Patients and tissue sample
A total of 26 tumor tissues and matched adjacent normal tissues were collected from 26 patients who had undergone surgery to treat NSCLC at the Affiliated Wujiang Hospital of Nantong University. Fresh tissue samples were confirmed via histopathological examination and then immediately stored in a ‑80 ˚C refrigerator prior to subsequent experiments. Our project was approved by Research Ethics Committee of Affiliated Wujiang Hospital of Nantong University.
Cell culture and transfection
All cell lines involved in this study were purchased from ATCC and cultured in our central lab. Human NSCLC cell lines, including A549 and H1975, and human bronchial epithelioid cells 16HBE were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were then incubated in a humidified cell incubator maintained with 5% CO2 at 37 ℃. Cells were plated in a 6-well dish and grown at 50%-60% density. All the transient transfections were conducted with Lipofectamine 2000 Reagent. The sequences were as followed: miR-221-3p mimics, 5′-AGCUACAUUGUCUGCUGGGUUUC-3′,5′-AACCCAGCAGACAAUGUAGCUUU-3′; miR-221-3p inhibitor, 5′-GAAACCCAGCAGACAAUGUAGCU-3; and negative control, 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′. The sequences of siRNA and its negative control were as follows: Axin2, 5′-GCAGAGGGACAGGAATCAT-3′, and the negative control, 5′-GCAGGGACAAGGTAGACAT-3′.
Total RNA was extracted from patient tissue and cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. cDNA was synthesized using PrimeScript® RT reagent Kit. RT-qPCR was performed using SYBR Premix ExTaqTM (Takara Bio, Inc.) on the platform of Applied Biosystems 7500 (Applied Biosystems; Thermo Fisher Scientific, Inc.). U6 and GAPDH were used as internal controls for miRNA and mRNA, respectively. Data analyses were performed using the comparative CT (ΔΔCT) approach to calculating relative gene expression. Primers were synthesized by Sangon Biotech Co., Ltd. as follows: miR-221-3p, forward, 5′-CGGCTACATTGTCTGCCTG-3′ and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′; and U6 forward, 5′-CGCTTCGGCAGCACATATAC-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The reverse universal miR qPCR primers were included in the PrimeScript™ miRNA RT-PCR kit (cat. no. RR716; Takara Biotechnology Co., Ltd.). The relative expression levels of miR-221-3p and were calculated using the 2−ΔΔCq method. All experiments were conducted in triplicate.
Colony formation assay
Colony formation assay was carried out to evaluate the proliferative potential of A549 and H1975 after transfections. Briefly, A549 and H1975 NSCLC cells with a density of 1 × 103 cells/well were seeded in six-well plates and cultured at 37 ℃ with 5% CO2. The medium was replaced with fresh culture medium every two or three days for two weeks. Subsequently, cells were fixed with 4% paraformaldehyde for 20 minutes and stained using 10% crystal violet for 30 minutes.
Wound healing assay
Wound healing assay was used to assess the cell migration ability of A549 and H1975 NSCLC cells in vitro. The cells were cultured into six-well plates and incubated for 24 hours to full confluence. Before scratching, the medium was replaced with fresh culture medium without FBS. A scratch was then created using a sterile plastic tip, and the cells were incubated for 48 hours at 37 oC ℃. The closure of the scratch was analyzed under the microscope and images were captured using an Olympus light microscope (Olympus Corporation, Tokyo, Japan).
EdU assay was performed to detect the proliferation of A549 and H1975 NSCLC cells. Cells were seeded in six-well plates and incubated for 48 hours after different treatments in a humidified cell incubator maintained with 5% CO2 at 37 ℃. After incubation with 10 μM EdU for 2 hours, cells were fixed in 4% paraformaldehyde. After that, Hoechst 33342 was used to stain the nuclei. Finally, cells were stained using a Cell-hour Light EdU Apollo 488 in vitro Imaging Kit according to the manufacturer’s recommendations.
Cell apoptosis assay
Cell apoptosis was performed using the Annexin V Apoptosis Detection Kit I. Briefly, the treated A549 and H1975 NSCLC cells were collected, washed twice with cold 1 × PBS and resuspended in 100μL binding buffer, followed by incubation with Annexin V-FITC and propidium iodide for 15 minutes at room temperature in the dark. Next, 200 μL of binding buffer was added. FACS Calibur was used to calculate the percentage of apoptotic cells.
Transwell migration and invasion assays
Migration and invasion assays were performed using transwell chambers. In migration assay, A549 and H1975 NSCLC cells at a density of 5 × 104 cells/well were added into the upper chamber. In invasion assay, Matrigel was inoculated into the upper chamber to form a gel at 37 ℃, and then A549 and H1975 NSCLC cells were seeded into the upper compartments at a density of 1 × 105 cells/well. For transwell migration and invasion assays, the lower compartments were filled with 600 μL of medium with 20% FBS. After incubation for 48 hours, cells that had not migrated or invaded were removed from the upper surface while the cells that had migrated or invaded to the lower surface of the membrane were fixed with 4% paraformaldehyde and stained in 10% crystal violet. All experiments were conducted in triplicate.
Western blot (WB) assay
Proteins were separated by electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% milk at room temperature for one hour, then incubated with diluted antibodies against MMP-2, MMP-9, GAPDH, Bcl-2, Bax, Caspase-3, Caspase-7, Axin2, MMP-7, β-catenin, and β-actin. All antibody dilutions were 1:1000. After being washed, blots were incubated with secondary antibody and visualization by enhanced chemiluminescence. All experiments were conducted in triplicate.
A549 NSCLC cells were incubated overnight at 4 °C with primary antibodies against β-catenin. The following day, cells were washed in PBS three times (5 minutes each wash) and incubated with secondary antibodies for 2 hours at room temperature. After incubation, cells were rinsed three times (5 minutes each wash) in PBS and mounted in 50% triglyceride. All experiments were conducted in triplicate.
All statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Data were presented as the mean standard deviation from three independent experiments. Significant differences between means were analyzed by the two-tailed, unpaired, non-parametric Mann-Whitney test, and differences were considered significant at P < 0.05.