Human thyroid cancer cell lines (TPC-1 and BCPAP) were obtained from ATCC (American Type Culture Collection) (Manassas, VA, USA), and cultured in DMEM (Dulbecco’s modified Eagle’s medium) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin and 100 µg/mL streptomycin (Hyclone, South Logan, UT, USA), and incubated at 37 °C in a humidified atmosphere containing 5% CO2.
LEF-1, β-catenin and LINC01278 overexpression vectors were bought from Kang-cheng Biotechnology Co (Guangzhou, China). Small interfering RNA (siRNA) against LEF-1, β-catenin and LINC01278 were designed and purchased from RiboBio Co., Ltd. (Guangzhou, China). All vectors and siRNAs were transfected into TPC-1 and BCPAP cells using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.
Analysis of online database
The online PROMO algorithm analysis (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) was used to predict the binding of transcription factors to the LINC01278 promoter sequence[14, 15].
Luciferase reporter assay
The wild promoter sequence of LINC01278 (WT) and mutant promoter sequence of LINC01278 (MUT) in which the LEF-1-binding site was mutated were cloned into a pGL3-Basic vector (Promega), respectively. The recombinant pGL3-Basic vector was co-transfected into TPC-1 and BCPAP cells with LEF-1 overexpression (LEF-1) or negative control (NC) plasmid. After 48 h, the cells were lysed, and the luciferase activity was analyzed using Dual-Luciferase Reporter Kit (Promega) and Varioskan Lux detection system (Thermo Scientific), which was standardized to Renilla.
Chromatin immunoprecipitation (ChIP) assay
Cells were fixed and crosslinked in 1% formaldehyde for 10 min at 37 °C and incubated with protease inhibitors. Chromatin was isolated and enzymatically fragmented using an EZ-Zyme Chromatin Prep Kit (17375, Merck). Rabbit anti-LEF-1 antibody (EPR2029Y, 1:50, Abcam) or nonspecific IgG (1:200, Sigma) was used to precipitate DNA crosslinked with the LEF-1. The immunoprecipitated promoter fragment containing the LEF-1 response element was probed by PCR using primers targeting the regulatory region of LINC01278 gene and visualized by agarose gel electrophoresis.
Cells were lysed using RIPA buffer. The lysate was separated by 10 − 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). The membrane was blocked with 5% nonfat milk and incubated with anti-LEF-1 (1:1000, ab137827, Abcam), anti-β-catenin (ab16051, 1:1000, Abcam), anti-CCND2(ab230883, 1:1000, Abcam), anti-CyclinD1 (ab226977, 1:1000, Abcam), anti-MYC (ab32072, 1:1500, Abcam), anti-SOX4 (ab86809, 1:1000, Abcam), and anti-GAPDH (ab8245, 1:1000, Abcam) antibodies overnight at 4◦C. Then the membrane was washed with TBST three times and incubated with the secondary antibody (Abcam, Cambridge, MA) for 1.5 h at room temperature. The ECL chromogenic solution was used to display the chemiluminescence of the bands. The Quantity One 4.4.0 software was used for densitometry determination.
Quantitative real-time PCR
Total cellular RNAs were isolated and purified using TRIzol reagents (Invitrogen) and cDNA synthesis was performed using PrimeScript RT reagent Kit (TaKaRa, Otsu, Shiga, Japan) according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq TM (TaKaRa) in the ABI 7900HT Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA). The comparative cycle threshold (CT) (2-CT) method was used to measure the LINC01278 level, and GAPDH was used as an internal control. The sequence of primers was as follows: LINC01278, F: 5’-CTGTTGCCCTCCTTCACCTA-3’, R: 5’-TGGTCTACAGGGAGTGCAAG-3’; GAPDH, F: 5’-TGTTCGTCATGGGTGTGAAC-3’, R: 5’-ATGGCATGGACTGTGGTCAT-3’.
RNA immunoprecipitation (RNA IP)
Cells were fixed and crosslinked in 1% formaldehyde, and lysed using RIPA buffer supplemented with protease-inhibitor cocktail and RNase inhibitor. The cell lysates were incubated with magnetic beads conjugated with anti-β-catenin antibody (Millipore). Mouse IgG (Millipore) was used as the negative control. The immunoprecipitated RNA was extracted from the eluate, and the enrichment of LINC01278 or control ACTB was analyzed by quantitative RT-PCR.
Statistical analysis was performed using SPSS 22.0 software (SPSS, Armonk, NY, USA). The data came from at least three independent experiments, evaluated by Student’s t test, and presented as mean ± SD. P < 0.05 was considered as statistically significant.