Cell culture medium components were purchased from Life Technologies (Grand Island, NY, USA) unless otherwise indicated. Polyclonal antibodies against cytokeratin 19 (Ck-19) and Ki-67 were purchased from Abcam (Cambridge, UK). All other chemicals (biotechnology grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Human cholangiocyte (H69) culture
The H69 cells between the 25th and 30th passage were maintained in Dulbecco’s Modified Eagle Medium (DMEM): DMEM/F12 (1:1) containing 10% FBS, an antibiotic mixture, 1.8 × 10−4 M of adenine, 5 μg/ml of insulin, 5.5 × 10−6 M of epinephrine, 2 × 10−9 M of triiodothyronine, 5 μg/mL of transferrin, 1.64 × 10−6 M of epidermal growth factor (EGF), and 1.1 × 10−6 M of hydrocortisone. Cells were cultured at 37 °C in a humidified 5% CO2 incubator.
Animal care and experimental procedures were performed in compliance with the national guidelines outlined by the Korean Laboratory Animal Act (no. KCDC-122-14-2A) of the Korean Center for Disease Control and Prevention (KCDC). The KCDC-Institutional Animal Care and Use Committee (KCDC-IA-CUC) ethics committee reviewed and approved the ESP preparation protocols (approval identification number, KCDC-003-11). New Zealand albino rabbits (male, 10 weeks old) were infected with ~ 250 metacercariae twice (with an interval of 1 week) via intragastric intubation. After 12 weeks, C. sinensis adult worms were collected from sacrificed rabbit livers for the preparation of ESPs.
Preparation of ESPs
The ESPs from C. sinensis adult worms were prepared as previously described . In brief, adult worms were recovered from bile ducts and washed several times with cold phosphate-buffered saline (PBS) to remove any host contaminants. Five fresh worms were cultured in 1 ml of prewarmed PBS containing antibiotic mixture and protease inhibitor cocktail (Sigma-Aldrich) for 3 h at 37 °C in a 5% CO2 incubator. The culture fluid was then pooled, centrifuged, concentrated with a Centriprep YM-10 filter unit (Merck Millipore, Billerica, MA, USA), and filtered through a sterile 20-μm syringe membrane. Even though these ESPs contained small sizes of peptides with < 10 kDa (Additional file 1: Figure S1), small peptides functionally important for the host interaction might be still excluded during the filtering process. The protein concentration of the ESPs was measured using DC Protein Assays (Bio-Rad, Hercules, CA, USA). ESP aliquots were stored at − 80 °C until use.
Cell seeding in three-dimensional SpheroFilm and ESP treatment
SpheroFilm microwells were obtained from InCyto Co. (Chonan, Korea). The SpheroFilm was placed at the bottom of 60-mm culture dish (Falcon, Corning, NY, USA). Before cell seeding, 100% ethanol was added to the plate and repeatedly pipetted to remove air bubbles from the wells. Once the ethanol was removed, the wells were washed with PBS and then incubated with complete culture medium at 37 °C in 5% CO2 for 24 h. The cells were detached from the culture dish with 0.05% trypsin/EDTA solution, counted, and adjusted to 1 × 106 cells/ml. The cell suspension was poured into SpheroFilm microwells and plated on the culture dish, and the culture dish was centrifuged at 100×g for 3 min to settle the cells inside each section of the microwell. After cell seeding, the culture dish was washed to prevent remaining cells outside the microwells from adhering and growing. The spheroid in each microwell was fully formed at day 5 of the culture period. Fully formed spheroids were incubated with serum-/EGF-free culture (conditional) medium for 24 h before ESP treatment. ESPs at a concentration of 4 μg/ml were added into the medium, and the culture medium containing ESPs was changed every 2 days, as described for previous ESP-treated H69 three-dimensional culture . Approximately 50 spheroids were harvested at 5 and 10 days after ESP treatment for extraction of total RNA.
Approximately ten spheroids were collected from the microwells at the indicated time points for immunocytochemical analysis. Spheroids were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, placed in optimal cutting temperature compounds (Sakura Finetek, USA Inc., Torrance, CA, USA), and frozen using dry ice. The frozen spheroid sections (5 µm thick) were blocked with 3% bovine serum albumin (BSA) in 0.1% Triton X-100 + PBS (PBST) for 1 h, washed with PBST, and incubated overnight at 4 °C with PBS containing 1% BSA and the primary antibodies (1:50 dilution for CK-19, 1:500 dilution for Ki-67), followed by anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (1:1000 dilution; Invitrogen, Carlsbad, CA, USA) for 1 h. After two washes with PBS, fluorescent spheroid images were taken using a confocal laser-scanning microscope (LSM 780; Carl Zeiss, Jena, Germany).
