Cell lines
The normal human thyroid follicular epithelial cell line (Nthy-ori 3-1) was procured from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK). Human thyroid cancer cell lines (IHH-4 and 8505C) were procured from Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan), SW1736 cell line was procured from Cell Lines Service GmbH (CLS; Eppelheim, Baden Wurttemberg, Germany), TPC-1 cell line was procured from TOKU-E Company (Tokyo, Japan), and CGTH-W-3 cell line was procured from ATCC (Manassas, VA, USA). Apart from TPC-1 cell line cultured in Ham’s F-12 nutrient mixture (Gibco, Rockville, MD, USA), all of the cells lines were routinely grown in RPMI1640 medium (Gibco). 10% FBS (Gibco) and 1% antibiotics (Gibco) were acquired for cell culture purposes under 37 °C and 5% CO2.
Total RNA isolation and RT-qPCR
Total RNAs were extracted by use of TRIzol Reagent (Invitrogen, Carlsbad CA, USA), followed by convertion into cDNA via PrimeScript Reverse Transcriptase Kit (Takara, Shiga, Japan). The quantitative assay was undertaken utilizing SYBR Green PCR Kit (Takara). The expression of RNA was calculated with 2−ΔΔCt method and normalized to GAPDH, U6 or β-actin expression. The efficiency of primers were shown in Additional file 1.
Western blot
Total proteins of IHH-4 and TPC-1 were isolated using RIPA lysis buffer. Then, SDS–polyacrylamide gel electrophoresisthe was performed to treat total proteins, followed by transferance onto the polyvinylidene fluoride (PVDF) membranes. Subsequently, the membranes were blocked with 5% non-fatted milk at room temperature for 2 h. Afterwards, the membranes were subjected to incubation with the primary antibodies against PLAGL2, E-cadherin, N-cadherin, Snail, and GAPDH overnight at 4 °C. After the elution, the membranes were incubated with the secondary non-specific antibodies for one hour at room temperature. The levels of proteins were then visualized and recorded. GAPDH served as the internal reference.
Cell transfection
The shRNAs for ILF3-AS1 and control shRNAs were procured from Genechem (Shanghai, China). The sequence of PLAGL2 was inserted into pcDNA3.1 vector (Invitrogen) for overexpressing PLAGL2, the empty vector was used as negative control. Besides, the miR-4306 mimics, miR-4306 inhibitor and their negative control (NC mimics and NC inhibitor) were procured from GenePharma (Shanghai, China). IHH-4 and TPC-1 cells were seeded in 6-well plates (1 × 106 cells/well) for transfection lasting 48 h, by use of Lipofectamine 3000 (Invitrogen). 10 ul mimics or inhibitor was used for transfection at the concentration of 100 nM.
Colony formation assay
After transfection, IHH-4 and TPC-1 cells in 6-well plates were seeded at the density of 500 cells per well. Following culture for 2 weeks, the colonies were subjected to the treatment of 4% PFA for 30 min for fixation, and then stained for 5 min by using 0.5% crystal violet solution. Colonies were imaged and then counted.
EdU assay
BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Shanghai, China) was employed to carry out EdU assay in IHH-4 and TPC-1 cells with the utilization of Alexa Fluor 594. The transfected cells were fixed and permeabilized. Afterwards, the cells were stained by EdU medium, with the counterstaining of DAPI solution. After being washed in PBS, cells were observed by inverted microscope for analysis (Olympus, Tokyo, Japan).
TUNEL assay
The transfected IHH-4 and TPC-1 cells were washed in PBS, and then fixation of cells was carried out using 4% PFA. TUNEL reagent (Merck KGaA, Darmstadt, Germany) was acquired to stain the apoptotic cells. Finally, the optical microscopy (Olympus) was applied for analysis.
Flow cytometry analysis
In brief, collected PTC cells were washed by use of PBS. Then, cells were double-stained with Annexin V-labeled with 7AAD and PE purchased from BD Biosciences (San Jose, CA, USA). Apoptosis rate in different stages was measured by a Cytoflex flow cytometer (Beckman Coulter, CA, USA) and assessed by FlowJo software (Tree Star, USA).
Wound healing assay
PTC cells were inoculated in 6-well plates when reached to 100% confluence. Cells were wounded with 1 mL pipette tips. Wound healing status were monitored and photographed at 0, 24 h after scratching.
