Sample collection and cell culture
The paired tumor tissues and adjacent normal tissues (n = 42) were collected from ESCC patients who underwent surgery resection at the Shangqiu first People’s Hospital. This study was approved by the Ethics Committee of Shangqiu first People’s Hospital and informed consents were signed by all patients. The normalized RNA-seq data of Esophageal Carcinoma (ESCA) were downloaded from the TCGA data portal website (https://cancergenome.nih.gov/).
Human immortalized esophageal epithelial cell line HET-1A and human ESCC cell lines (ECA109 and EC9706) were purchased from ATCC (Manassas, VA, USA). All cells were cultured in PRMI-1640 medium (Gibco, Rockville, MD, USA) supplemented with 10% FBS (Gibco) at 37 °C with 5% CO2. DDP-resistant variants (ECA109/DDP and EC9706/DDP) of ECA109 and EC9706 cells were established using a repetitive pulsatile treatment with constant concentrations of cisplatin [17]. The degree of chemotherapy resistance of DDP-resistant variants was evaluated before transfections.
Cell transfection
Empty pcDNA3.1 vector (Vector) was obtained from Genepharma (Shanghai, China). TUG1 or PDCD4 overexpressing vector pcDNA3.1-TUG1 or pcDNA3.1-PDCD4 (TUG1 or PDCD4), small interfering RNAs against TUG1 (si-TUG1 #1 or si-TUG1 #2) or PDCD4 (si-PDCD4) and their scramble negative control (si-con) were chemically synthesized by Genepharma (Shanghai, China). All cell transfections were performed using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from ESCC tissues and cells using Trizol reagent (Invitrogen) and then reversely transcribed into cDNA using PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). TUG1 and PDCD4 expression levels were detected by quantitative real-time PCR with SYBR Green Master Mix (TOYOBO, Osaka, Japan) using an Applied Biosystems 7500 Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA). The primes were as follows: TUG1 forward, 5′-TAGCAGTTCCCCAATCCTTG-3′, TUG1 reverse, 5′-CACAAATTCCCATCATTC CC-3′; PDCD4 forward, 5′-GGCCTCCAAGGAGTAAGACC-3′, PDCD4 reverse, 5′-AGGGGTCTACATGGCAACTG-3′. Data were analyzed using the comparative Ct method (2−ΔΔCt) with GAPDH as an internal control.
Drug sensitivity assay
The cell viability of ECA109/DDP and EC9706/DDP cells and their parental cells was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, Missouri, USA) assay. DDP sensitivity was determined using the IC50 value (half maximal inhibitory concentration).
Flow cytometric analysis
Cell apoptosis was evaluated using Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN Biotech, Nanjing, China) as described previously [18]. Briefly, ECA109/DDP and EC9706/DDP cells with different transfection were treated with 20 μM DDP for 48 h, followed by double stained with Annexin V-FITC and PI under a dark condition. Cell apoptotic rates were evaluated by FACSan flow cytometry (BD Biosciences, San Jose, CA, USA).
Subcellular fraction assays
The separation of the nuclear and cytosolic fractions of ECA109 cells was performed using the PARIS Kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions.
RNA pull-down assays
TUG1 and anti-sense-TUG1 was transcribed with TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) and then labeled with Thermo Scientific Pierce RNA 3′ Desthiobiotinylation Kit (Thermo Fisher Scientific). Pierce Magnetic RNA-Protein Pull down Kit (Thermo Fisher Scientific) was used to perform RNA pull down assay. Briefly, labeled RNAs were bound with Streptavidin Magnetic Beads and then incubated with ECA109/DDP cell protein lysates. Then the RNA-binding proteins were eluted for the further western blot analysis.
RNA immunoprecipitation (RIP) assays
RIP experiments were performed using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to the manufacturer’s protocol. EZH2 and IgG antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The co-precipitated RNAs were purified and analyzed by qRT-PCR analysis.
Chromatin immunoprecipitation (ChIP) assays
Chromatin immunoprecipitation assay was performed to confirm the interaction between TUG1 and PDCD4 gene using EZ-ChIP kit (Millipore). The chromatins were immunoprecipitated with antibodies against EZH2 (Cell Signaling Technology), H3K27me3 (Millipore) or IgG (Millipore). Finally, the immunoprecipitated chromatin was purified and analyzed by qRT-PCR analysis. Primers for PDCD4 promoter region were 5′-GGTCTGGGAAGCTCCGATTT-3′ (forward) and 5′-GCAGTTGGTGGTCATCCTCA-3′ (reverse).
Luciferase reporter assay
PDCD4 promoter sequences were inserted into pGL3-Basic luciferase plasmid (Promega, Madison, WI, USA) to generate PDCD4 promoter reporter vector. Then, PDCD4 promoter reporter was transfected into ECA109/DDP cells using Lipofectamine 2000 (Invitrogen) along with phRL-TK vector (Promega) and (Vector or TUG1) or (si-con or si-TUG1). Luciferase Reporter assay system (Promega) was performed to detect luciferase activity in ECA109/DDP cells 48 h post-transfection.
Western blot analysis
Western blotting was performed according to our previously reported protocol [19]. The primary antibodies anti-EZH2, anti-PDCD4 and anti-GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).
Animal experiments
The animal experiment was performed according to the national standard of the care and use of laboratory animals and got the approval of the Ethics Committee of Shangqiu first People’s Hospital. ECA109/DDP cells were infected with sh-TUG1 or sh-con lentivirus, followed by the sieving using puromycin (Sigma-Aldrich, St. Louis, MO, USA) for nearly 7 days to construct stable lentivirus-transfected ECA109/DDP cell line. Then, ECA109/DDP cells (1.0 × 107) stably infected with sh-TUG1 or sh-con were subcutaneously injected into the tail veins of BALB/c-nude mice (4 weeks old) from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). One week later, mice were intraperitoneally injected with 6 mg/kg DDP or same volume of PBS every week according to indicated groups (n = 5 each group): sh-con + PBS, sh-TUG1 + PBS, sh-con + DDP, sh-TUG1 + DDP. The tumor sizes were measured every week. After 42 days, the mice were killed, and the tumor weights were detected. qRT-PCR and western blot assays were performed to detect TUG1 expression and PDCD4 protein levels.
Statistical analysis
All data were presented as means ± standard deviation (SD). Student’s t-test and one-way ANOVA were used to calculate the statistic difference using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Differences were considered statistically significant when P value < 0.05.