Expression analysis in TCGA datasets and clinical specimens
The mRNA-seq data of PTC patients were provided by The Cancer Genome Atlas (TCGA; http://www.cancer.gov/about/nci/), and expression profile (GSE3678) from Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) database. 46 cases of PTC had been pathological diagnosis at the Harbin Medical University Cancer Hospital from October 2018 to September 2019. Paraffin embedded tissue samples and adjacent non-cancerous tissues were frozen after collection, followed by being stored in liquid nitrogen until use. This study has obtained the approval from the Ethics Committee of the Harbin Medical University Cancer Hospital.
Cell lines and transfection
Human PTC cell lines (PTC-1, NPA87, KTC-3, BCPAP) and primary thyroid follicular epithelial cell line of normal human (Nthy-ori 3 − 1) were provided by the Institute of BeNa Culture Collection (Beijing, China). These cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) in a humid incubator at 37◦ C with 5% CO2.
We obtained the short hairpin RNA (shRNA) target KIFC1 or negative control (NC) from GeneChem (Shanghai, China). The shRNA sequences: shKIFC1-1: 5'- GCAACATCCGTGTATTCTGCC − 3'; shKIFC1-2: 5'- CCAGGGCTATCAAATAAAGAA-3'. Retrovirus particles were produced in NPA87 and KTC-3 by transient transfection plasmids harboring KIFC1 shRNA-1, shKIFC1- shRNA-2. The cells were transfected with shRNAs by virtue of Lipofectamine 2000 transfection reagent (Invitrogen, USA) based on the protocol of manufacturers. qRT-PCR and Western blot analysis measured the silencing efficiency exhibited by KIFC1. The cells were gathered and divide into three groups for subsequent experiments as follows: KIFC1 shRNA-1 group, KIFC1 shRNA-2 group and NC shRNA group.
CCK-8 assay
The proliferation capacity of cells was evaluated with the cell-counting kit 8 (CCK8). PTC cell line NPA87 and KTC-3 were plated in 96-well black bottom well plates at 1.5 × 103 for 24 h. CCK-8 reagent (Dojindo, Kumamoto, Japan) was added incubation for 2 hours at 37℃. A microplate reader ((SpectraMax M2, Molecular Devices, CA, USA) assisted in evaluating the optical density (OD) at the 450 nm absorbance.
Colony formation assay
We first seeded PTC cells into 6-well plates (500 cells/each well), and then incubated them at different time for colony formation after transfection. Staining solution with 1% formaldehyde, 0.05% crystal violet, and 10% methanol buffered with PBS were used for staining the fixed colonies. Then, getting images by Nikon microscope (Tokyo, Japan) and performing it in triplicate.
Cell migration and invasion assay
Putting the PTC cells into Boyden’s chambers (24-well insert; with pore size of 8 µm; BD Biosciences, San Jose, CA, USA) were used in the migration and invasion assay. Matrigel (BD biosciences) assisted in coating membranes in the invasion assay. We plated cells in the top chamber of the medium with serum-free, and filled the lower chamber with 10% FBS as the chemoattractant. After 48 hours of incubation, 4% paraformaldehyde (PFA) and crystal violet were used to fix and stain cells, respectively.
Wound-healing assays
Here, we seeded PTC cells were in 6-well plates and cultivated them until confluent. A sterile 100 ml pipette tip facilitated scratch the monolayer for creating artificial wounds, and a culture media was used to wash cells. A microscope (Nikon, Japan) was applied for capturing wound areas’ images at 0 h and 24 h.
Luciferase Reporter Assays
The luciferase reporter assay was performed as previously described [16]. The activity in Nis and Tg promoter region was assessed by transfected with pNis 2.8 [17] and pGI-Tg [18] plasmids in PTC cell lines. In brief, 4 × 104 cells were seeded in 24-well plates. After 72 hours, the 300 ng reporter plasmid puls 30 ng pRL (Renilla luciferase) were used to transfect cells by virtue of Lipofectamine 2000 (Invitrogen, USA). Dual-Glo Luciferase Assay (Promega) measured the luminescence following the transfection.
RNA isolation and Real-time PCR
The real time-PCR technique assessed the mRNA expression. Briefly, total RNA was isolated using RNeasy Mini kit (Invitrogen, Carlsbad, CA, USA) following the instruction of manufacturers, and a reverse transcription kit (Applied Biosystems, CA, USA) assisted in synthesizing cDNA. The reactions were performed by A Super Real PreMix Plus (SYBR Green) Kit (TaKaRa, Biotechnology Co. Dalian, China) and real-time PCR analysis (Applied Biosystems, CA, USA). The primers were shown below:
KIFC1 Fwd: 5′-ACTACAGTGCCACAGACA‐3′ and Rev: 5′‐CCTGATGTGCCAGACTTC‐3′;
ALDH2 Fwd: 5’- ATGGCAAGCCCTATGTCATCT − 3’ and Rev: 5’- CCGTGGTACTTATCAGCCCA − 3;
SOX2 Fwd: 5’- GCCGAGTGGAAACTTTTGTCG-3’ and Rev: 5’- GGCAGCGTGTACTTATCCTTCT-3;
MMP2 Fwd: 5'-TCGGAATGGGACAGACCTACT-3′ and Rev: 5’- TCAAAGGGGTCACATTGCTCC − 3′;
MMP9 Fwd: 5'-AGACCTGGGCAGATTCCAAAC-3′ and Rev: 5’- CGGCAAGTCTTCCGAGTAGT-3′;
GAPDH Fwd: 5’-ACAACTTTGGTATCGTGGAAGGA-3’ and Rev: 5’-GCCATCACGCCAGTTTC-3
GAPDH was used as the positive control. The 2−ΔΔCt method was adopted to calculate the mRNA expression.
Protein extracts and western blot analysis
Protein samples were obtained from cells and homogenized tissues. The concentration of protein was determined using Bradford (Bio-Rad, Hercules, CA, USA). Then, 30 µg of protein per sample and transferred onto PVDF membrane (Millipore, Billerica, MA, USA). 5% non-fat milk was used to block membranes, and were incubated overnight at 4 °C with the primary antibodies. The following antibodies were used: KTFC1 (sc-9553), E-cadherin (sc-3195), N-Cadherin (sc-13116), Snail (sc-3437), ZEB1 (sc-3396), MMP2 (sc-9126) and MMP9 (sc-2867) were provided by Cell Signaling Technology (Danvers, MA). ERK (sc-4659), p-ERK (sc-4660), JNK (sc-4334), p-JNK (sc-4335) and GAPDH (sc-166) antibodies were offered by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). ALDH2 (sc-KGAA3d5), SOX2 (sc-KGAA5d1), p38 (sc-KGAA253) was purchased from KeyGen Biotechnology (KeyGen, China). Then incubate HRP-conjugated secondary antibodies at room temperature for 1 hour.
Statistical analysis
Statistical analyses were performed using SPSS 22.0 software. Data are represented in the form of means + standard deviation (S.D). Student’s t-test was used to compare the two groups. Multiple-group statistical analyses were performed by one-way analysis of variance (ANOVA). P value < 0.05 is considered with statistical significance.