Clonorchis sinensis granulin promotes malignant transformation of ... - Parasites & Vectors

Cell culture and reagents

HIBEC lines and a human monocytic leukemia cell line (THP-1) were obtained from the Center of Hepato-Pancreato-Biliary Surgery in the First Affiliated Hospital of Sun Yat-sen University. Both HIBECs and THP-1 cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (Gibco, USA) with 5% CO₂ at 37 °C. THP-1 cells were treated with 200 ng/mL of phorbol-12-myristate-13-acetate (PMA) (HY-18739; MedChemExpress, USA) for 48 h and then treated with 20 ng/mL of IL-4 (NBP2–35131; Novus, USA) to generate M2 polarization or treated with 100 ng/mL of lipopolysaccharide (HY-D1056; MedChemExpress) to generate M1 polarization.

Recombinant Maltose Binding Protein (MBP) and MBP-CsGRN were obtained from our previous study [14]. The CsGRN fragment was amplified using forward primers 5′-ATA AGG ATC CCG GAG CAC AGGTGTAG-3′ (EcoRI) and reverse primers 5′-CGC GGA TCC TGT AAA TATA ACC AGA CTT G-3′ (BamHI), under the following conditions: 30 s at 94 °C for denaturation, 30 s at 60 °C for annealing, and 1 min at 72 °C for extension for 30 cycles. CsGRN was cloned into the protocadherin (pCDH)-luc vector plasmid (identifier, CS-HLUC-pCDH; GeneCopoeia, China) to construct the recombinant plasmid pCDH-luc-CsGRN. The recombinant plasmids were purified using the endotoxin-free Maxiprep Kit (Qiagen, USA) and then transformed into Escherichia coli DH5α for amplification (Promega, USA). Finally, the recombinant plasmid was then extracted with E.Z.N.A. Plasmid Mini Kit I (Omega, USA).

Animal studies

BALB/c wild-type mice aged 6–8 weeks were purchased from the Laboratory Animal Center of Sun Yat-sen University. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-sen University (no. SYSU-IACUC-2022-000370). The protocol was as follows: each mouse was intravenously injected with 2 ml saline dissolved in 20 μg pCDH-luc vector or 20 μgpCDH-luc-CsGRN plasmid once every week, a total of four times, respectively. The plasmid solution was rapidly injected, i.e. within 8 s, through the lateral tail vein into five mice per group. The pCDH-luc vector was used as the control. The mice were checked once a week to determine whether they were suffering from abdominal distension or other health problems. Four weeks after injection, all of the mice were sacrificed with anaesthetic at the end of the experiment. The serum and proteins from the liver tissues were extracted for the following analyses.

Detection of the location of pCDH-luc-CsGRN in mice

The location of the injected CsGRN plasmid in the mice was detected by an in vivo imaging system (IVIS 100; Xenogen, Alameda, CA). Briefly, 4 weeks after injection of the pCDH-luc vector or pCDH-luc-CsGRN, the animals were anaesthetized. After treatment with 100 μl of D-luciferin (HY-12591A; MedChemExpress) solution (10 mg/ml) through intraperitoneal injection, the CsGRN plasmid was observed by fluorescence imaging and analyzed by IVIS 100.

Establishment of the co-culture system

Transwell chambers (0.4/8 μm PET; Millipore) were used for cell co-cultivation. After preconditioning the HIBECs for 24 h with 10 μg/ml of MBP-CsGRN, the supernatant was collected and used to create the co-culture system with THP-1 cells. HIBECs were cultured in the lower chambers. THP-1 cells were cultured in the upper chambers. The THP-1 cells treated with 10 μg/ml MBP-CsGRN are henceforth referred to as the THP-1-MBP-CsGRN group. The THP-1 cells treated with the supernatant of HIBECs in the MBP-CsGRN group are henceforth referred to as the THP-1-HIBEC-MBP-CsGRN group. The THP-1 cells treated with 200 ng/ml of PMA for 48 h and 20 ng/ml of IL-4 for 48 h are henceforth referred to as the THP-1-PMA/IL-4 group.

