Clonorchis sinensis granulin promotes malignant transformation of human intrahepatic biliary epithelial cells via interplay with M2 macrophages through regulation of STAT3 phosphorylation and MEK/ERK pathway | Parasites and vectors

Cell tradition and reagents

HIBEC strains and a human monocytic leukemia cell line (THP-1) have been obtained from the Hepatopancreatobiliary Surgical procedure Middle within the First Affiliated Hospital of Solar Yat-sen College. Each HIBEC cells and THP-1 cells have been 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 have been handled with 200 ng/mL of phorbol-12-myristate-13-acetate (PMA) (HY-18739; MedChemExpress, USA) for 48 hours after which handled with 20 ng/mL of IL-4 (NBP2 –35131; Novus, USA) to generate M2 polarization or handled with 100 ng/mL lipopolysaccharide (HY-D1056; MedChemExpress) to generate M1 polarization.

Recombinant maltose binding protein (MBP) and MBP-CSGRNs have been obtained from our earlier examine [14]. THE CSThe GRN fragment was amplified utilizing ahead primer 5′-ATA AGG ATC CCG GAG CAC AGGTGTAG-3′ (EcoRI) and reverse primer 5′-CGC GGA TCC TGT AAA TATA ACC AGA CTT G-3′ (BamHI), within the following circumstances : 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 assemble the recombinant plasmid pCDH-luc-CSGRN. The recombinant plasmids have been purified utilizing the endotoxin-free Maxiprep package (Qiagen, USA) after which reworked into Escherichia coli DH5α for amplification (Promega, USA). Lastly, the recombinant plasmid was then extracted with EZNA Plasmid Mini Equipment I (Omega, USA).

Animal research

BALB/c wild-type mice aged 6 to eight weeks have been bought from the Laboratory Animal Middle of Solar Yat-sen College. The animal use protocol was reviewed and permitted by the Institutional Animal Care and Use Committee (IACUC), Solar Yat-sen College (No. SYSU-IACUC-2022-000370). The protocol was as follows: every mouse was intravenously injected with 2 ml of saline answer dissolved in 20 μg pCDH-luc vector or 20 μgpCDH-luc-vector or 20 μgpCDH-luc-CSGRN plasmid as soon as every week, a complete of 4 occasions, respectively. The plasmid answer was injected quickly, i.e. inside 8 s, through the lateral tail vein in 5 mice per group. The pCDH-luc vector was used as a management. The mice have been checked as soon as every week to find out in the event that they have been affected by belly distension or different well being issues. 4 weeks after injection, all mice have been sacrificed with anesthetic on the finish of the experiment. Serum and proteins from liver tissues have been extracted for the next analyses.

Detection of the place of pCDH-luc-CSGRN in mice

The placement of the injection CSGRN plasmid in mice was detected by an in vivo imaging system (IVIS 100; Xenogen, Alameda, CA). Briefly, 4 weeks after injection of pCDH-luc vector or pCDH-luc-CSGRN, the animals have been anesthetized. After therapy with 100 μl D-luciferin answer (HY-12591A; MedChemExpress) (10 mg/ml) by intraperitoneal injection, the CSThe GRN plasmid was noticed by fluorescence imaging and analyzed by IVIS 100.

Institution of the co-culture system

Transwell chambers (0.4/8 μm PET; Millipore) have been used for cell co-cultivation. After preconditioning of HIBECs for twenty-four hours with 10 μg/ml MBP-CSGRN, the supernatant was collected and used to create the co-culture system with THP-1 cells. HIBECs have been grown within the decrease chambers. THP-1 cells have been cultured within the higher chambers. THP-1 cells handled with 10 μg/ml of MBP-CSGRNs are hereafter known as THP-1-MBP-CSGRN group. THP-1 cells handled with HIBEC supernatant in MBP-CSThe GRN group is hereinafter known as THP-1-HIBEC-MBP-CSGRN group. THP-1 cells handled with 200 ng/ml PMA for 48 h and 20 ng/ml IL-4 for 48 h are hereinafter known as the THP-1-PMA/IL-4 group.

Measurement of cell proliferation

Cell proliferation was estimated utilizing the EdU-488 Incorporation Assay Equipment (C0071S; Beyotime, China) and a colony formation assay. Handled cells have been labeled with EdU-488 for two hours following the product protocol. 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei, for 10 min. The variety of EdU-488-incorporated cells was noticed by fluorescence microscopy (Leica DMI8; Leica, Wetzlar, Germany) and analyzed by ImageJ software program (Nationwide Institutes of Well being, USA). The colony formation assay was used to research the long-term results of CSGRN on HIBEC proliferation. Briefly, 1000 cells have been cultured in 24-well plates and handled with 10 μg/ml of recombinant MBP-CSGRN or MBP for 14 days. The mounted cells have been then stained with crystal violet (Sigma-Aldrich, St. Louis, USA) after 30 min in 4% paraformaldehyde. After washing with H2Or triplicate, colony numbers have been decided by ImageJ software program (NIH).

