Patients and tissue samples
Thyroid tissues were obtained from 97 patients at the time of initial surgery at the Medical Center of Breast and Thyroid Disease of the Affiliated Hospital of Zunyi Medical College (Guizhou, China) and stored at − 80 °C. The study protocol was approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical College, and human thyroid tissues were obtained following provision of informed consent by the patients. All tissue samples were examined and the diagnosis was confirmed by two pathologists.
Cell lines and cell culture
The BCPAP, TPC-1 and K1 cell lines were provided by Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 supplemented with 10% FBS and incubated at 37 °C in a 5% CO2 atmosphere.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA was extracted from BCPAP cells using TRIzol reagent (Takara Bio Inc., Otsu, Japan), according to the RNAiso Plus kit instructions. Total RNA was reverse-transcribed to cDNA using Prime ScriptTMRT reagent kit (Takara Bio Inc.). RT-qPCR was performed using SYBR®Premix Ex TaqTM II (Takara Bio Inc.). GAPDH was used as endogenous control. The primers for RT-qPCR were as follows: LASS2: forward, 5′-ATCGTCTTCGCCATTGTT-3′ and reverse, 5′-CGGTCACTGCGTTCATCT-3′; GAPDH: forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′ (197 bp). The levels of LASS2 were analyzed by the 2−∆∆Ct method.
Paraffin-embedded specimens were cut into 3-μm sections for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). The assays were performed as previously described , and the primary antibody used was anti-LASS2 at a dilution of 1:200 (Abcam, Cambridge, UK).
The scoring of LASS2 expression was also performed as previously described . The intensity distribution (ID) score for LASS2 was evaluated by the sum of the percentage of positive cells (0: < 5%; 1: 5–25%; 2: 26–50%; 3: 51–75%; and 4: 76–100%) and the staining intensity was graded from 0 to 3 (0, negative; 1, weak; 2, moderate; and 3, strong). ID scores ≥ 6 were considered to reflect high expression, and those ≤ 4 low expression.
All histological stainings were evaluated by two pathologists, and the scores were calculated by two observers.
Construction of Adv-LASS2-GFP recombinant adenovirus vector
The LASS2 recombinant eukaryotic expression of LASS2-GFP (as insert segment) and pShuttle-CMV recombinant shuttle vector, were constructed and digested with HindIII/NotI, respectively, and then ligated with T4 DNA ligase. Subsequently, transformed plasmids were extracted and sequencing was performed to obtain pShuttle-LASS2-GFP recombinant shuttle plasmid. Next, the pAdxsi vector and pShuttle-LASS2-GFP were separately digested with I-Ceul and I-Scel, then ligated and transformed. To obtain pAdxsi-LASS2-GFP viral plasmid, extensive extraction of the viral plasmids was performed, followed by packaging, collection and amplification. Finally, a recombinant Adv-LASS2-GFP was obtained, with a titer of 1.2 × 1010 PFU/ml.
The BCPAP cells were lysed with RIPA lysis buffer (1% NP-40, 0.1% SDS, 50 mM DTT) containing protease inhibitor cocktail on ice. After centrifugation, the supernatant was collected in 1.5-ml centrifuge tubes. The cell lysate was loaded on 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels after being heated at 100 °C for 3 min for denaturation and then transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk at 37 °C for 2 h. The blocked membranes were washed with PBST buffer 2–3 times and incubated with the primary antibodies for 2 h at room temperature. After the membranes were washed four times with PBST buffer, they were incubated with a corresponding secondary antibody in PBST buffer at 4 °C overnight, followed by washing four times with PBST. The blots were detected using Enhanced Chemiluminescence Detection kit (KGP116, KeyGen BioTECH, Jiangsu, China). The primary antibodies used in the experiment were anti-p53 (ab31333, 1:1000), anti-CDK4 (ab137675, 1:2000), anti-cyclin D1 (ab137875, 1:5000) and p21 (ab109520, 1:1000), all purchased from Abcam; Anti-p-p53 (#9284, 1:1000) was purchased from CST; Anti- GAPDH (SC-365062, 1:800) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The secondary antibody used in the experiment were: goat anti-rabbit, goat anti-mouse IgG (1:4000 for both).
