The cDNA of PTCSC3 was amplified from normal human thyroid tissues (surgical specimen from benign thyroid lesion). The sequences of the forward and reverse primers were 5′-GTACGGTAC CCTCCTTCAGACTTCTCAGTACTC-3′ and 5′-CGACTC GAGATTGCTACTGTGAGCATAACCTAC-3′, respectively. Subsequently, PTCSC3 was subcloned into a pcDNA3 vector (Invitrogen Life Technologies, Carlsbad, CA, USA) to create the expression plasmid for PTCSC3 and the products were confirmed by polymerase chain reaction (PCR) and sequencing.

Thyroid tumor cell lines BCPAP, FTC133 and 8505C were cultured in RPMI-1640 medium supplemented with 10% calf serum, 0.1 mM non-essential amino acids, 1 mM sodium-pyruvate and 1% penicillin-streptomycin in a 37°C humidified incubator with 5% CO₂. The thyroid cancer cell lines were assessed for PTCSC3 expression and no endogenous expression was observed. BCPAP, FTC133 and 8505 cells, which are of papillary, follicular and anaplastic cancer origin, respectively, were transfected with the PTCSC3 expression construct and the empty vector (pcDNA3) as the control, respectively, using 2 μl Lipofectamine 2000 reagent (Invitrogen Life Technologies). At 24 h post-transfection, the cells were harvested and total RNA was extracted using TRIzol reagent and Ambion^® DNase I (Invitrogen Life Technologies). An Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to assess its integrity and a high-capacity reverse transcriptase kit (Applied Biosystems, Foster City, CA, USA) was applied to produce cDNA. To confirm successful transfection, reverse transcription (RT)-PCR was performed to detect PTCSC3 expression (forward primer, 5′-TCAAACTCCAGGGCTTGAAC-3′; reverse primer, 5′-ATTACGGCTGGGTCTACCT-3′). The study was approved by the ethics committee of Xiangya Hospital, Central South University, Changsha, China.

To study the changes in the biological characteristics of the thyroid cancer cell lines following PTCSC3 transfection, cell proliferation was analyzed with the MTT assay and apoptosis and cell cycle with flow cytometry, as previously described (13). For each group, cells in the logarithmic phase were used in all experiments and incubated at 37°C in 5% CO₂. For the MTT assay, cells were incubated with 0.5 mg/ml MTT for 4 h. The formazan crystals produced by the living cells in the culture were dissolved with 100 ml dimethyl sulfoxide and the absorbance [optical density (OD) value] was measured at 570 nm using a 96-well plate reader at various time points (0, 24, 48, 72 and 96 h). For flow cytometry, the prepared cells were collected and digested into single-cell suspensions using 0.25% trypsin. Then, the cells were centrifuged at 500 × g for 5 min and washed with phosphate-buffered saline (PBS; 0.01 M, pH 7.4) twice. Seventy percent ethanol stored at 4°C was used to fix the cells for 24 h before they were fully shaken and dispensed. The plates were incubated with 0.5% Triton X-100 (Sigma, St. Louis, MO, USA) and 0.05% RNase (Sigma) in 1 ml PBS at 37°C for 30 min and then centrifuged at 1,500 × g for 5 min. The cells were stained with 50 mg/ml propidium iodide (Sigma) at room temperature for 30 min and the cell number was adjusted to 1×10⁶/ml. Samples were immediately analyzed using a FACSCalibur flow cytometer (Becton-Dickinson, Mountain View, CA, USA).

Bioinformatic analysis was carried out based on the online software, PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html). Briefly, the PTCSC3 sequence, NR_049735.2, was obtained from PubMed and entered into the PITA system. As PTCSC3 is a non-protein coding RNA, it is unlikely that the open reading frame (ORF) is occupied by ribosomes. We uploaded the full sequence of PTCSC3, including the 5-untranslated region (UTR), ORF and 3-UTR, into the PITA system. Only the top 20 miRNAs presenting targeting sites in PTCSC3 were selected for further analysis.

To investigate the expression of miR-574-5p in thyroid cancer cells following transfection with PTCSC3, a quantitative RT-PCR technique was used. Briefly, total RNA was isolated from tissues using the TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. Quantitative real-time PCR was performed on an ABI 7300 real-time PCR system (Applied Biosystems) using SYBR-Green mix (Applied Biosystems). Relative gene expression was calculated using the ΔΔCt method, following the manufacturer’s instructions. All reactions were carried out in triplicate. The primer sequences were: glyceraldehye 3-phosphate dehydrogenase (GAPDH), 5′- GGTGATGCTGGTGCTGAGTATGT-3′ and 5′-AAGAATGGGAGTTGCTGTTGAAGTC-3′; PTCSC3, 5′-TCAAACTCCAGGGCTTGAAC-3′ and 5′-ATT ACGGCTGGGTCTACCT-3′; miR-574-5p, 5′-GGGGTG AGTGTGTGTGTG-3′ and 5′-TGCGTGTCGTGGAGTC-3′. For each plate, a dissociation curve was obtained to monitor any additional double stranded DNA. GAPDH was used as an internal control and the formula ΔΔCt, where ΔΔCt = Ct(gene) - Ct(GAPDH), was used to calculate the relative mRNA level.

Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Comparisons were performed using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

In order to assess the inhibitory effect of PTCSC3 on thyroid cancer cells, a cell growth assay was performed. The MTT assay revealed that absorptance values of thyroid cells (BCPAP, FTC133, 8505C) transfected with PTCSC3 at 48, 72 and 96 h were significantly lower than those transfected with the empty plasmid (P<0.01; Fig. 1). Additionally, cell cytometry revealed significant cell cycle arrest at G1/S and M/G2 phases and an increased rate of apoptosis in thyroid cancer cells (BCPAP, FTC133, 8505C) transfected with PTCSC3 compared with those transfected with the empty plasmid (P<0.01; Table I).

As the majority of lncRNAs share the same biogenesis mechanisms and have a similar structure with mRNAs, including the 5-UTR, ORF and 3-UTR, it is logical to assume that PTCSC3, a lncRNA, may be targeted by certain miRNAs that are upregulated in thyroid cancers. We carried out bioinformatic analyses aiming to screen miRNAs that match the 3-UTR of PTCSC3 using the online software, PITA (14). Since the ORF of mRNA is occupied by ribosomes, the majority of miRNAs target the 3-UTR of mRNAs. However, certain miRNAs target ORF of mRNAs, including the targeting of c-Myc by miR-85-3p (15). Therefore, we uploaded the full sequence of PTCSC3, including 5-UTR, ORF and 3-UTR, into the PITA system, to identify miRNAs that may target PTCSC3. As shown in Table II, twenty miRNAs have putative targeting sites in PTCSC3. Among these miRNAs, miR-574-5p is the top candidate, since it has the highest score (−30.64) according to the PITA results(14). Additionally, the matching between miR-574-5p and PTCSC3 (Fig. 2) appears perfect according to the results of miRNA-mRNA matching.

Based on the bioinformatics study, we next confirmed this targeting in thyroid cancer cells. To investigate the possible binding of miR-574-5p with PTCSC3, miR-574-5p expression in thyroid cancer cells was determined by quantitative RT-PCR. As shown in Fig. 3, a dramatic decrease of miR-574-5p expression in all three thyroid cancer cell lines following transfection with PTCSC3 was detected (P<0.01). These data demonstrate that overexpression of PTCSC3 significantly downregulates miR-574-5p expression in thyroid cancer cells that are of papillary, follicular and anaplastic origin.