Patients and specimens
One hundred and twelve patients with histologically proven ICC, who underwent curative hepatectomy at the First Affiliated Hospital of Sun Yat-sen University, in Guangzhou, Guangdong, China between June 2006 and June 2012, were included in this study. The inclusion criteria of the study were as follows: (1) histologically diagnosed ICC; (2) curative resection of tumors; and (3) absence of distant metastases. The patients who met one of the following criteria were excluded from the study: (1) perihilar cholangiocarcinoma (Klatskin tumor); (2) mix tumors of HCC and ICC; (3) R1 or R2 resection or laparotomy with tumor biopsy; and (4) receiving neoadjuvant chemotherapy and/or radiotherapy.
Of the 112 patients included in this study, 63 (56.2%) were men, and 49 (43.8%) were women, with a median age of 57 years (range, 28–79 years). Eighty-six patients (76.8%) had a single tumor; the remaining 26 patients (23.2%) had multiple tumors. Tumor size ranged from 1.0 to 16.0 cm in diameter (median, 5.7 cm). Fourteen patients (12.5%) had adjacent organ invasion. Forty-nine patients (43.8%) had lymph node metastasis. Seventy patients (62.5%) had an elevated level of serum CA19-9 (>37 U/L), and 43 (38.4%) had an elevated serum level of carcino-embryonic antigen (CEA) (>5 µg/L). All patients underwent curative hepatectomy with regional lymph node dissection. According to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (7th edition) [16], of the 112 ICC patients, 43 (38.4%) had tumor-node-metastasis (TNM) stage I disease, 10 (8.9%) had TNM stage II disease, 6 (5.4%) had TNM stage III disease, and 53 (47.3%) had TMN stage IV disease.
Patients were followed up every 1–3 months, ending in March 2014. Tumor recurrence/metastasis was diagnosed on the basis of dynamic imaging results (i.e., contrast-enhanced computed tomography and/or contrast-enhanced magnetic resonance imaging and/or contrast-enhanced ultrasonography), and serum CA19-9 and CEA levels.
For 48 patients, tumorous and adjacent non-tumorous tissues were collected, snap-frozen instantly in liquid nitrogen, and stored at −80 °C for molecular biological analysis. Paraffin-embedded ICC specimens of 112 patients that were used for immunohistochemical staining were obtained from the Department of Pathology, First Affiliated Hospital of Sun Yat-sen University.
This study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. Written informed consent was obtained from each patient.
Real-time quantitative polymerase chain reaction (RT-qPCR)
Using RNAiso (TaKaRa, Dalian, Liaoning, China), total RNA was extracted from 48 pairs of tumorous and adjacent non-tumorous tissues as well as cultured cells. Using the PrimeScript® RT Reagent kit (TaKaRa), reverse transcription was performed with 0.5 μg of total RNA. RT-qPCR was performed to examine the mRNA level of EYA4 using TaKaRa SYBR® Premix Ex Taq™ Kit (TaKaRa) and the ABI PRISM® 7900HT RT-qPCR System (Applied Biosystems, Carlsbad, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as endogenous control. Primers were listed as follows: EYA4 sense 5′-GAATAACACAGCCGATGG-3′, antisense 5′-CCAGGTCACTATCAGGAG-3′; GAPDH sense 5′-GCACCGTCAAGGCTGAGAAC-3′, antisense 5′-TGGTGAAGACGCCAGTGGA-3′. Thermocycling conditions for PCR were as follows: an initial cycle of 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 95 °C for 5 s, and 65 °C for 30 s, and finally 72 °C for 5 min for the extension. ΔCt (difference in cycle threshold) was calculated for each sample (ΔCt = CtTarget gene − CtGAPDH), and relative quantities were compared. All reactions were repeated in triplicate.
Immunohistochemical (IHC) staining
Paraffin-embedded tissues were cut into 4-μm sections and mounted on glass slides. IHC analysis was performed on 112 ICC tissues to detect EYA4 protein. Briefly, tissues were deparaffinized in dimethylbenzene, rehydrated in graded alcohol, and then incubated in 3% H2O2 to block endogenous peroxidase activity. Antigen retrieval was achieved by treating the tissues with citrate buffer in a pressure cooker. Tissues were subsequently incubated with rabbit anti-human EYA4 polyclonal antibodies (dilution 1:25; Abcam, Cambridge, UK) at 4 °C overnight. A ChemMate™ Envision™ Detection Kit (Dako, Glostrup, Denmark) was used to detect and visualize the bound primary antibodies. Human skeletal muscle tissue was used as positive control for EYA4 antibody (recommended by supplier), and the rabbit IgG antibody (Biosynthesis, Beijing, China) was used as negative control in IHC staining. Staining intensity (negative, 0; mild, 1; moderate, 2; and severe, 3) and proportion of positive cells (negative, 0; ≤10, 1; >10 and ≤33%, 2; >33 and ≤66%, 3; and >66%, 4) were quantified, respectively [17]. Summation of the scores of the two parameters represents protein expression level. Each slide was scored by two observers independently, and the average of their scores was recorded as the IHC score.
