Patients and histological methods
We re-examined the gallbladders from 80 GBC patients who underwent surgery from 2012–2017 (mean age: 58 years; range: 32–74 years; 24 male, 56 female), 80 control patients (included 60 gallstone patients, 20 normal gallbladders), composed of liver transplant donors with no biliary disease, liver rupture, liver cancer with no biliary disease were included. There were no obvious macroscopic and pathological abnormalities in the gallbladder specimens resected.(mean age: 52 years; range: 11–77 years; 36 male, 44 female) from 2012 to 2017. These tissues were obtained at the time of laparotomy. These samples were divided into two equal portions. One part of the sample was immediately transferred into liquid nitrogen for DNA and RNA isolation, and the second part was fixed in 10% buffered formalin solution for the histopathological evaluation. The experiment was conducted with the understanding and written consent of each subject. The research method complies with the stipulated standards set by the Declaration of Helsinki. The study procedure was authorized by the Ethics Committee of Xian Jiaotong University.
Bile sample collection
Gallbladder bile samples were collected from all patients at the hospital after they were diagnosed with GBC and control. After cholecystectomy, collect at least 1ml of bile from each patient by gallbladder suction, inject it into a 1.5 mL epppendorf safety lock tube, and close the lid. For identification of bacterial infections, the samples remained stored at − 80°C until processing and testing. Before collection, all patients underwent serum chemistry and complete blood count.
DNA extraction from bile samples
DNA was extracted from approximately 5 mg of bile samples. To determine whether the bacteria were included, the researchers took two 2–3 mg tablets from each case. According to the manufacturer's instructions, DNA was extracted using the QIAamp DNA Mini Kit Organization Protocol (Qiagen, Hilden, Germany). The extracted DNA was purified using AMPure XP kit (Beckman Coulter, Inc., CA, USA) and quantified using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). Combine the extracts (200 µL in total) and extract 5 µL of the mixture for PCR analysis.
PCR amplification of Helicobacter DNA
Next, in order to evaluate the sensitivity of the PCR system for the helicobacter 16S rRNA gene, the following studies were performed. We harvested H.pylori, H.hepaticus, and H.bilis and extracted and quantified bacterial DNA. We then used the amount of 100 ng bacterial DNA and their serial dilutions as a template for the PCR. We prepared five sets of primers: Helicobacter universal, H.pylori, H.hepaticus, and H.bilis. Beginning, all DNA samples were amplified to detect the presence of Helicobacter genus DNA.The DNA extracts were amplified in GeneAmp 2700 Thermocycler (Applied Biosystems, Foster City, CA, USA) and detected by semi nested PCR targeting 16S rDNA of Helicobacter pylori as described above (Supplementary Table 1).
Subsequently, the samples positive by Helicobacter genus-specific PCR were analyzed to determine the presence of H.pylori, H.hepaticus, and H.bilis. In order to confirm the specificity of the primers, these primers were used to amplify DNA purified from H.pylori, H.hepaticus, and H.bilis.Using Helicobacter genus-specific PCR-positive samples, these primers encoded by H.pylori 16S rRNA, H.hepaticus 16S rRNA and H.bilis 16S rRNA were analyzed (Supplementary Table 1).The following conditions were used for each amplification: 94℃/1 min, 53℃/1.5 min, and 72℃/1 min for 40 cycles for the H.pylori 16S rRNA gene; 94℃/1 min, 60℃/2 min, and 72℃/2 min for 40 cycles for the H.hepaticus 16S rRNA gene; and 94℃/1 min, 55℃/1.5 min, and 72℃/2.5 min for 40 cycles for the H.bilis 16S rRNA gene.
Five microliters of the PCR products were electrophoretically separated in a 1% agarose gel containing ethidium bromide and visualized with UV light. Distilled water was used for DNA extraction and PCR solution preparation.When the extracted DNA sample was replaced by distilled water as a negative control, no bands were obtained.
Amplification of non-Helicobacter bacteria
Enterobacteriaceae, Bacterioides-Prevotella and Enterococcus group-specific PCRs were performed. Except for the annealing temperature, the reaction mixture and amplification conditions for non-Helicobacter PCR detection were the same as the first step of the semi-nested Helicobacter PCR. Except for the annealing temperature, the reaction mixture and amplification conditions of the non- Helicobacter PCR detection are the same as the first step of half-nested Helicobacter PCR.The annealing temperatures and primers used for detection of Enterobacteriaceae, Bacterioides-Prevotella and Enterococcus group were as described before [53] and described (Supplementary Table 1). As positive controls, Escherichia coli (CCUG 17620) was used in all PCR reactions. PCR products were visualized by 1.5% agarose gel electrophoresis.
