cycle analysis, cells were harvested 48 h after transfection, washed twice with cold PBS, fixed in ice-cold 70% ethanol, incubated with propidium iodide and RNase A, and then analyzed by fluorescence-activated cell sorting (FACS).
All experiments were performed four times and the average percentages of cells are shown.

Cell migration and invasion assays
Migration assays were carried out in modified Boyden chambers (BD Biosciences, San Jose, CA, USA) with 8 μm pore filter inserts in 24-well plates. 24h after transfection, 2×10 5 cells suspended in serum-free medium were added to the upper chamber. Medium containing 20% FBS were added to the lower chambers as a chemoattractant. After 24 h transfection, the non-filtered cells were gently removed with a cotton swab. Filtered cells located on the lower side of the chamber were stained with crystal violet, air-dried and photographed. For analysis of invasive capacity, the transwell migration chambers were coated with Matrigel (BD Biosciences, San Jose, CA, USA) and incubated at 37°C for 3 h, allowing it to solidify. After 24 h of transfection, 4×10 5 cells suspended in serum-free medium were added to the upper chamber. The remaining steps were the same as migration assays. Three independent experiments were performed.

5-Aza-CdR and trichostatin A treatment of cell lines
ES cell lines A673 and SK-ES-1 were with 5-Aza-CdR (Sigma-Aldrich, USA) at 5 μmol/L for 3 days or 300 nmol/L trichostain A (TSA, Sigma-Aldrich, USA) for 24 hours. For the combination treatment, cells were treated with 5-Aza-CdR for 48 hours firstly. Then TSA was added, and the cells were treated for an additional 24 hours. Culture medium containing drug was replaced every 24 hours. RNA of cell lines was purified with TRIzol reagent following the instructions from manufacturer.