Patients and tissue samples
Eighty papillary thyroid cancer tissues and adjacent normal tissues were collected from 80 patients with PTC who received operation on the First Affiliated Hospital of Nanjing Medical University (NMU). None of the patients underwent any other treatment but surgery. All collected tissue samples were immediately snap frozen in liquid nitrogen and stored at −80 °C until required. Our study was approved by the Ethics Committee of the First Affiliated Hospital of NMU.
Four PTC cell lines (K-1, TPC-1, B-CPAP and IHH-4) and a normal thyroid follicular epithelium cell line (Nthy-ori3-1) were purchased from the American Type Culture Collection (ATCC, Virginia, USA). K-1, B-CPAP and Nthy-ori3-1 cell were cultured in RPMI1640 medium (Gibco, Carlsbad, CA, USA), while TPC-1 cell was cultured in DMEM with high glucose (Gibco, Carlsbad, CA, USA). Mixture (1:1) of RPMI1640 and DMEM was used to culture the IHH-4 cell line. 1% antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) and 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) were added to all of the above culture media. All cell lines were incubated in a humidified atmosphere at 37 °C containing 5% CO2. All cell lines have been authenticated by short tandem repeat analysis, as well as tested for mycoplasma contamination before conducting this study.
RNA extraction and quantitative real-time PCR analysis
Total RNA was extracted from tissues and cultured cell lines using TRIzol reagent (Invitrogen, MA, USA). A PrimeScript RT reagent kit (Takara, Kyoto, Japan) was used to synthesize cDNA. MiRNAs were reverse transcribed after polyadenylation using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, MA, USA). Quantitative real-time PCR (qRT-PCR) was performed with AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). Results were calculated using the 2-ΔΔCT method and normalized to the expression of GAPDH for mRNA or U6 for miRNA. Primers used in the study were listed in Supplementary Table 1 (Additional file 1: Table S1).
PTC cells were transfected with 50 nm small interfering RNAs (siRNAs) and plasmid vectors when they grew to 30-40% density using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). In this study, siRNAs, miR-612 mimics, miR612 inhibitor and correspondent negative control were purchased from Genepharma. After 48h post-transfection, the cells were harvested for performing the following experiments. Plasmid vectors encoding FER1L4 and biologically active short hairpin RNAs (shRNA) targeting FER1L4 or CDH4 were generated either (Genepharma, Shanghai, China). Stable cell lines were obtained by using 2 μg/ml puromycin (Sigma-Aldrich, St-Louis, Missouri, USA) for about three weeks. Nucleotide sequences mentioned above were listed in Supplementary Table 2 (Additional file 1: Table S2).
Cell proliferation assay
In order to investigate the effect on cellular proliferation of corresponding treatment to K-1 and TPC-1 cells, cell counting kit 8 (CCK8) assay, colony formation assay and 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay were applied. Detailed information has been described before .
Four weeks old female BALB/c nude mice were purchased from the Animal Center of NMU, and all experiments were approved by the Committee on the Ethics of Animal Experiments of the Nanjing Medical University. For the tumorigenicity studies, a total of 20 mice were randomly assigned, and stable transfected cells (1×106 cells/100 μl of phosphate buffer saline) were subcutaneously injected into the flank of nude mice. The tumor volume was measured every week and calculated by the formula: volume = (length × width2)/2.
Immunohistochemical (IHC) analysis
All specimens were fixed in 4% formalin and then embedded in paraffin. After blocking endogenic peroxides and proteins, these sections were incubated with primary antibodies specific for Ki-67 (Abcam, Cambridge, MA, USA) or CDH4 (Abclonal, Wuhan, China) at 4°C overnight, lastly were counterstained with hematoxylin after incubating with the secondary antibodies at 37 °C for 1 h. Random images were obtained using a light microscope (Olympus Corp. Tokyo, Japan).
Cell migration and invasion assays
Transwell chambers (Corning, New York, NY, USA) coated with or without Matrigel (BD Bioscience, Franklin Lakes, NJ, USA) were used to evaluate the function of genes on cellular migration and invasion. Meanwhile, cell motility was also examined by wound healing assay. Detailed information has been described before .
Flow cytometric analysis
Treated cells were collected for Flow cytometric analysis. According to the protocol (MultiSciences, Hangzhou, China), APC-Annexin V and Propidium Iodide (PI) were used to stain cells, and the rate of apoptosis was analyzed by a flow cytometer (FACScan, BD Biosciences, USA). For cell cycle analysis, treated cells were stained by PI-staining solution, and then the percentages of cells in G0–G1, S, and G2–M phase were counted.
Western blot assay and antibodies
Western blot assay was performed following previous protocol. The primary antibody used were listed in Supplementary Table 3 (Additional file 1: Table S3).
Subcellular fractionation and Fluorescence in situ hybridization (FISH)
The separation and purification of cytoplasmic and nuclear RNA were implemented using the PARIS Kit (Life Technologies, USA) according to the manufacturer’s instructions. For the FISH assay, the Cy3-labeled FER1L4 probes used in our study were synthesized (RiBo Ltd, Guangzhou, China). Briefly, the prepared cells were incubated with specific probes at 37 °C overnight after fixation and permeabilization. Finally, the nuclei were stained by DAPI and observed using a confocal laser scanning microscope (Zeiss LSM5 Live, Oberkochen, German).
Dual-luciferase reporter assay
The sequences of FER1L4 and CDH4 3′-UTR containing wild-type or mutated miR-612 binding sites were synthesized and loaded into a pGL3 luciferase reporter vector (Promega, Wisconsin, USA). Then, TPC-1 cells (4×105) were co-transfected with miRNA mimics and luciferase reporter vectors. After 48h of incubation, the luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega, Wisconsin, USA). Relative luciferase activity was normalized to Renilla luciferase.
RNA immunoprecipitation (RIP) assay
To detect whether FER1L4 influences miR-612-dependent RNA-induced silencing complex, RIP assays were conducted using the Magna RIPTM RNA-binding protein immunoprecipitation kit (Millipore, USA). Briefly, the prepared cells were lysed and incubated with anti-Ago2 (Abcam, Cambridge, MA, USA) or IgG antibody at 4 °C overnight. Then, cell lysates were incubated with the protein A magnetic beads for 4h. Finally, the coprecipitated RNAs were collected for qRT-PCR analysis.
Gene expression data of PTC was extracted from TCGA database and used to explore abnormally expressed lncRNA. MiRNAs targeted by FER1L4 were predicted using starbase (http://starbase.sysu.edu.cn/), miRcode (http://www.mircode.org/) and RegRNA2.0 (http://regrna2.mbc.nctu.edu.tw/). Meanwhile, miRWALK (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk/) was applied to determine the downstream targets of miR-612.
The data was described by the mean ± standard deviation (SD) in three independently experiments. For comparing statistical differences, Student’s t-tests, Pearson Chi-square test, Wilcoxon test were performed as appropriate using SPSS v22.0 and GraphPad Prism 6. Spearman’s correlation analysis was selected to analyze the correlations among FER1L4, miR-612 and CDH4. P value less than 0.05 was considered statistically significant.