Measurement of inflammatory cytokines, including IL-6, TNF-α and TGF-β in serum by ELISA
The blood samples were centrifuged at 3000 rpm for 15 minutes. And the supernatant was collected and stored at − 80°C for biochemical analysis. The serum was then used to evaluate the levels of cytokines by commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. Interleukin (IL)-6, tumor necrosis factor (TNF)-α and interleukin (IL)-1β ELISA kits were purchased from Beyotime Biotechnology (Beyotime Biotechnology, Shanghai, China). Subsequently, the IL-6,TNF-α, IL-1β levels in the sample were measured respectively, according to the manufacturer's protocol. A total of 20 µl serum and 80 µl sample diluent were added to the wells of a 96-well plate, gently mixed and incubated at 37°C for 30 min.Wash the plate 5 times with 100µl of washing buffer, add 100µl of enzyme-labeled reagent after 30 seconds, and incubate at 37°C for 30 minutes except for the blank wells.The plate was washed with 100 µl washing buffer 5 times for 30 sec, and 100 µl DAB substrate was added at 37°C for 15 min. A total of 50 µl of stop solution was then added.The absorbance of the resultant yellow colour was determined at 450 nm. These results were calculated by generating a standard curve using a logistic fit of four parameters. All ELISA analysis were carried out using Thermo Scientific Multiskan FC™, and SkanIt 3.1 software was used for data reduction.
CACUL1, ITGBL1 and NRF2 expression vector construction and transfection
Stable transfection of cells to up-regulate CACUL1, ITGBL1 and NRF2 expression was performed as previously described. Briefly, CACUL1(NM_153810.5), ITGBL1(NM_001271754.2) and NRF2(NM_001145412.3) were inserted into the pcDNA3.1 (+) mammalian expression vector (Invitrogen, Carlsbad, CA, USA). The cells were seeded in DMEM media containing 10% fetal bovine serum (GIBCO, Carlsbad, CA, USA). 24 hours later, the cells were transfected with the Transfast™ Transfection Reagent (Promega, Madison, WI, USA). The ratio of DNA to Transfast™ Transfection Reagent was1:2. After 1 month of selection with 400µg/ml G418 (Sigma-Aldrich, St.Louis, MO, USA), the clones were screened for up-regulation of CACUL1, ITGBL1 and NRF2 expression. The pcDNA3.1 (+) expression vector was used as a negative control.
Cell culture
The human GBC cell lines SGC-996, GBC-SD, and HEK293T cells were purchased from the Cell Bank of the Chinese Academic of Sciences (Shanghai,China). The cells used in all experiments were in the logarithmic growth phase. These cells were cultured in DMEM media containing 10% fetal bovine serum (FBS) (GIBCO, Carlsbad, CA, USA) (pH7.4) at 37°C in a humidified atmosphere containing 5% CO2, and the culture medium was changed every 2-3d.
Cell scratch wounding assay and invasion assay
In the wound healing test, cells were inoculated into 12-well plates and cultured at 37°C for 24 h, then wounds were created in a single layer of cells using a 10 µ L pipette tip.The cells were washed to remove cell debris and cultured in DMEM without FBS at 37°C.Images were taken at different time points following wounding. The wound area was measured by photographing two selected fields at the time of wounding (time 0) and 24 h after wounding. Image J software (NIH, Bethesda, MD, USA) was used to calculate the percentage of wound healing.
The cells transfected with ITGBL1 and control were examined for their invasive ability in vitro in BD BioCoat Matrigel chambers (Transwell, BD Biosciences, San Jose, CA, USA). Briefly, 1×105 cells transfected with ITGBL1 and control were seeded into the top chamber with a matrigel coated filter, and 750 µl DMEM containing 5% fetal bovine serum was used as a chemoattractant. After incubating for 24 hours, removed the non-invasive cells on the upper surface of the membrane, fixed the invasive cells on the lower surface of the membrane, and stained with 5% crystal violet (Sigma-Aldrich, St.Louis, MO, USA). The number of cells intruded through the matrigel was standardized to those that threaded the non-matrigel to acquire an index of relative percentage of invasion, indicating the invasive ability of cells to in vitro. Cells were counted in five fields for triplicate at 10×magnification. The cells were photographed through phase-contrast microscopy (Leica Microsystems, Bannockburn, IL).
RNA isolation and RT-PCR
As earlier described, RNA isolation and RT-PCR were performed.Total RNAs were isolated from cultured cell lines and tissue samples using a Qiazol RNeasy Mini kit (QIAGEN, Maryland, USA). The final volume of the RNA was 50µl and stored at − 80°C. Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the purity and integrity of RNA.cDNA was synthesized using PrimeScript™ 1st Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). Gene-specific primers were used to determine the relative amount of mRNA.ITGBL1(NM_001271754.2), F: 5'- GGCTGGTATGGGAAGAAGTGT-3'; R: 5'-CGATCTCCTGGAGGATAGCA-3'. ITGA9(NM_002207.3): F:5'-GACCGCGATGATGAGTGGAT-3'; R: 5'-CATCAATGGTGGACGGGTGA-3'. CACUL1(NM_153810.5): F:5'- ACATGGGTCGGATTTTCCCC-3'; R: 5'- AGCAGCTGGTGACAGACATC-3'. Three independent PCR tests were performed from each sample.
