Cell lines and tissue samples
EC cell lines (Ishikawa and ECC), and the normal cell line (SHT290) were procured from the Cell Resource Center of Chinese Academy of Medical Sciences (Beijing, China), then cultivated in the Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY). Cell culture was implemented with 10% fetal bovine serum (FBS; Gibco) as medium supplement, in the 95% air/5% CO2 incubator at 37 °C.
RNA extraction and real-time quantitative PCR (RT-qPCR)
The total cellular RNAs were severally extracted on the basis of the protocol of TRIzol reagent (Invitrogen, Carlsbad, CA). 3 μg of total RNAs were converted into the cDNA by applying the Reverse Transcription Kit (Toyobo, Osaka, Japan). Gene expression was quantified by ABI Step-One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA) with the SYBR® Premix Ex Taq™ II (Takara). As for Data calculation, 2−ΔΔCT method was used, with GAPDH and U6 as controls.
Cell transfection
The short hairpin RNAs (shRNAs) against OIP5-AS1 were constructed by Genepharma (Shanghai, China) for gene silencing analysis, as well as its control, sh-NC. The pcDNA3.1/OIP5-AS1, pcDNA3.1/SLC7A5 and the control pcDNA3.1 vectors were commercially obtained from Ribobio (Guangzhou, China) to up-regulate target genes. Besides, the miR-152-3p mimics/inhibitor, as well as the corresponding negative control (NC mimics/inhibitor) were designed at Genepharma. Plasmids were transfected into EC cell for 48 h via Lipofectamine 2000 (Invitrogen).
Colony formation
Clonogenic cell samples were cultivated as 500 cells per well into the 6-well plates for 14 days, followed by the treatment with the 0.5% crystal violet staining solution in 4% paraformaldehyde. Clones were determined and counted.
Flow cytometry analysis of apoptosis
Flow cytometry analysis was performed for the examination of cell apoptosis. The cells were stained in Annexin V-labeled with 7AAD and PE solution (BD Biosciences, San Jose, CA) for a quarter of an hour in the dark. After being rinsed in the pre-chilled phosphate-buffered saline (PBS) and double-staining, cell samples were assayed by FACS cytometry (BD Biosciences) finally.
Western blot
30–50 mg of the cellular total proteins were subjected to RIPA lysis buffer for isolation and then to 12% SDS-PAGE gel for separation. Subsequently, the separated protein samples were electro-blotted onto the PVDF membranes (Millipore, Billerica, MA), and then treated with the 5% skimmed milk solution for blocking. Samples were cultivated with the primary antibodies against the loading control GAPDH and Bcl-2, Bax, Cleaved caspase-3, Total caspase-3, as well as four EMT markers, slug, Vimentin, N-cadherin, E-cadherin (at 1: 2000 dilution, Abcam, Cambridge, MA) for a whole night. Followed by rinsing in the Tris-buffered saline (TBST), samples were incubated with the secondary antibodies (at 1: 5000; Abcam) for 2 h. Blots were finally detected by Immobilon® Western Horseradish peroxidase substrate (Millipore).
Transwell assays
6 × 104 cell samples in the 100 ml of medium free of FBS were placed into the upper part of the Transwell chamber (Corning Life Sciences, Corning, NY). 600 ml of conditioned medium was placed into the lower chamber. Followed by 24 h of incubation at 37 °C, migrating cell samples were fixed for 15 min and treated with 0.5% crystal violet staining for 30 min. The up‐right fluorescence microscope (200×) was applied for counting. Cell invasion was analyzed with Transwell chamber coated by the Matrigel membrane (BD Biosciences).
FISH assay
Cell samples were first fixed for 15 min, then rinsed in PBS and air-dried. 40 nm of the FISH probe synthesized for OIP5-AS1 was procured from Ribobio and mixed with the cell samples in the hybridization buffer. As for the counterstaining of nucleus, Hoechst solution was adopted. The final images were captured using the fluorescence microscope.
RNA pull down
The assay was conducted by employing the Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA) in light of the user manual. The wild-type (WT) or mutated (Mut) miR-152-3p sequences with the OIP5-AS1 binding sites were constructed, followed by the biotinylation into Bio-miR-152-3p-WT/Mut probes. The cells, after being lysed, were subjected to the 1 h-incubation with the probes and streptavidin agarose magnetic beads. After RNA extraction and purification, RT-qPCR was finally conducted for determining OIP5-AS1 enrichment in pull-downs.
Dual-luciferase reporter assays
OIP5-AS1-WT/Mut or SLC7A5-WT/Mut fragments covering the miR-152-3p interacting sequences were constructed and subjected to the subcloning into the pmirGLO Dual-Luciferase Expression Vector (Promega, Madison, WI). Afterwards, the reporter vectors were subjected to the co-transfection into EC cells along with the specific plasmids for 48 h. Dual-Luciferase Reporter Assay System (Promega) was employed for luciferase activity.
RNA immunoprecipitation (RIP)
The assay was implemented on a basis of the instruction of EZ-Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore). The cultured cell samples were lysed in the RIP lysis buffer, then collected and treated with magnetic beads bound to the antibodies of Ago2 or IgG (Millipore) in RIP buffer. Following RNA extraction, the purified RNAs were assayed by RT-qPCR.
Statistical analysis
All assays were subjected to independent bio-repetition in triplicate. All the experimental results were represented as the mean ± standard deviation (SD). Data analysis was developed by Student's t-test and one-way or two-way analysis of variance (ANOVA) employing the Prism 5.0 (GraphPad Software Inc., La Jolla, CA). Significant level was set as the p-value less than 0.05.