Cell lines
Human ICC cell lines RBE and SSP-25 were obtained from the Cell Resources Center of Shanghai Institutes for Biological Science, Chinese Academy of Science (Shanghai, China). Cells were cultured in RPMI-1640 (Gibco BRL, Rockville, MD, USA) and supplemented with 10% fetal bovine serum (Gibco BRL).
Western blotting
Total cell lysates were prepared using a KeyGEN Total Protein Extraction Kit (Nanjing, Jiangsu, China). Aliquots of 10–20 μL cell lysates were electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAGE), and proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Cambridge, UK). The membrane was blocked, incubated with primary antibody at 4 °C overnight, and then incubated with secondary antibody at room temperature for 30 min. Rabbit anti-human EYA4 polyclonal antibodies (dilution 1:300; Abcam), GAPDH monoclonal antibody, and goat anti-rabbit secondary antibody (dilution 1:3000; Biosynthesis) were used to detect EYA4 and GAPDH protein. GAPDH was used as loading control. Bands were visualized using an ECL kit (KeyGEN, Nanjing, China) and exposed to Kodak X-OMAT film (Carestream Health Inc., Rochester, NY, USA).
Establishment of stable EYA4-overexpressing transfectants of ICC cells
The pReceiver-M02 empty vector and the recombinant pReceiver-M02/EYA4 overexpression plasmids (U0188) were purchased from Genecopoeia (Rockville, MD, USA). The recombinant EYA4-expressing plasmid was transfected into RBE and SSP-25 cells at 70%–80% confluence using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). Plasmid pReceiver-M02 was used as control (FulenGen, Guangzhou, Guangdong, China). After 2 weeks of G418 selection, stable transfected cells were subjected to limited dilution. Survival clones with the highest expression of EYA4 in both cell lines were used for further studies. Stable EYA4-overexpressing transfectants and vector transfectants of RBE and SSP-25 cells were designated as RBE-EYA4, RBE-Vector, SSP-EYA4, and SSP-Vector, respectively.
Cell proliferation assay
Cells were seeded into 96-well plates at the density of 2000 cells per well. Cell proliferation was detected at 24, 48, 72, and 96 h after seeding using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Absorbance values at 450 nm (A450) were recorded as representation of cell viability. Each experiment was done in triplicate, and four individual experiments were performed.
Foci formation assay
Cells were seeded into 6-well plates at the density of 1000 cells per well and cultured for 7 days. After fixing in 4% paraformaldehyde and staining with 1% crystal violet, foci with more than 50 cells were counted. Four independent experiments were performed.
Cell invasion assay
Cell invasion assay was performed with BD BioCoat™ Tumor Invasion System (BD Biosciences, Bedford, MA, USA). Approximately 5 × 104 cells/chamber were seeded into the rehydrated chamber. After 22 h of incubation, cells that invaded to the bottom surface of the chamber were fixed and stained as mentioned above, then counted under the microscope. Four assays were performed.
In vivo experiments using xenograft ICC in NOD/SCID mice
The cultured RBE and SSP-25 cells were trypsinized and resuspended to a density of 1 × 108 cells/mL. Then, 1 × 107 RBE or SSP-25 cells in 100 µL were inoculated subcutaneously into both flanks of 4–5-week male NOD/SCID mice (HFK Bioscience Co. Ltd, Beijing, China). After the xenograft tumors reached 6 mm in diameter (or volume >100 mm3), the RBE and SSP-25 tumor-bearing mice were randomized into EYA4, vector, and blank groups (in each group, there were three mice bearing 5 tumors) and subjected to the following treatments, respectively. (1) EYA4 group: intra-tumoral injection of lipofactamine 2000, EYA4-expressing plasmids, and Opti-MEM; (2) Vector group: intra-tumoral injection of lipofactamine 2000, vector-plasmids, and Opti-MEM; and (3) Blank group: intra-tumoral injection of lipofectamine 2000 and Opti-MEM. Each injection contained 20 μg plasmid (1 μg/μL) mixed with 40 μL lipofectamine 2000 and 20 μL Opti-MEM in EYA4 and vector groups; in blank group, 40 μL lipofectamine 2000 mixed with 2 μL Opti-MEM was injected each time. The treatment was repeated every 5 days and given a total of 6 times. Body weights of mice and the tumor size were monitored every 3 days. Tumor volume was calculated as follows: volume (mm3) = length × width2/2. On post-treatment day 30, the mice were euthanized, and their tumors were removed and weighed. The in vivo experiments were approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University.
Statistical analysis
Results were presented as mean ± standard deviation (SD) or median (range). Statistical analysis was performed using SPSS 17.0 software (IBM, Chicago, IL, USA). DFS was calculated from the date of surgery to the date that tumor recurrences or metastases were confirmed. OS was defined as the interval between the date of surgery and death or the date of last follow-up. DFS and OS were calculated using the Kaplan–Meier method. The associations between EYA4 protein expression and clinicopathologic parameters were analyzed using the Mann–Whitney U test. Univariate and multivariate Cox regression analyses were used to identify independent prognostic factors. Inter-group comparisons of foci formation, cell invasion ability, mice weight, and xenograft tumor size and weight were done using Student’s t test. Cell proliferation and the xenograft tumor growth curve were compared among groups by repeated measures of analysis of variance. Two-sided P < 0.05 values were considered statistically significant.