Western blotting
Total protein of GBC tissue and cells was extracted using a total protein extraction buffer (Beyotime, China) and the protein concentration was measured using a BCA Protein Assay Kit (Pierce Biotechnology, US), according to the manufacturer's instructions. Samples were separated by 10% SDS-PAGE gel electrophoresis and then transferred to nitrocellulose membrane (Millipore, US). Membranes were blocked with 5% (w/v) nonfat milk (BD, US) in Tris-buffered saline (TBS) and incubated with ITGBL1 antibody (dilution 1:1000, Cat # PAB20310, rabbit polyclonal antibody, Abnova, USA),CACUL1 antibody [N1C3] (dilution 1:1000, GTX118514, rabbit polyclonal antibody, GeneTex, USA), NRF2 antibody(A-10) (dilution 1:800, sc-365949; mouse monoclonal antibody, Santa Cruz Biotechnology, USA), ITGA9 antibody (dilution 1:800, PA5-103444, rabbit polyclonal antibody, Thermo Scientific, USA), VEGF-C antibody(E-6) (dilution 1:1000, sc-374628, mouse monoclonal antibody, Santa Cruz Biotechnology, USA), β-actin antibody (C4) (dilution 1:1000, sc-47778, mouse monoclonal antibody, Santa Cruz Biotechnology, USA) antibody diluted in TBS containing 1% (w/v) bovine serum albumin at 4℃ overnight. Bound secondary antibodies were detected by Odessey Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
ITGBL1 luciferase reporter gene constructs and luciferase assay
The region approximately 2.0 kb upstream from the transcriptional start site of the ITGBL1 was cloned into the pGL3 luciferase vectors (Promega, Madison, WI, USA). The mutants of the ITGBL1 promoter reporter constructs were generated using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The ITGBL1 luciferase reporter gene constructs or the mutation constructs were cotransfected with the NRF2 plasmid into cells for 48h; pGL3-basic (Promega, Madison, WI, USA) was used as a negative control, while cells transfected with phRL-TK (Renilla luciferase) (Promega, Madison, WI, USA) were used as an internal control. Firefly luciferase activity was normalized to the corresponding Renilla luciferase activity.
Luciferase reporter assay Approximately 8000 HEK293T cells per well were plated into 96-well plates and were co-transfected with 50 ng of the Firefly luciferase reporter and 5 ng of the pRL-CMV Renilla luciferase reporter using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). After 48 hours of incubation, the double luciferase reporter test (Promega, Madison, WI, USA) was used to quantify the activity of firefly and renilla luciferase. The data are expressed as the relative ratio of the firefly luciferase activity to the renilla luciferase activity.
ITGBL1 shRNA transfection
ITGBL1 shRNA plasmids (psiHIV-U6-shRNA) were obtained from OmicsLink™ shRNA Expression Clone Datasheet from GeneCopoeia, Inc. ITGBL1-shRNA-transduced cells with particles for shRNA targeting ITGBL1 or a scrambled nontarget negative control were used based on the manufacturer’s protocol. The transfection process was described above. After 1 month of selection with 2 µg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA), the clones were screened for downregulation of ITGBL1 expression.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed as described previously. Briefly, cells were fixed with 1% formaldehyde to cross-link the protein and DNA. The chromatin of the samples by ultrasound on ice was sheared into fragments with an average length of about 250 bp. Upon sonication, the samples were rotated in a cold microcentrifuge at the highest speed for 12 minutes.
The samples were precleared using a 50% slurry of protein A/G-Sepharose beads (Sigma-Aldrich Co., Ltd, Poole, Dorset, UK) prepared in immunoprecipitation (IP) buffer for 1h at 4°C. After centrifugation, the supernatant was taken for IP detection. The precleared supernatants were immunoprecipitated overnight at 4°C using 10µg anti-NRF2 antibody (H-300): (sc-13032; rabbit polyclonal, Santa Cruz Biotechnology, USA), and normal rabbit IgG antibodies (the negative control). After 3 hours of incubation at 4°C, the immunoprecipitated DNA/protein complex was combined with protein A/G-Sepharose beads and washed. Proteins were eliminated using proteinase K(200 µg; Promega, Madison, WI, USA) in the presence of 10% SDS by overnight incubation at 37°C. After phenol extraction, the DNA was precipitated and suspended in water as a template for PCR. Amplify unprecipitated (input) genomic DNA as input control. The PCR products were analyzed on a 2% agarose gel stained with ethidium bromide.
