Study subjects and patient tissue samples
A total of 31 paired ATC and adjacent thyroid tissue samples were obtained from patients that experienced surgery in the Shengjing Hospital of China Medical University from 2013 to 2016. The research had been supported by the Ethics Committee of Shengjing Hospital. Relevant clinical information was gathered from patients’ records and informed written consent had been gained. All the samples (including cancer and normal thyroid tissue) are diagnosed by three pathologists to confirm the histologic diagnosis.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The overall RNA was extracted by adopting TRIzol reagent. The miRNA expression was implemented by adopting the TaqMan miRNA assay. The related quantification of Gene expression was normalized through the 2−ΔΔCt strategy related to U6. Every experiment was carried on in triplicate.
Total protein of tissues and cells was separated by electrophoresis on 8% polyacrylamide gels and then electrotransferred to polyvinylidene fluoride (PVDF) membranes. After that, the PVDF membranes was incubated with primary anti-SOCS1 (12,000, Abcam, Cambridge, MA, USA) as well as ß-actin (12,000, Abcam, MA, USA) antibody at 4 °C for a night, followed by 2-h incubation with a secondary antibody. Image J software was adopted for quantifying the integrated density of the bands.
Transient transfection and cell culture
The human ATC cell lines 8305c (Cat NO. SCSP540) and FRO (Cat NO. SCSP577) were obtained from Cell bank of typical culture preservation Committee of Chinese Academy of Sciences. 8305c cell line was originally derived from anaplastic thyroid carcinoma of an adult female. FRO cell line was also originally derived from anaplastic thyroid carcinoma of an adult female. These cell lines have been authenticated by using Single Tandem Repeat (STR) profiling method. There is no mycoplasma contamination in 8305c and FRO cell lines. 8305c cells were cultured within MEM (the medium of the modified Eagle) supplemented in a humidified 5% CO2 incubator with 10% fetal bovine serum (FBS) at room temperature. FRO cells were cultured within DMEM (the medium of the modified Eagle) with 10% fetal bovine serum (FBS). MiR-155 ASO, mimic and negative controls were bought from Genecopoeia. Empty vector (EV) as well as SOCS1-specific siRNA and overexpression vector were gained from Shanghai Genechem Co., LTD. Cells were transfected by adopting the Lipofectamine 2000 kit (Invitrogen) in accordance with the instructions of the manufacturer.
Cell proliferation assays
Counting Kit-8 (CCK-8) (Invitrogen) was used to evaluate the cell proliferation ability. Cells was seeded at a density of 2000 cells/100 μL within 96-well plates and the absorbance was calculated in an ELx-800 Universal Microplate Reader (BioTek Instruments, Winooski, VT, USA) at 450 nmat 0, 24, 48, 72 and 96 h.
Cell invasion assays
Cell invasion assays were carried out by transwell chambers coated with Matrigel (BD Biosciences, Bedford, MA, USA). Briefly, 5 × 104 cells in 200 μL of medium were placed with an 8-μm pore size polycarbonate filter in the upper chamber (BD Biosciences). The lower chamber was added with culture medium containing 10% FBS. The cells which invaded the lower chamber membranes was stained with 0.1% crystal violet after being incubating for two whole days(Merck, Darmstadt, Germany) for 30 min, and photographed under a microscope (Olympus, Tokyo, Japan).
Dual-luciferase reporter assay
Luciferase assays were implemented by adopting the luciferase reporter assay system in accordance with the instructions of the manufacturer. The mutated (Mut) or wild-type (WT) SOCS1 3′-UTR sequence including the miR-155 targeting site was inserted into pGL3 (Invitrogen) for the purpose of constructing pGL3-luc-SOCS1. 8305c and FRO cells were transfected with pGL3-luc-SOCS1 (50 ng) and seeded in 96-well plates, miR-155 negative or mimic control (5 pmol) as well as renilla luciferase (5 ng) by adopting Lipofectamine 2000 (Invitrogen). The dual luciferase assay detected the luciferase activity after two whole days (Promega).
8305c and FRO cells were harvested after transfection at 48 h, and 7-aminoactinomycin Das well as annexin-V (7-AAd) antibody was added, followed by incubation at room temperature. After 15 min, flow cytometry assay was implemented through adopting an LSR II flow cytometer (BD Biosciences). The outcomes were discussed by adopting Flow Jo software. Annexin V as well as 7-AAd double positive cells was defined to be apoptotic cells. 7-AAd negative as well as annexin V positive cells were regarded to be in the primary apoptosis procedure.
Colorimetric caspase-3 assays
8305c and FRO cells were lysed primarily, and then protein concentration had been decided. An overall of 100 μg protein was incubated for two hours by using 10 μL of Ac-DEVD-pNA (Abcam, America) at room temperature, and the absorbance at 405 nm was calculated by adopting a microplate reader (BioTek Instruments).
Information was shown to be mean ± SD and discussed by adopting the student t-test. A paired t-test was adopted for paired samples. Pearson test was used to define the relationship between miR-155 and SOCS1. Statistical analyses were conducted with SPSS 17.0 (SPSS Inc., USA) software. P < 0.05 was regarded statistically important.