The human studies involved in this study were approved by the Joint Chinese University of Hong Kong- New Territories East Cluster Clinical Research Ethics Committee (CUHK-NTEC CREC). An informed consent for the human tissues for research purposes only was obtained from all patients recruited in this study. The animal work in this study was approved (17/088/MIS-5-B) by the animal experimentation ethics committee of CUHK.
A total of 84 pairs of HCC tissues and the corresponding adjacent para-tumor liver tissues were collected from patients who underwent surgery in the Prince of Wales Hospital. All the patients were diagnosed with HCC. These collected tissues were either fixed in 10% formalin for histological evaluation or snap-frozen in liquid nitrogen or stored at -80 °C until experimentation.
Primers, plasmids, and antibodies
The primers used in this study were listed in Supplementary Table S6. The pcDNA3.1-FOXP3, pcDNA3.1, Flag-pcDNA3.1-FOXP3, Flag-pcDNA3.1 were produced in our Lab. CS2-flag-Smad2 (Addgen plasmid #14042) and CS2-flag-Smad3 (Addgen plasmid #14052) was a gift from Joan Massague, pcDNA3-Flag-Smad4 (Addgen plasmid #80888) was a gift from Aristidis Moustakas, Pbv-Luc (Addgen plasmid #16539) and c-Myc promoter del-1 (Addgen plasmid #16601) was a gift from Bert Vogelstein. SiRNA-FOXP3 (sc-43569) and siRNA-Control (sc-43569) were from Santa Cruz Biotechnology. The primary antibodies for western blot were as follows: FOXP3 (150D/E4, Invitrogen, 1:1000), FOXP3Δ3 (16J4G6, Novus,1:1000), PCNA (PC10, Santa Cruz, 1:2000), Bax (B-9, Santa Cruz, 1:1000), Bcl2 (C-2, Santa Cruz, 1:1000), p53 (DO-1, Santa Cruz, 1:1000), Smad2 (A-11, Santa Cruz, 1:1000), Smad3 (38-Q, Santa Cruz, 1:1000), Smad2/3 (A-3, Santa Cruz, 1:1000), p-Smad2(s467, Abcam, 1:1000), p-Smad3(1D9, Santa Cruz, 1:1000), Smad4 (B-8, Santa Cruz, 1:1000), c-Myc (9E10, Santa Cruz, 1:1000), and GAPDH (6C5, Santa Cruz, 1:2000). The primary antibodies for immunohistochemistry were listed below: FOXP3 (150D/E4, Invitrogen, 1:100), FOXP3Δ3 (16J4G6, Novus,1:100), Ki-67 (Ki67, Santa Cruz, 1:50), c-Myc (9E10, Santa Cruz, 1:100), Smad2/3 (A-3, Santa Cruz, 1:100), and p-Smad3 (Santa Cruz, 1:100).
Nested PCR to determine the isoforms of FOXP3
RNA was isolated from 62 paired samples according to standard protocol (Trizol, Invitrogen), after reverse transcription, we performed two-round nested PCR (R004A, Takara). First-round PCR, we designed a pair of primers (FOXP3-1) to amplify the FOXP3 transcript. The primer (FOXP3-2) was used to amplify the region from exon 2 to exon 5, and primer (FOXP3-3) was used to amplify the region from exon 7 to exon 9. PCR products were cloned into T-A vectors and the sequence was checked by BGI HONGKONG company. Thus, the FOXP3 and its isoforms were confirmed by their sequences. All primers used in this study were listed in the Supplementary Table S6.
Quantitative real-time PCR
After the determination and confirmation of the isoforms, we designed the specific primers to determine the levels of FOXP3 and FOXP3Δ3 in HCC. For the FOXP3, the forward primer was located in the junction between exon2 and exon3 (primer FOXP3-4). For the FOXP3Δ3, the forward primer was located in the junction between exon2 and exon4. The quantitative real-time PCR was performed to determine the mRNA level according to the protocol (RR820A, Takara) in the Quant-studio 12 K Flex Real-time PCR system.
The IHC staining assay was performed according to the standard protocol on five-micrometer formalin-fixed paraffin sections by using the primary antibodies as indicated above. After staining, we invited a pathologist and an investigator who were blind to the study design to score the staining intensities according to the immunoreactive score system.
Cell lines and culture conditions
Two HCC cell lines: Hep3B, PLCR/PRF/5, and HEK293T cell line were obtained from the American type culture collection. Huh7 was obtained from the Japanese Collection of Research Bioresource Cell Bank (JCRB0403). All cells were cultured in DMEM with 10% fetal bovine serum and 1% antibiotic at 37°C in humidified air with 5% CO2.
The generation of FOXP3 overexpression stable cells
Hep3B and PLC/PRF/5 cell lines were used to generated FOXP3 overexpression stable cell lines. The plasmid pcDNA3.1-FOXP3 and corresponding vectors were transfected with lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacture’s protocol. After transfected, the cells were selected by G418 at 500 ng/ml for two weeks.
Knockdown FOXP3 by siRNA
The expression of FOXP3 was knocked down by siRNA in Huh7. siRNA-FOXP3 and siRNA-control were transfected on to the cells with the aid of lipofectamine 2000.
