MiR

Dulbecco’s Modified Eagle Medium (DMEM), Lipofectamine LTX and PLUS reagents as well as Lipofectamine 3000 transfection reagent were products of Thermo Fisher Scientific (Waltham, MA, USA). 24-well transwell plates (8.0-μm pore polycarbonate membrane inserts) were purchased from Corning (New York, NY, USA). Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ). The pCMV3-CORO1C-WT-GFPSpark vector, pCMV3-CORO1C-Mut-GFPSpark vector (the binding site of miR-206 to CORO1C was replaced with UGCGAGG), NC mimic (5′-UUUGUACUACACAAAAGUACUG-3′) and miR-206 mimic (5′-TGGAATGTAAGGAAGTGTGTGG-3′) were purchased from Sino Biological Inc. (Beijing, China). The E.Z.N.A. Total RNA Kit I was purchased from Omega Bio-Tek (Doraville, GA, USA). The iQ SYBR Green Supermix Kit for q-RT-PCR was a product of Life Technologies (Grand Island, NY, USA). Antibodies against vimentin (D21H3), E-cadherin (24E10), N-cadherin (D4R1H), ZO-1(D6L1E) and Snail (C15D3), CORO1C (D6K5B) and β-actin (13E5) were purchased from Cell Signaling Technology (Danvers, MA). Other reagents used in this study were products of Sigma-Aldrich (St. Louis, MO).

We collected 50 pairs of NSCLC tissues and corresponding normal mucosa tissues from the General Hospital of Guangzhou Military Region, Guangzhou, China. Tissues were obtained from the patients who underwent surgery to remove NSCLC tissues in the General Hospital of Southern Theater Command. All patients’ diagnoses were confirmed histopathologically. The study was conducted under the supervision of the General Hospital of Southern Theater Command following the World Medical Association, and all patients have signed informed consent.

Total RNA of the frozen NSCLC tissues and cell lines was extracted using Trizol reagent. The cDNA of mRNA was generated with a Transcriptor First Strand cDNA Synthesis kit, and the cDNA of miRNA was generated with all-in-One miRNA qRT-PCR detection kit. The oligonucleotide primers used to detect miR-206 and CORO1C are presented in Supplementary Material 1.The PCR conditions were as follows: predenaturing at 95 °C for 5 min, then 45 cycles (95 °C for 10 s, 60 °C for 20 s, 72 °C for 20 s), followed by 72 °C for 10 min. All these processes were performed with a Roche LightCycler 480 real-time PCR machine. GAPDH and U6 snRNA were set as endogenous controls for mRNA and miRNA. ΔCt was the result that normalized to U6 or GAPDH Ct values. Then ΔΔCt was calculated by normalizing the ΔCt of the control sample. The values of the target gene are presented as the relative fold change of the housekeeping gene, which was set as 1. Fold change of the gene was calculated by the equation: 2−ΔΔCt.

Human lung cancer cell lines including A549, NCI-H365, NCI-H460, SPCA-1 and XL-1, as well as human lung epithelia cells BEAS-2B, were products of the American Type Culture Collection (ATCC, Manassas, VA, USA). All the cells were cultured with DMEM containing 10% FBS, 100 U/ml penicillin sodium (PS), and 100 mg/ml streptomycin sulfate in a 37 °C incubator containing 5% CO2.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate cell vitality as previously described [22]. Briefly, after culturing for 24 h, the cells that were seeded in the 96-well plates were transfected with NC mimic or miR-206 mimic for various times (24 h, 48 h, 72 h and 96 h). Then the cell viabilities were measured with MTT assay using a Multi-Detection Microplate Reader (BMG Labtech, Ortenberg, Germany).

Briefly, 5 × 105 A549 cells were seeded in 6 plates and grown to approximately 100% confluence. Then the cells were starved for 6 h with 2% FBS DMEM, followed by scratching using a 10-μl pipette tip. Next, the cells were exposed to NC mimic or miR-206 mimic for an additional 8 h in 2% FBS DMEM. The cells of the same field were photographed at 0 h and 8 h with an Olympus IX70 inverted microscope (Shinjuku, Tokyo, Japan). The Image-Pro Plus 6.0 (IPP 6.0) software (Rockville, MD) was used to calculate the migratory cells and the experiment was carried out in triplicate.

We conducted the transwell migration assay and transwell invasion assay to evaluate the effect of miR-206 on the vertical migration ability and invasion ability of A549 cells using a 24-well transwell chamber. The cells suspended in non-serum DMEM were seeded in the upper chamber of the transwell with a density of 2 × 104 per well, and 600 μL of fresh complete DMEM (10% FBS) were added to the lower chamber. The cells were treated with NC mimic and miR-206 mimic at the same time. The cells in the upper chamber were fixed with 4% paraformaldehyde for 30 min after incubating for 24, following by staining with 0.1% crystal violet. After wiping of the non-migratory cells in the inner side of the upper chamber, the migratory cells adhering to the lower surface of the membrane were observed and photographed using an Olympus IX70 inverted microscope. Migratory cells were analyzed by IPP 6.0 software. Regarding the transwell invasion assay, the upper chambers were precoated with Matrigel. Other processes were the same as in the transwell migration assay.

The miR-206 mimic or NC mimic was transfected into A549 cells using Lipofectamine RNAiMAX transfection reagent (Invitrogen) in the reference of the manufacturer’s instructions.

Regarding vector transfection, the NC vector or CORO1C vector was transfected into adherent A549 cells using Lipofectamine 3000 transfection reagent following the manufacturer’s instructions. Transfected cells were subjected to western blotting assay and q-RT-PCR assay to detect the transfection efficacy.

Adherent 293 T cells were cotransfected with pCMV3-CORO1C-WT-luc vector/pCMV3-CORO1C-Mut-luc vector (expressing firefly luciferase) and pGL4.73 [hRluc/SV40] vectors (expressing Renilla luciferase) using Lipofectamine LTX and PLUS reagents according to the manufacturer’s protocol. The cells were applied for luciferase signals analyses on a TECAN Infinite F500 platform (Männedorf, Switzerland). The relative luciferase activity was the value that normalized to Renilla luciferase. Three experiments were performed.

We conducted western blotting assay according to a previously described protocol [23] with some modifications. The A549 cells that were transfected with NC mimic or miR-206 mimic for 24 h were collected, lysed with RIPA buffer and subjected to Western blotting assay.

5- to 6-week old male BABL/c (nu/nu) mice obtained from Vital River Laboratory Animal Technology Co, Ltd. (Beijing, China) were housed in a specific pathogen-free room. All the animal studies were conducted under the supervision of the General Hospital of Southern Theater Command animal care and use committee. A549 cells suspended in 200 μl of PBS were inoculated subcutaneously into the backs of male mice. After about 10 days, tumor-bearing mice were randomized to the NC mimic group and miR-206 mimic group (five mice per group) and the tail was intravenously injected with NC mimic or miR-206 mimic once every two days for a total of 7 times. A slide caliper was used to measure the tumor volumes, and the formula a × b2 × 0.5 (a represents the longest diameter and b is the shortest diameter) was used to calculate the tumor volume. The mice were anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital sodium. After that, the tumors were removed and weighed. Then the tumors were cryopreserved for further q-RT-PCR experiments. During the experiment, no adverse outcomes occurred, such as blistering or ulceration of tumor.

The data analysis was processed with GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA) and all data were presented as the mean ± standard error of the mean (SEM). Significant differences between two groups were analyzed with two-tailed unpaired Student’s t-test, and the difference of multiple groups was calculated using one-way ANOVA (Tukey’s post hoc test). P