Upregulation of the long noncoding RNA UCA1 affects the proliferation, invasion, and survival of hypopharyngeal carcinoma

HSCC specimens and corresponding adjacent normal tissues were obtained from 53 HSCC patients who underwent surgery in the Department of Otolaryngology at the Qilu Hospital of Shandong University from March 2011 to December 2011. All HSCC specimens were confirmed by pathological diagnosis, and all patients underwent hypopharyngeal tumor resection plus ipsilateral modified neck dissection. None of the 53 patients received chemoradiotherapy or biotherapy before surgery. All patients received radiotherapy after surgery.

The clinicopathological information were obtained from patients’ history record including patient age, overall survival duration, tumor cell differentiation, T category, clinical disease stage, and lymph node status. The TNM staging system follows the standard of the International Union Against Cancer. Patients returned for follow-up visits once every 3 months until March 2016; 4 patients were lost to follow up. Before undergoing surgery, all patients provided their written informed consent for their tissues to be used in the study. The study protocol was approved by the Ethics Committee of the Qilu Hospital of Shandong University.

The only available HSCC cell line, FaDu, was purchased from the Shanghai Institute of Biology and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) in a humidified atmosphere containing 5% CO₂ at 37°C.

To extract total RNA from human tissue samples, we used Trizol reagent (Takara, Otsu, Japan) according to the manufacturer’s instructions. We used a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) to measure RNA concentration and purity. We subjected 2 μg of RNA from each tissue sample to cDNA synthesis using a reverse transcription kit (Takara). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with SYBR Green Real-Time PCR Master Mix (Toyobo, Osaka, Japan) and the LightCycler 480 II System (Roche, Basel, Switzerland). UCA1 and β-actin were amplified in triplicate using an annealing temperature of 60°C. Signals were quantified using the ΔΔCt method and normalized to β-actin levels.

DNA oligonucleotides to produce plasmid-based small hairpin RNA (shRNA) were cloned into the pLeno–green fluorescent protein (GFP) vector. The shRNA target sequences for UCA1 were GGACAACAGTACACGCATA (sh-1) and TTAATCCAGGAGACAAAGA (sh-2); a scrambled sequence, which does not share obvious homology with human gene sequences, was used as a negative control. The pLeno-GFP vector and packing vectors (pRsv-REV, pMDIg-pRRE, and PMD2G) were cotransfected into 293T cells using Lipofectamine 2000 according to the manufacturer’s instructions. After 48 h, the supernatant was collected, concentrated by centrifugation, and filtered through 0.45-μm cellulose acetate filters. The titer was measured by counting the GFP-positive cells under a fluorescence microscope (Olympus, Tokyo, Japan). FaDu cells in 6-well plates were infected with the constructed lentiviruses at a multiplicity of infection of 60. The infection efficiency was determined by counting the GFP-positive cells under a fluorescence microscope 96 h after infection. The efficiency of UCA1 silencing was subsequently quantified by qRT-PCR (Fig. 1).

The Annexin V–allophycocyanin apoptosis detection kit (eBioscience, San Diego, CA, USA) was used to identify apoptotic cells. Briefly, FaDu cells were seeded in 6-well plates following 5 days of lentiviral infection at a multiplicity of infection of 60. After being trypsinized and washed twice with phosphate-buffered saline (PBS), the FaDu cells were stained with Annexin V–fluorescein isothiocyanate and propidium iodide (PI) successively in the staining buffer at 4°C in the dark and then detected by flow cytometry with the FACSCalibur system (BD Biosciences, San Diego, CA, USA).

FaDu cells were seeded in 60-mm dishes and infected with the lentivirus for 24 h. Cells were harvested and washed twice with cold PBS and then fixed with ice-cold 70% ethanol for 1 h at 4°C. After centrifugation at 1500 r/min for 5 min, the cells were washed twice with PBS and resuspended with 0.5 ml of PBS containing PI (50 μg/ml) and Rnase (100 μg/ml). The cells were then stained at room temperature in the dark for 15 min, and the cell cycle distribution was assessed by flow cytometry and analyzed using ModiFit 3.0 software (Becton Dickinson).

We used a colony formation assay to assess the effect of UCA1 knockdown on FaDu cell colony formation. Briefly, stably transfected FaDu cells were sufficiently trypsinized and suspended in DMEM supplemented with 10% FBS. The cells were then seeded in 6-well plates in triplicate (800 cells/well) and maintained in a humidified atmosphere containing 5% CO₂ at 37°C. The medium was changed every 3 days. After culture for 14 days, cell colonies were washed with PBS, fixed with 4% paraformaldehyde for 30–60 min, and then stained with Giemsa (ECM550 Chemicon, CA, USA) for 20 min. Only colonies containing more than 50 cells were counted.

To assess the invasiveness of FaDu cells, we used transwell chambers (Corning, Tewksbury, MA, USA) coated with Matrigel (BD Biosciences). Fully trypsinized, transfected cells (1 × 10⁵ cells in 100 μl of serum-free DMEM) were plated into the upper chamber. DMEM (500 μl) supplemented with 20% FBS was added to the lower chamber. After incubation in a humidified atmosphere containing 5% CO₂ at 37°C for 72 h, FaDu cells that had invaded the lower chamber were fixed with methanol and stained with Giemsa. We used an inverted microscope (magnification 200×) to count the invading cells manually.

All experiments were repeated at least 3 times. Data were presented as the mean ± the standard deviation and analyzed with the SPSS software program (version 20.0). P values < 0.05 were considered statistically significant. The relationship between clinicopathological characteristics and UCA1 expression was analyzed using the chi square test or Fisher exact test. The Student t test was used to assess differences between two experiment groups. We used the Kaplan-Meier method to create survival curves for patients with high or low UCA1 expression and used a log-rank test to compare overall survival rates.