Immunohistochemical (IHC) staining and scoring
Gallbladder specimens fixed in 10% buffered formalin (40% formaldehyde, sodium dihydrogen phosphate, disodium hydrogen phosphate anhydrous) were processed after 24h and embedded in paraffin. 4 micrometer-thick sections were cut and stained with hematoxylin–eosin (HE). IHC was performed on paraformaldehyde-fixed paraffin sections. CACUL1 antibody [N1C3] (dilution 1:400, GTX118514, rabbit polyclonal antibody, GeneTex, USA) and ITGBL1 antibody (dilution 1:400, Cat # PAB20310, rabbit polyclonal antibody, Abnova, USA) was used in IHC with streptavidin peroxidase-conjugated method. Briefly, deparaffinized sections of formalin-fixed tissues were microwaved (5 min), incubated for 15 min in 1 mM citrate buffer (pH 6.0) at 90°C, treated with 0.3% hydrogen peroxide in methanol for 20 min at room temperature to quench endogenous peroxidase activity, incubated with 1% Bull serum albumin (BSA) in phosphate-buffered saline (PBS) for 30 min to block nonspecific binding sites. The sections were treated and incubated overnight at 4°C with diluted in 1% BSA in PBS primary CACUL1 and ITGBL1 antibodies. The sections were then incubated at room temperature for 1 h with a peroxidase-coupled IgG (Dianova, Hamburg, Germany) and diluted in 10% ABO type human blood serum. Following washes, diaminobenzidine was added (Vector Laboratories, Burlingame, CA, USA) to the slides for 5 min, the slides were washed, and the nuclei were counterstained using Harris hematoxylin (Lerner Laboratories, New Haven, CT, USA). The primary antibodies were replaced with PBS or normal serum from the same species as the negative control. The sections were observed and photographed under a microscope (Carl Zeiss, Germany). Immunoreactivity of CACUL1 and ITGBL1 were evaluated according to both the ratio of positive-staining cells and staining intensity. The percentage of positive tumor cells was graded as per the following criteria: −, < 5%; +, 5–25%; ++, 25–50%; +++, > 50%. Patients with different CACUL1 and ITGBL1 expressions in GBC tissues were divided into the low-expression group (− or +) and the high-expression group ( + + or +++).
Microvascular and lymphatic microvessel density was assessed. IHC stained with CD34 antibody (1:500; sc-74499, mouse monoclonal antibody, Santa Cruz, USA) and D2-40 antibody(1:400, sc-166906, mouse monoclonal antibody, Santa Cruz, USA) to analyze the microvascular and lymphatic microvessel density. The endothelial cell or endothelial cell cluster that was separated from adjacent microvascular and lymphatic microvessel, tumor cells, and other connective tissue elements was considered a single, countable microvascular and lymphatic microvessel. Ten microscopic fields (×400) were counted for evaluation of microvascular and lymphatic microvessel density in each specimen, and we calculated the mean value of ten microscopic fields for the analyses.
In vivo Assays for Tumor Growth and Metastasis
The human GBC cell line SGC-996 (1×107) transfected with ITGBL1, siITGBL1 and Control were implanted subcutaneously into the flank of nude mice (male BALB/c nu/nu, 4–6 weeks) (Institute of Materia Medica, CAS, Shanghai, China). Tumor growth was monitored with tumor volume, which was calculated as described: V (mm3) = width2 (mm2) × length (mm)/2. The mice were sacrificed 8 weeks later, and the lungs and tumor nodes were removed. Each lung tissue block was sliced serially and stained with HE. Two pathologists independently calculated and assessed the incidence and classification of lung metastasis.
These sections were made for every tissue block of the tumor nodes and IHC stained with CD34 antibody (1:500; sc-74499, mouse monoclonal antibody, Santa Cruz, USA) and D2-40 antibody (1:400, sc-166906, mouse monoclonal antibody, Santa Cruz, USA) to analysis the microvessel density of tumor nodes. The anesthetic and surgical procedures used, and of peri-operative care were very careful and in line with the ethic standard, that animals did not suffer unnecessarily at any stage of an experiment, whether acute or chronic. These methods were approved by the Animal Care and Use Committee of Xi'an Jiaotong University.
Statistical analysis
All statistical analyses were done using SPSS 19.0 for windows (IBM Corporation, Armonk, NY, USA) and GraphPad Prism 5 software (San Diego, CA, USA). The Chi-square test and Fisher's exact probability method were used to analyze the correlation between CACUL1 and ITGBL1 expression and clinicopathological parameters in GBC patients. The survival curve was evaluated by Kaplan-Meier method, and the log-rank test was used for analysis. Cox proportional hazards regression model was used to conduct univariate and multivariate analysis to identify the factors that have a significant influence on survival. P < 0.05 was considered statistically significant.