Subcellular localization of LASS2 by confocal laser scanning microscopy in BCPAP cells
BCPAP cells were transfected with Adv-LASS2-GFP for 48 h. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde for 30 min. Autofluorescence from the LASS2 protein was observed under a confocal microscope (LSM510; Carl Zeiss AG, Oberkochen, Germany) with excitation at 488 nm and a 525-nm GFP filter. Images were acquired at a ×200 magnification.
Cell proliferation assay
For the proliferation assay, BCPAP cells were treated with Cell Counting Kit-8 reagent (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer’s instructions. Absorbance at 450 nm was measured on a microplate reader at the designated time points after treatment. The assay was performed in triplicate.
Colony formation assay
For the colony formation assay, the transfected cells were plated at 1000 cells/well in a 6-well plate and incubated for 2 weeks. The colonies were fixed and then stained with 0.5% (w/v) crystal violet solution for 30 min at room temperature. The colonies were photographed and counted. The assay was performed in triplicate.
Cell cycle analysis
BCPAP cells transfected with Adv-GFP or Adv-LASS2-GFP were harvested 48 h after culture, washed with PBS and fixed in 500 µl of 75% cold ethanol at 4 °C overnight, and then washed with PBS, stained by 100 µl propidium iodide (3.8 × 10−2 sodium citrate, pH 7.0) containing RNase A (10 mg/ml) for 30 min in the dark at 37 °C. The cell populations in the G0–G1, S and G2-M phases were measured by flow cytometry (Beckman Coulter, Inc., Brea, CA, USA), and analyzed using ModiFit software. All the samples were assayed in triplicate.
Annexin-V APC/7-AAD double-staining to detect apoptosis
Following transfection with Adv-GFP or Adv-LASS2-GFP, BCPAP cells were harvested with 0.25% trypsin (without EDTA). Complete medium was added to the cells to inactive trypsin, and the cells were washed twice with PBS (centrifugation at 800g, 5 min). The cells were subsequently resuspended in 500 µl of binding buffer. After 5 µl of Annexin V-APC was added and mixed well, 5 µl of 7-AAD was added and mixed well. The cells were incubated at room temperature for 15 min in the dark, then immediately analyzed using a flow cytometer (FACSCalibur, BD Biosciences, San Diego, CA, USA) using CellQuest software.
The amount of DNA fragmentation was determined using a commercial BIOTIN labeling dUTP TUNEL kit (KeyGen BioTECH, Jiangsu, China) according to the instructions of the manufacturer. Briefly, BCPAP cells on the slides were washed with PBS three times, and then fixed with 4% paraformaldehyde for 30 min, permeabilized with 1% Triton X-100 at room temperature for 15 min, and then treated with 3% H2O2 containing methanol for 15 min. After the enzymatic reaction, cells were washed with PBS, incubated with a mixture of TdT solution and fluorescein isothiocyanate dUTP solution at 37 °C for 60 min in a humidified chamber, then incubated with 100 µl streptavidin–horseradish peroxidase at 37 °C for 30 min in a humidified chamber. After washing with PBS, the cells were stained with DAB solution, followed by counterstaining with hematoxylin, and observed under a light microscope. Cells with brown granules in the nuclei were considered as TUNEL-positive. Cells were counted (high-power field, magnification ×200) under an optical microscope (Olympus, Tokyo, Japan) to determine the mean percentage of positive cells.
All statistical analyses were performed using SPSS 21.0 software (SPSS version 21.0., IBM Corp., Armonk, NY, USA). GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) was used for graphs. The Chi squared test or Fisher’s exact test was used to evaluate the association between clinicopathological characteristics and LASS2 expression. Student’s t-test was performed to analyze differences between groups. All values represent at least three independent experiments and are expressed as the mean ± standard deviation. P < 0.05 was considered to indicate statistically significant differences.