Subjects and sample collection
This study was conducted on 60 subjects consecutively recruited from the characteristic medical centre of Chinese People's Armed Police Force between July 2018 and December 2018. Subjects provided informed consent, and the study protocol was approved by the institute's committee on human research. The study included 20 newly diagnosed patients with PTC, 20 patients diagnosed with benign thyroid lesions and 20 normal controls. The diagnoses of PTC and benign thyroid lesions were confirmed by histopathological evaluation of the ultrasound-guided biopsy sample preoperatively and by samples retrieved postoperatively. The normal patients were those who came to our hospital for physical examinations. All of them underwent thyroid function tests and thyroid ultrasound tests, and the results were negative. The normal thyroid tissues were located 1cm away from the benign lesion of thyroid, which was resected during the operation. No any prior chemotherapy or radiotherapy was received by any patient preoperatively. No other malignant tumours were found in any of the patients.There was no history of microbial infection in the past three months. Blood samples were collected three days before thyroidectomy, and the specimens were immediately snap-frozen in liquid nitrogen and then refrigerated at -80˚C. Patients data were collected, including age, sex, TNM staging, extrathyroidal invasion, multifocality and lymph node metastasis.
All procedures used were in accordance with the Logistics University of the Chinese People's Armed Police Force human welfare guidelines. The protocols were approved by the Ethics Review Committee of the Logistics University of the Chinese People's Armed Police Force (Tianjin, China).
All of the following RNA isolation protocols were performed in an RNA-dedicated work area with RNase/DNase-free water and RNase-free labware. Total RNA was extracted from whole blood samples with Quick-RNATM Whole Blood (ZYMO RESEARCH, USA) and from thyroid samples with Quick-RNA™ MicroPrep (ZYMO RESEARCH, USA), according to the instructions provided in the kit. The RNA was then purified and concentrated with RNA Prep Buffer and RNA Wash Buffer. The total RNA concentrations and quality of each sample were then assessed with a NanoDrop ND1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). In addition, OD260/OD280 ratios between 1.8 and 2.1 were acceptable, while OD260/OD230 ratios less than 1.8 were unacceptable.
Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Following RNA extraction from whole blood and thyroid samples, cDNA was synthesized with reverse transcriptase according to the instructions provided in the kit (TIAN GEN, China). PCR was conducted in a 10-μl reaction volume consisting of the following: 1 μl cDNA, 5 μl Premix, 0.3 μl primer forward, 0.3 μl primer reverse, 0.2 ul ROX and 3.2 μl H2O. The qPCR reaction was performed on a SYBR Green 7500 Real-Time PCR System (Applied Biosystems) as follows: one 15-min cycle at 95˚C, forty cycles of 10 s at 95˚C and 32 s at 60˚C. β-actin served as a control. PCRs were conducted in triplicate. Relative E2F3 expression was calculated with the 2−ΔΔCt method.
HE staining & Immunohistochemistry
Formalin-fixed paraffin-embedded biopsy specimens were sectioned into sections (5 µm). After that, sections were deparaffinized with xylene, rehydrated with graded alcohol solutions and stained with hematoxylin and eosin (HE). Finally, sections were examined microscopically.
Immunohistochemical detection of E2F3 was performed with formalin-fixed paraffin-embedded biopsy specimens. Sections (5 µm) were deparaffinized with xylene and rehydrated with graded alcohol solutions. Endogenous peroxidase activity was quenched by boiling the sections in 10 mM citrate buffer (pH 6.0) for 3 min, followed by cooling at room temperature for more than 20 min. Sections were incubated with anti-E2F3 antibody (1:200; cat. no. ab50917; Abcam, Cambridge, UK) overnight at 4°C. Sections were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (PV6001, Zhongshan Goldenbridge Biotechnology Co, Ltd, Beijing, China) for 30 min at 37°C, and the colour was developed with 3, 3'-diaminobenzidine for 1min. The negative control involved the omission of primary antibody. All slides were analysed by two independent observers. The images were acquired using NIS-Elements BR 4.30.00 64-bit software by Nikon DS Ri2 Microscopy (Kodak, Rochester, NY, USA).
The expression levels of E2F3 in different groups were systematically assessed. E2F3-positive cells were identified at low magnification (×10 ocular and ×10 objective) as “hot spots”. We selected ten hot spots per section to observe at higher magnification (×10 ocular and ×40 objective, high power field). Using a grid in the ocular lens (Olympus 100×), all positively stained cells in every high-power field were counted. The mean percentage of positively stained cells was used to evaluate the expression of the protein in a section. The fraction of E2F3-positive cells was scored as 0 (0% positive cells), 1 (1%–25% positive cells), 2 (26%–50% positive cells), 3 (50%–75% positive cells) or 4 (>75% positive cells). The intensity of E2F3 immunostaining was scored as 0 (negative), 1 (weak), 2 (intermediate) or 3 (strong). The intensity score (0–3) was multiplied by the fraction score (0–4), and a final score was assigned as follows: 0 (negative, -), 1–4 (weak expression, +), 5–8 (moderate expression, ++), and 9–12 (strong expression, +++).
Quantitative data are descriptively expressed as the median and interquartile range (IQR). Comparison between the ranks of more than 2 independent groups, in case of nonparametric data, was done by Kruskal Wallis test, while that of 2 independent groups was done using the Wilcoxon rank sum Test. The chi-square test was used to compare the counting data. To assess E2F3 staining intensity, we used the nonparametric Mann-Whitney test for the comparison of groups. A receiver operator characteristic (ROC) curve was applied to assess the overall diagnostic value for discriminating PTC from benign hyperplastic diseases and normal tissue. “P < 0.05” was used for statistical significance.