Total RNA from each time point was extracted using Trizol (Invitrogen) and purified using RNeasy columns (Qiagen, Valencia, CA, USA) according to the manufacturers’ protocol. After processing with DNase digestion and clean-up procedures, RNA samples were quantified, aliquoted, and stored at − 80 °C until use. For quality control, RNA purity and integrity were evaluated by denaturing gel electrophoresis (OD 260/280 ratio) and analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).
Total RNA was amplified and purified using an Ambion Illumina RNA amplification kit (Ambion, Austin, TX, USA) to yield biotinylated cRNA, according to the manufacturer’s instructions. In brief, 550 ng of total RNA was reverse-transcribed to cDNA using a T7 oligo(dT) primer. Second-strand cDNA was synthesized, in vitro transcribed, and labeled with biotin-NTP. After purification, the cRNA was quantified using an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). Seven hundred fifty nanograms of labeled cRNA samples was hybridized to each human HT-12 expression v.4 bead array for 16–18 h at 58 °C, according to the manufacturer’s instructions (Illumina, Inc., San Diego, CA, USA). Detection of array signals was carried out using Amersham FluoroLink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK), following the bead array manual. Arrays were scanned with an Illumina Bead Array Reader confocal scanner according to the manufacturer's instructions. The quality of hybridization and overall chip performance were monitored by visual inspection of both internal quality control checks and the raw scanned data. Raw data were extracted using the software provided by the manufacturer (Illumina GenomeStudio v2011.1, Gene Expression Module v1.9.0). Two independent experiments were performed to select commonly regulated genes showing good reproducibility and reliability with a mean average.
The construction of the RNA-sequencing library was performed using a TruSeq RNA Sample Preparation Kit v.2 (Illumina, Inc., cat. no. RS-122-2002). In brief, 100 ng of total RNA from each sample was exposed to poly-T oligo-attached magnetic beads to isolate poly-A mRNA following mRNA fragmentation. The cleaved RNA fragments were constructed onto a double-stranded cDNA. Then, the double-stranded library was purified using AMPure XP beads to remove all reaction components. The end repair, base addition, adapter ligation, and PCR amplification steps were performed according to the manufacturer’s instructions. Libraries were analyzed by an Agilent 2100 Bioanalyzer using a high-sensitivity DNA chip (Agilent Technologies). Then, the cDNA libraries were used for paired-end sequencing using an Illumina NextSeq 500 (Illumina, Inc.).
Validation of microarray and RNA-Seq data by quantitative real-time (RT)-PCR
Equal amounts of 100 ng of total RNA were reverse-transcribed into cDNA using a reverse transcription system (Promega). Significantly regulated genes from the microarray and RNA-Seq were quantified using an Applied Biosystems 7500 Real-Time PCR System with Fast SYBR Green Master Mix (Applied Biosystems), in accordance with the manufacturer’s protocols. The GAPDH level was measured and used to normalize the relative abundance. Table 1 shows the primer pairs designed with the PrimerDesigner program, based on the cDNA sequences in the GenBank database. Data were analyzed using the Ct method.
Bioinformatic analysis for the microarray and RNA-Seq
The entire analysis pipeline of RNA-Seq was coded using R software (ver. 3.6), which was controlled by systemPipeR (ver.1.18.2). Raw sequence reads were trimmed for adaptor sequence and masked for low-quality sequences using systemPipeR. Transcript quantification of RNA-Seq reads was performed with GenomicAlignments (ver.1.20.1) using reads aligned to Ensemble v95 Homo sapiens transcriptome annotation (GRCh.38.95) using Rsubread (ver. 1.24.6). The FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values were calculated using the ‘fpkm’ function of DESeq2 (ver. 1.24.0) and were processed with the robust median ratio method. Transcript reads were normalized by the ‘voom’ function of Limma (ver. 3.40.6). To analyze a transcript as DE, EdgeR (ver. 3.26.7) calculates the results based on the normalized counts from entire sequence alignments. Significantly DE transcripts with fold changes greater than the raw FPKM value (> 2) and adjusted P-value (< 0.01) in all experimental comparisons were selected and used for further analysis. Gene annotation was added by the online database using Ensembl biomaRt (ver. 2.40.4), and visualization was performed using the R base code and the gplots package (ver. 18.104.22.168). For differentially expressed gene (DEG) sets, hierarchical cluster analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Gene enrichment and functional annotation analysis for the significant probe list was performed using GO (http://geneontology.org) and DAVID (https://david.ncifcrf.gov/). All data analysis and the visualization of DEGs was conducted using R version 3.0.2 (www.r-project.org). For the statistical analysis, values were presented as mean ± SEM of three independent experiments. Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism software version 5.01 (California, USA). Differences between groups were considered to be significant at P < 0.05.
Experimental values were presented as mean ± SEM of three independent experiments. Data were analyzed by Student’s t-test or two-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism software version 5.01 (San Diego, CA, USA). Differences between groups were considered to be significant at P < 0.05.