Measurement of cell migration

Cell migration was detected by a wound therapeutic assay and a Transwell assay. For the cell migration impact of CSGRN on HIBEC, the latter have been inoculated into 6-well plates at 80% density and wounds have been created utilizing 10 L plastic pipettes. Then the cells have been handled with 10 μg/ml of recombinant MBP-CSGRN or MBP for twenty-four hours, adopted by statement and imaging by mild microscopy (Leica DMI3000B; Leica). Handled HIBEC (5.0 × 104 ) have been grown in Transwell 24-well higher chambers (Costar, New York) for twenty-four h, whereas medium containing 10% fetal bovine serum was added to the decrease chambers. The migrating cells have been then stained with 10% crystal violet (Sigma-Aldrich) for 10 min and counted below a lightweight microscope (Leica DMI3000B; Leica).


Mouse livers have been frozen in 10% formalin for twenty-four hours at room temperature, dehydrated and transparentized, then embedded in paraffin and sectioned into 5 μm thick slices. Hematoxylin and eosin staining was adopted by immunohistochemical (IHC)/immunofluorescence (IF) analyzes of the sections.


Tissue samples have been sectioned into 5 μm thick slices and incubated with H2OR2 at room temperature for 10 min, then blocked with 3% bovine serum albumin (BSA) at 37 °C for 30 min. The tissues have been then incubated with main antibodies at 4°C in a single day after which with secondary antibodies at room temperature for 1 hour. The next main antibodies have been bought from Cell Signaling Know-how (CST, Boston, USA) and Abcam Shanghai Buying and selling Company (Abcam, Shanghai, China): cytokeratin 19 (CK19), MCP-1, CD206 and INOS. HRP-conjugated secondary antibodies have been bought from Proteintech Group, USA. Immunohistochemical staining was taken utilizing Caseviewer software program (3DHISTECH, Hungary).

IF sensible

Liver tissue samples have been mounted in 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% Triton X-100 (9002-93-1; Beijing Biotopped Science & Know-how, China) for 10 min, after which incubated with 3% BSA in PBS at RT for 30 min. The tissues have been then incubated with main 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 in a single day 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. Nuclei have been stained with DAPI (Abcam, Cambridge, USA) for 10 min at room temperature. Liver tissue samples from every animal have been imaged six occasions utilizing laser scanning confocal microscopy (LSM780; Zeiss, Oberkochen, Germany). For every mouse liver, the imply sign worth of six pictures is offered because the true worth.

Western blot and enzyme immunosorbent assay

After cells or liver homogenates have been harvested and washed twice in chilly PBS, proteins have been extracted utilizing radioimmunoprecipitation assay answer (Beyotime, Shanghai, China), and protein focus was measured utilizing a Bicinchoninic acid protein evaluation package (Thermo, Shanghai, China). Western blot was carried out in accordance with a earlier examine [14]. All main 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) have been bought from Cell Signaling Know-how (Boston, USA) . Enzyme-linked immunosorbent assay (ELISA) kits (Multi Sciences, Hangzhou, China) have been used to find out the degrees of IL-6 and IL-10 in THP-1 cells and co-culture medium.

Stream cytometry

THP-1 cells and liver macrophages in every group have been 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 minutes. Liver macrophages have been detected in mice by gently grinding liver tissue, filtering the homogenate via an 80 μm nylon mesh filter, and isolating liver mononuclear cells with 40% to 80% Percoll (17-0891 -01; GE Healthcare, UK). The remoted cells have been stained with the above floor markers after being blocked with anti-mouse CD16/32 (TruStain FcX PLUS; BioLegend) following crimson blood cell lysis (420301; BioLegend, USA). After staining, cells have been washed thrice 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). Information have been analyzed utilizing FlowJo v10 (TreeStar, Ashland, USA).

Quantitative polymerase chain response

Complete RNA from liver homogenates was extracted utilizing TRIzol answer, in line with the producer’s protocol (Invitrogen, Carlsbad, USA). A quantitative polymerase chain response (qPCR) package (Vazyme, Nanjing, China) was used to quantify the extent of gene expression. PCR circumstances have been as in a earlier examine [13]. The primer sequences have been 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′.

statistic evaluation

All outcomes are from three unbiased experiments and are offered as imply ± SD. GraphPad Prism 8.0 software program (San Diego, CA) was used for statistical evaluation. Analyzes of statistical variations have been performed utilizing Scholar’s T-test and ANOVA.

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