Measurement of cell proliferation

Cell proliferation was estimated by employing the EdU-488 incorporation assay kit (C0071S; Beyotime, China) and a colony formation assay. The treated cells were labeled with EdU-488 for 2 h following the product protocol. 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei, for 10 min. The number of cells with EdU-488 incorporation was observed by fluorescence microscopy (Leica DMI8; Leica, Wetzlar, Germany) and analyzed by ImageJ software (National Institutes of Health, USA). The colony formation assay was used to analyze the long-term effects of CsGRN on HIBEC proliferation. Briefly, 1000 cells were cultivated in 24-well plates and treated with 10 μg/ml of recombinant MBP-CsGRN or MBP for 14 days. The fixed cells were then stained by crystal violet (Sigma-Aldrich, St. Louis, USA) after 30 min in 4% paraformaldehyde. After washing with double-distilled H₂O three times, the colony numbers were determined by ImageJ software (NIH).

Measurement of cell migration

Cell migration was detected by a wound-healing assay and Transwell assay. For the cell migration effect of CsGRN on HIBECs, the latter were inoculated into 6-well plates at 80% density and wounds were created using 10-L plastic pipettes. Then the cells were treated with 10 μg/ml of recombinant MBP-CsGRN or MBP for 24 h, followed by observation and imaging by light microscopy (Leica DMI3000B; Leica). Treated HIBEC (5.0 × 10⁴ ) were cultured in 24-well Transwell upper chambers (Costar, New York) for 24 h, while medium containing 10% fetal bovine serum was added to the lower chambers. The migrant cells were then stained with 10% crystal violet (Sigma-Aldrich) for 10 min and counted under a light microscope (Leica DMI3000B; Leica).

Histopathology

The mouse livers were frozen in 10% formalin for 24 h at room temperature, dehydrated and transparentized, then embedded in paraffin and sectioned into 5-μm-thick slices. Hematoxylin and eosin staining was followed by immunohistochemical (IHC)/immunofluorescence (IF) analyses of the sections.

Immunohistochemistry

Tissue samples were sectioned into 5-μm-thick slices and incubated with 3% H₂O₂ at room temperature for 10 min, then blocked with 3% bovine serum albumin (BSA) at 37 °C for 30 min. The tissues were then incubated with primary antibodies at 4 °C overnight and then with secondary antibodies at room temperature for 1 h. The following primary antibodies were purchased from Cell Signaling Technology (CST, Boston, USA) and Abcam Shanghai Trading Corporation (Abcam, Shanghai, China): cytokeratin 19 (CK19), MCP-1, CD206 and INOS. The HRP-conjugated secondary antibodies were purchased from Proteintech Group, USA. The immunohistochemical staining was imaged using Caseviewer software (3DHISTECH, Hungary).

IF assay

The liver tissue specimens were fixed in 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% Triton X-100 (9002–93-1; Beijing Biotopped Science & Technology, China) for 10 min, and then incubated with 3% BSA in PBS at room temperature for 30 min. The tissues were then incubated with primary antibodies [CD68, 1:100; CD163, 1:100, CK19, 1:100; phosphorylated (p-) STAT3 (p-STAT3), 1:200; p-JAK2, 1:100; p-ERK, 1:100; p-MEK, 1:100; Abcam, Cambridge, UK] at 4 °C overnight and with secondary antibodies [Alexa Fluor 488 conjugated goat anti-rabbit immunoglobulin G (IgG); Alexa Fluor 594 conjugated goat anti-mouse IgG (Invitrogen, Waltham, USA)] at room temperature for 1 h. The nuclei were stained with DAPI (Abcam, Cambridge, USA) for 10 min at room temperature. The liver tissue specimens of each animal were imaged six times using confocal laser scanning microscopy (LSM780; Zeiss, Oberkochen, Germany). For each mouse liver, the average signal value of six images is presented as the real value.

