The PTC samples
80 cases of PTC samples were obtained from the Pathology Department of the Affiliated Shenjing Hospital of China Medical University. All participants fully understand and agree with the written informed consent before enrolment in this study. The participants did not receive any chemotherapy or radiation therapy before curative surgical resection. The ethical approval was obtained from the Medical Research Ethics Committee of China Medical University. Formalin-fixed paraffin-embedded sections of PTC were stained with hematoxylin and eosin (HE), and diagnosed according to the guidelines of classification of endocrine tumors (2017) and the TNM staging system of UICC by two senior pathologists. The gender, age, differentiation, tumor size and extension, lymph node metastasis and stage status were determined accordingly and summarized in Table 1.
80 cases of PTC paraffin sections were deparaffinized and rehydrated conventionally. After the recovery of antigen, the sections were incubated with 3% H2O2 to block endogenous peroxidase, followed by 5% non-immune serum to avoid unspecific binding of antibody at 37°C for 30min. Then the rabbit polyclonal antibody specific for HPSE (1:200 dilution, PeproTech, USA) was added on sections overnight at 4°C. The next day, sections were incubated with goat anti-rabbit IgG and streptavidin-peroxidase (SP) complex at 37°C for 30 min (SP kit, Maxim, China), and then developed with 3,3’-diaminobenzidine (DAB). The non-immune goat IgG instead of primary antibody was used as negative control. Two senior pathologists evaluated the immunostained sections separately. The obvious brown particles in cytoplasm were regarded as positive expression of HPSE. The intensity of HPSE staining (0=negative, 1=weak, 2=intense) and the percentage of positive cells (≤50%=1, >50%=2) were assessed in at least 5 high power fields (×400 magnification). Then they were multiplied to obtain a final score of each section as 0, 1, 2 or 4, and all the sections were finally determined as low expression: score ≤2 (including negative: score 0); Or high expression: score >2.
HPSE overexpression and RNA interference
Human PTC of B-CPAP and KTC-1 cells were purchased from Chinese Academy of Sciences and cultured in RPMI 1640 medium (Gibco, USA), supplemented with 10% FBS, under the condition of 37℃ and 5% CO2. The overexpression plasmid for HPSE and the empty vector were purchased from GeneChem (China). The shRNAs specific for HPSE and the scrambled non-targeted shRNA were also purchased from GeneChem (China). All the plasmid were transfected into B-CPAP or KTC-1 cells using Lipofectamine 3000 (Invitrogen, USA). The experiments for cells were repeated at least three times.
Quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted using RNApure kit (Aidlab, China) and reverse transcribed into cDNA using GoScript Reverse Transcription System (Promega, USA). qPCR was performed using the GoTaq qPCR Master Mix (Promega, USA) in the Roche LightCycler 480 Real Time PCR instrument. Primers were used as follows: HPSE forward: 5’-AGT GGG TGT GGG TGA TTT CC-3’; reverse: 5’-GGC TCC TGG GTG AAG AAG TC-3’. GAPDH forward: 5’-CAG GAG GCA TTG CTG ATG AT-3; reverse: 5’-GAA GGC TGG GGC TCA TTT-3’. The length of PCR products were 193bp and 138bp, respectively. The qPCR was performed for 40 cycles with an initial denaturation at 95℃ for 2min, amplification at 95℃ for 15s, and annealing at 60℃ for 1min. GAPDH was used as reference gene, and the results were analyzed using 2-ΔΔCt method.
Cells were lysed in RIPA containing protease inhibitor (Promega, USA) and phosphatase inhibitor (APPLYGEN, China) cocktail, and centrifuged for the supernatant extraction. 50μg total protein was used for electrophoresis and transmembrane routinely. Rabbit anti-HPSE (1:1000, PeproTech, USA), p-FAK (1:500, Sangon Biotech, China), p-PDK1, p-PKCα/βII (1:1000, Cell Signaling Technology, USA), p-Akt, cleaved caspase-3 (1:1000, Beyotime, China) or mouse anti-p-ERK (1:200, Santa Cruz, USA), β-actin (1:500, ZSGB-BIO, China) antibodies were used as primary antibodies and incubated with membranes at 4℃ overnight. Then the goat anti-rabbit (1:4000) or anti-mouse IgG (1:2000, both from ZSGB-BIO, China) was used, and membranes were developed using ECL (Millipore, USA). The software of Image J was used to evaluate the integrated optical density (IOD) of protein bands. The ratio of IOD target protein and IOD β-actin of the same specimen was calculated as the relative expression level of target protein.
EdU incorporation assay
The kFlour555 Click-iT EdU imaging kit (KeyGEN BioTECH, China) was applied to explore cell proliferation. The staining was performed briefly as follows: cells were cultured with diluted EdU of 20μmol/L for 2h. Cells were fixed with 4% paraformaldehyde, and neutralized by glycine solution of 2mg/mL. After washed with PBS, cells were permeated with 0.5% Triton X-100, and then incubated with prepared Click-iT reaction cocktail in the dark. Hoechst33342 was used to counterstain nucleus. Under an inverted fluorescent microscopy, 5 random fields of 200× were focused and cells with red or blue fluorescence were counted in the same fields. Cell proliferative rates were represented as red/blue.
Cell apoptosis assay
The cell apoptosis was examined by flow cytometry using an Annexin V-PE/7-AAD apoptosis detection kit (KeyGEN BioTECH, China). Cells were digested and collected using trypsin without EDTA. Cells (1×105) were washed twice in PBS and incubated in mixture of 5μL 7-AAD in 50μL binding buffer for 10min in the dark. After gently mixed with 450μl binding buffer, cells were added with 1μl Annexin V-PE and incubated for 10min away from light. Results are representative of three individual experiments.
Wound healing assay
Cells were passaged 24h before scarification and seeded in 6 well plate (1×106). 200μL pipettes were used to draw a straight line, which was vertical to the bottom of plate, using ruler as a guide. The detached cells were removed by PBS. Cells in plate were kept on being cultured in medium without FBS and cell migration was monitored at 12h and 24h. 5 random fields of 100× were selected at 24h, and images were captured. The blank area (A) in the scratch was measured using Image J soft, and the wound healing rate was calculated as (A0h-A24h)/A0h×100%.
Cell invasion assay
The cell invasion was performed in a transwell chamber (Costar, USA) of 24-well with an 8μm pore size insert, which was precoated with Matrigel (BD, USA). Cells (1×104) were seeded in the upper chamber, and cultured in medium with 1% FBS for 24h. Cells were allowed to migrate towards medium containing 10% FBS in the bottom chamber. Cells on the upper membrane surface were erased with a cotton swab, and the migratory cells attached to the lower membrane surface were fixed with 4% paraformaldehyde and stained with crystal violet. The number of invasive cells was counted in 5 random fields of 200× under a microscope. Data shown are representative of three individual wells.
The statistical software of SPSS 13.0 was applied to perform data analysis. One-way ANOVA was used to evaluate the differences between cells with various plasmid transfection. Multiple comparisons were made using LSD-t test within groups. All data were shown as mean±SD and data were regarded statistically significant when the p value <0.05.