The total protein of the cells was isolated by the RIPA lysis buffer (20-188, Merck, Germany) with the protease inhibitor cocktail (Roche, Basel, Switzerland), and phenylmethylsulphonyl fluoride (Roche). The Bio-Rad Bradford protein assay was used to determine protein concentration. Before electrophoresis, the protein was denatured with the 1× protein loading dye at 95°C for 10 min. Samples containing 20 μg of protein were electrophoresed. Then, the protein was transferred on to 0.22 μm polyvinylidene difluoride (PVDF, BioRad). After transferred, the PVDF membrane was blocked in TBST containing 5% non-fat milk for 1 h at room temperature with shaking, and followed by incubating with primary antibodies at the indicated concentration at 4 °C overnight with shaking. We washed the membrane with the TBST and incubated with the corresponding secondary antibodies. The enhanced chemiluminescent substrate and western blot film plates from Kodak (Rochester, NY) were used to detect protein levels on the membranes in the darkroom. The protein expression level was semi-quantified by the internal control GAPDH (Santa Cruz Biotechnology). Then the grayscale ratio was determined by Adobe Photoshop CS5 (San Jose, CA). All experiments were performed in triplicate.
Cell proliferation assays
An MTT kit (Sigma) was used to assay the cell proliferation according to the manufacturer’s instruction. FOXP3-overexpressing cells, Hep3B and PLC/PRF/5, and FOXP3-knockdown cells Huh7 and their corresponding control cells were seeded into 96-well plates, and cultured for different periods(24 h, 48 h, and 72 h). 10 µl 5 mg/ml MTT was added to each well, and the OD570 and OD630 were determined after 4 hours of incubation. The cell viability was represented by OD570 minus OD630.
Colony formation assays
After 1000 FOXP3-overexpressing cells, Hep3B and PLC/PRF/5, and FOXP3- knockdown cells Huh7 and their corresponding control cells were seeded into 6-well plate for 14 days, colonies containing more than 50 cells were counted.
Migration and invasion assays
At 24 h after transfection, approximately 5 × 103 transiently transfected and the control cells were suspended in 200 μl of serum-free media and cultured in the upper chamber (8 μm pore size, Millipore) for the migration assay. For the invasion assay, the chamber was coated with 20% Matrigel (Sigma) 24 h before culturing with cells. 800 ml of the cell growth medium was filled within the lower chambers. After 48-hour incubation, the cells that had migrated or invaded through the membrane from the upper chamber to the lower chamber were fixed by the 4% paraformaldehyde and stained with 0.05% crystal violet (Sigma). The cells in the below of chamber surface were photographed, and the cells in three random fields were counted.
Apoptosis was detected using the apoptosis Assay Kit (ab-176749 Abcam) following the manufacturer’s protocol by flow cytometry.
After the treatment with peptide 60 for 24 h, cells were fixed at room temperature in 3% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100, and blocked with BSA. After blocked, the cells were incubated with primary antibodies (FOXP3, Invitrogen, 1:100) and fluorochrome-conjugated secondary antibodies (goat α mouse-Cy2/Cy3, Jackson ImmunoResearch, 1:500) for 1 h each. The nuclei of the cells were stained by DAPI. Stained cells were analyzed with a fluorescence microscope (Carl Zeiss).
Dual-luciferase reporter assay
Hep3B and PLC/PRF/5 cells were transfected or co-transfected with various plasmids, as indicated in the figures. Cells were collected 48 h after transfection, and luciferase activities were detected by the dual-luciferase reporter assay kit (Promega, Fitchburg, WI). The luciferase activity was normalized to the control Renilla.
After HEK293T or PLC/PRF/5 cells were transfected or co-transfected with plasmids, as shown in the figures for 24 h, the nuclear protein was extracted for immunoprecipitation. The ANTI-FLAG® M2 Affinity Gel was added into the lysis and incubated with continuing slow rotation at 4 °C overnight. After washing and denatured, the samples were used for western blot analysis.
ChIP assay was performed with a Magna ChIP kit (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. PLC/PRF/5 cells were sonicated to shear the chromatin to a manageable size. The antibody for Flag antibodies (Invitrogen) was used for immunoprecipitation. End-point PCR was conducted to detect targeted DNA.
Female nude mice (4–6 weeks old, weighing 16–20 g) were provided by Laboratory Animal Service Center of CUHK. The mice were free to access to food and water on a 12-h light, 12-h dark cycle. Ten female nude mice were randomly subjected to two groups (five mice per groups) that were subcutaneously injected with 1 × 106 Hep3B-FOXP3 and Hep3B-control cells, respectively. The tumor size was measured every 3 days for up to 30 days by a micrometer. The tumor volume was calculated by length × width × width/2. After 30 days, the mice were euthanized by cervical dislocation, and the tumors were collected for further investigation. No adverse event of the animals was observed in this study. The experiments were repeated twice. All experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.
In this study, the continuous data were presented as the median ± SD, and discrete variables were presented as absolute values and relative frequencies. We used the paired t-test to compare the expression levels of FOXP3 and FOXP3Δ3 in tumor tissues and adjacent normal tissues. The clinicopathologic features were compared using Pearson’s chi-squared test or Fisher’s exact test. Kaplan–Meier plots were used for OS and DFS rates, then compared with the log-rank test. Univariable or multivariable Cox proportional hazard regression was performed to evaluate the predictive values of FOXP3 and other clinicopathologic features. The independent Student’s t-test was used to compare colony formation and gene expression between two groups. Repeated Measures ANOVA was used to compare the tumor growth rate between two groups in the in vivo assay. All the tests were performed by SPSS 22.0. P-values less than 0.05 were considered statistically significant.