Transwell invasion assay
For invasion assay, PTC cells were incubated in the upper chamber of transwell chamber coated with Matrigel at the density of 2 × 104 cells per well. The medium free of serum was added to the upper chamber, while the medium containing 10% FBS (Sigma-Aldrich, St. Louis, MO, USA) was supplemented to the lower chamber. Twenty four hours later, cells left on the upper surface of the membrane was removed. Cells in the lower chamber were stained with 0.5% crystal violet solution for 5 min and then were photographed under an inverted microscope (Leica, Germany).
Immunofluorescence (IF) assay
Cells of IHH-4 and TPC-1 were cultured until adhered to slides, and then washed in PBS three times and fixed for 10 min. Cells in 5% BSA were prepared to incubate with the primary antibodies against E-cadherin and N-cadherin, and then with secondary antibodies. Following washing in PBS, cells were stained in DAPI solution and examined by Olympus confocal imaging system.
In situ hybridization (ISH) assay
Enhanced Sensitive ISH Detection kit I (POD) (MK1030, Boster, USA) was used for ISH assay. After the fixation, the tumor tissues from the in vivo experiments were sliced and then treated with proteinase-K for incubation at 37 °C. After the hybridization of ILF3-AS1 probe, the slice was incubated with anti-DIG reagents. The signal of the probe was visualized using diaminobenzidine solution.
Caspase-3 activity assay
Caspase 3 Activity Assay Kit (C1116, Beyotime, Shanghai, China) was used for the experiment. The total proteins of transfected IHH-4 and TPC-1 were obtained through using lysis buffer. Then the proteins were incubated with reaction buffer and caspase substrate. Caspase-3 activity was measured at wave length of 405 nm.
In vivo study
Commercially obtained from the National Laboratory Animal Center (Beijing, China), Ten 6-week-old male BALB/c nude mice were fed in the SPF-grade laboratory. This in vivo experiment was approved by the Animal Research Ethics Committee of Hunan Provincial People’s Hospital. Ten nude mice were randomly and equally divided into two groups, and each mice was subcutaneously injected with TPC-1 cells into which sh-NC or sh-ILF3-AS1#1 was transfected. Twenty-eight hours later, tumor volume and weight were recorded every 4 days. Tumors were weighed after sacrificing mice.
Subcellular fractionation
Cytoplasmic and nuclear RNA Isolation of IHH-4 and TPC-1 cells were undertaken by PARIS™ Kit (Invitrogen), as instructed by supplier. Cells were treated with cell fractionation buffer, followed by centrifugation. The isolated RNAs were detected in cell cytoplasm and cell nucleus using RT-qPCR.
Fluorescence in situ hybridization (FISH)
IHH-4 and TPC-1 cells were fixed, and then air-dried for culturing with ILF3-AS1-specific FISH probe (Ribobio, Guangzhou, China) in line with user guide. After hybridization, cells were subjected to Hoechst staining and analyzed using Olympus microscope.
RNA pull down assay
RNA interaction was assayed in IHH-4 and TPC-1 cells by Pierce Magnetic RNA–Protein Pull-Down Kit procured from Thermo Fisher Scientific (Waltham, MA, USA). The cell protein lysates were prepared for mixing with biotinylated ILF3-AS1 or miR-4306 probes, following the adding of streptavidin agarose magnetic beads. Relative RNA enrichment in mixture was assayed by RT-qPCR.
Luciferase reporter assay
The ILF3-AS1 or PLAGL2 3′UTR fragments covering wild-type (WT) or mutant (Mut) miR-4306 binding sites were prepared for generating pmirGLO-ILF3-AS1-WT/Mut and pmirGLO-PLAGL2 3′UTR-WT. They were co-transfected into IHH-4 and TPC-1 cells with NC mimics or miR-4306 mimics for 48 h. The results were finally studied using luciferase reporter assay system acquired from Promega (Madison, WI, USA).
RNA immunoprecipitation (RIP)
As instructed by manufacturer (Millipore, Bedford, MA, USA), RIP assay was undertaken by Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit. After the lysis, cell lysates were incubated with control IgG antibody or human Ago2 antibody in magnetic beads for immunoprecipitation. RT-qPCR was finally used for analysis.
Statistical analyses
Data from three separately conducted assays were presented as the mean ± SD with GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). Student’s t-test was used to compare the difference between two groups, and one-way ANOVA to analyze the differences among more than two groups. The data obtained from experiments were defined to be statistically significant when p < 0.05.
