The study included 559 patients with PTC and 445 healthy controls. The subjects were enrolled from the West China Hospital of Sichuan University and were all unrelated Han nationality. Participants' information such as age, gender, and TNM staging were recorded in detail. Tumor stages were determined according to American Joint Committee on Cancer. The healthy volunteers were selected from the same hospital during the same period as the PTC cases were recruited. All controls were frequency matched to PTC cases based on age and gender. Among them, individuals suffering from thyroid disease or other tumors were excluded. Peripheral blood samples of all subjects were taken before undergoing any medical treatment such as surgery and radiotherapy. Furthermore, the fresh PTC clinical specimens and adjacent normal tissues were collected immediately after the operation and placed in liquid nitrogen for later use. Informed consent was obtained from all individuals included in the study. The research protocol was approved by the ethics committees of West China Second University Hospital, Sichuan University.
We selected SNPs according to the following criteria: (1) SNPs within TINCR enhancer that are annotated by ensemble database (http://www.ensembl.org/index.html); (2) SNPs with minor allele frequency more than 10% in Han Chinese. Only one SNP, rs8101923, followed the criteria and was selected for further analysis.
DNA extraction and SNP genotyping
According to the manufacturer's protocol, we used TIANamp Genomic DNA Kit (TianGen Biotech Co. Ltd., Beijing, China) to extract genomic DNA from blood samples, and used NanoDrop spectrophotometer (ND-1000) to determine the purity and concentration of DNA. The DNA fragment containing the SNP site (i.e., rs8101923) was amplified by polymerase chain reaction (PCR). The amplification primers used in this study were: 5'-AAGGTTGTGTGTGGGAGAGG-3' (forward), 5'-CATGACCTCTGGGGTGTCTT-3' (reverse). The PCR system was set to 10 μl mix: 5 μl 2X Power Taq PCR MasterMix, 10 μM for each primer, 0.5 μl DNA template, and Milli-Q water as a supplement to 10 μl. The PCR reaction conditions were: 94°C for 4 min, 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, 35 cycles, and 72°C for a final extension of 10 minutes. Subsequently, the PCR product was digested by Sty I (New England BioLabs, MA, USA), and genotyped by PCR-restriction fragment length polymorphism assay. After digestion, the G allele produced a 216 bp band, and the A allele produced 123 bp and 93 bp bands. In order to verify the genotyping results, we randomly selected the samples for Sanger sequencing, and the agreement rate was 100%.
Quantitative real-time RT-PCR (qRT-PCR)
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from 43 pairs of PTC tissues and adjacent normal tissues, and RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific Scientific, Rockford, IL, USA) was used to reverse transcribed total RNA into cDNA. LightCycler® 480 System (Roche Indianapolis, IN, USA) and QuantiNova™ SYBR® Green PCR Kit (Qiagen, Hilden, Germany) were used for determining the relative expression levels of TINCR. The TINCR primers used for qRT-PCR were described as follows 17: forward 5'-TGTGGCCCAAACTCAGGGATACAT -3' and reverse 5'-AGATGACAGTGGCTGGAGTTGTCA-3'. 18s was used as an internal control and amplified with the following primers: forward 5'-GCAATTATTCCCCATGAACG-3' and reverse 5'-GGCCTCACTAAACCATCCAA-3'. Each amplification reaction was completed in a total volume of 10 μl, which contained 0.7 μl primers, 5 μl Master mix, and 100 ng cDNA. The reaction conditions were set as follows: 95°C for 2 min, 95°C for 5 s, and 60°C for 10 s, 40 cycles.
Cell culture, plasmid construction, and luciferase reporter assay
TPC-1 and BCPAP cells were purchased from GuangZhou Jennio Biotech Co. Ltd. (Guangzhou, China). All cell lines were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum and 1% ampicillin/streptomycin and cultured under 5% CO2. The TINCR fragment containing the rs8101923 AA or GG was amplified by PCR, and the amplification primers were forward 5'-AGATGACAGTGGCTGGAGTTGTCA-3' and reverse 5'-TGTGGCCCAAACTCAGGGATACAT-3'. The PCR products were inserted into pmirGLO miRNA target expression vector (Promega) to construct pmirGLO-rs8101923A and pmirGLO-rs8101923G plasmids. For the luciferase reporter gene detection, we used the dual luciferase reporter gene detection system (Promega). Firefly and renilla luciferase activity was measured at 48 h after transfection according to the manufacturer's instructions.
Cell transfection and cell viability assay
The small interfering RNA (siRNA) targeting AP-2α (si1-AP-2α and si2-AP-2α) was constructed by Genepharma (Shanghai, China). The sequences of si1-AP-2α and si2-AP-2α were 5'-GCAAGAUCCUUACUCCCACTT-3' and 5'-CCUGCUCACAUCACUAGUATT-3', respectively. When cells reached 90% confluence, the medium was changed to serum-free and antibiotic-free medium, and transfection was performed using Lipofectamine 3000 (Invitrogen) according to the reagent manufacturer's protocol. At 24 h, 48 h, and 72 h after transfection, cell viability assay was performed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.
Chromatin immunoprecipitation (ChIP)
Cells were grown to approximately 80% to 90% confluence, cross-linked with 1% paraformaldehyde at 37°C for 10 minutes, and quenched in 125 mM glycine. The cell lysates were sonicated under conditions that produced fragments of 200 bp to 1000 bp. The material was clarified by centrifugation, diluted 10 times in dilution buffer, and pre-clarified with protein A-Sepharose beads. The pre-cleared supernatant containing chromatin was used for the immunoprecipitation reaction with the antibody against AP-2α (Abcam, Cambridge, MA, USA). Anti-mouse IgG was used as a control. After digestion with proteinase K and RNase, the immunoprecipitated genomic DNA was extracted with phenol: chloroform: isoamyl alcohol and precipitated with ethanol. For gene-specific ChIP analysis, PCR and Sanger sequencing were used to determine the DNA sequence enriched by immunoprecipitation.
SPSS 20.0 statistical software (SPSS Inc, IL, USA) was used for data analysis. The rs8101923 genotype frequency was obtained by direct counting. The Student's t-test used to compare the age of cases and controls. The gender comparison and Hardy-Weinberg equilibrium (HWE) were calculated using Chi-square (χ2) test. Results of qPCR were analyzed using Wilcoxon rank sum test. The unpaired Student’s t-test was used to analyze the luciferase data. The difference of the rs8101923 genotype distribution between cases and controls was analyzed using the χ2 check test. Odds ratio (OR) and 95% confidence interval (CI) were used to assess the correlation between the rs8101923 and PTC risk. P <0.05 was considered statistically significant.