Western blot and enzyme-linked immunosorbent assay

After cells or liver homogenates had been harvested and washed in cold PBS twice, proteins were extracted using a radioimmunoprecipitation assay solution (Beyotime, Shanghai, China), and the protein concentration was measured using a bicinchoninic acid assay protein assay kit (Thermo, Shanghai, China). Western blot was undertaken in accordance with a previous study [14]. All primary antibodies—anti-E-cadherin (1:2000), anti-vimentin (1:2,000), anti-N-cadherin (1:2000), anti-tight junction protein (ZO-1) (1:2000), anti-β-catenin (1:2000), anti-IL6 (1:2000), anti-cyclooxygenase-2 (COX-2) (1:2000), anti-MCP-1 (1:2000), anti-p-JAK2 (1:2000), anti-, p-STAT3 (1:2000), anti-c-Myc (1:2000), anti-p-MEK (1:2000), anti-p-ERK (1:2,000), anti-STAT3 (1:2000), anti-MEK (1:2000), anti-ERK (1:2000) and anti-GAPDH (1:2000) were purchased from Cell Signaling Technology (Boston, USA). Enzyme-linked immunosorbent assay (ELISA) kits (Multi Sciences, Hangzhou, China) were used to determined IL-6 and IL-10 levels in THP-1 cells and the co-culture medium.

Flow cytometry

THP-1 cells and hepatic macrophages in each group were harvested and stained with APC/CY7 anti-mouse CD45 (103116; BioLegend, USA), PE anti-mouse CD11b (101208; BioLegend), APC anti-mouse F480 (123116; BioLegend), PC7 anti-mouse CD206 (141720; BioLegend), PC7 anti-human CD206 (T7-782-T100, EXBIO) and PC5.5 anti-mouse MHC-II (107626; BioLegend) antibodies at 4 °C for 30 min. Hepatic macrophages were detected in the mice by grinding the liver tissues gently, filtering the homogenate through a 80-μm nylon mesh filter, and isolating hepatic mononuclear cells with 40% and 80% Percoll (17-0891-01; GE Healthcare, UK). The isolated cells were stained with the above surface markers after being blocked with anti-mouse CD16/32 (TruStain FcX PLUS; BioLegend) following red blood cell lysis (420301; BioLegend, USA). After staining, the cells were washed three times with PBS and resuspended with 200 μl of 10% BSA diluted with PBS, and analyzed by FACS with a BD FACS Aria II (BD Science, USA). The data were analyzed using FlowJo v10 (TreeStar, Ashland, USA).

Quantitative polymerase chain reaction

Total RNA from the liver homogenates was extracted using TRIzol solution, according to the manufacturer's protocol (Invitrogen, Carlsbad, USA). A quantitative polymerase chain reaction (qPCR) kit (Vazyme, Nanjing, China) was used to quantify the gene expression level. The PCR conditions were as in a previous study [13]. The primer sequences were as follows: CsGRN, F-5′-CGC GGA TCC TGT AAA TAT AAC CAG ACT TG-3′, R-5′-TTA CTC GAG CGG AGC ACA GGT GTA GTG AT-3′; iNOS, F-5′-GCA CAG GAA ATG TTC ACC TAC-3′, R-5′-CAC GAT GGT GAC TTT GGC TAG-3′; Arg1, F-5′-ACG GAA GAA TCA GCC TGG TG-3′, R-5′-GTC CAC GTC TCT CAA GCC AA-3′; STAT3, F-5′-CAG CAG CTT GAC ACA CGG TA-3′, R-5′-AAA CAC CAA AGT GGC ATG TGA-3′; Bcl-2, F-5′-GGT GGG GTC ATG TGT GTG G-3′, R-5′-C GGT TCA GGT ACT CAG TCA TCC-3′; TFF3, F-5′-CCA AGC AAA CAA TCC AGA GCA-3′, R-5′-GCT CAG GAC TCG CTT CAT GG-3′; c-Myc, F-5′-GTC AGT TCG GGA AGG CTG TA-3′, R-5'-AAT CGG AGT TGG AAT CAG TCA C-3′; GAPDH, F-5′-ACG ACC ACT TTG TCA AGC TC-3′, R-5′-GTG AGG AGG GGA GAT TCA GT-3′.

Statistical analysis

All the results are from three independent experiments and are presented as means ± SD. GraphPad Prism 8.0 software (San Diego, CA) was used for statistical analysis. Analyses of statistical differences were conducted using Student's t-test and ANOVA.

Adblock test (Why?)