Immunohistochemistry (IHC)
This study used archival tissues obtained from 105 consecutive patients who underwent surgery at the Department of Surgery 1, School of Medicine, University of Occupational and Environmental Health. All of these specimens were properly used after obtaining patients’ written informed consent, and this study was approved by the Ethics Committee of School of Medicine, University of Occupational and Environmental Health.
Each section was incubated in a 1:100 dilution of anti-HABP1 antibody (Monoclonal Anti-C1QBP antibody produced in mouse sc-23885 Sigma-Aldrich, St. Louis, MO) for an hour. The IHC reaction was quantified by multiplying staining intensity by the percentage of positive tumor cells. The staining intensity was graded as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The percentage (0 to 100%) of the extent of reactivity was scored as follows: 0 (no positive tumor cells), 1 (≤ 10%), 2 (10–50%), and 3 (≥ 50%) [28]. Each case was scored independently by two investigators in a blinded manner. Scores ≤ 4 were regarded as negative expression, and the remainder were classified as positive expression.
Cell culture and reagents
We used 2 PDAC cell lines, PANC-1 (American Type Culture Collection, Manassas, VA, USA), and NOR-P1 (RIKEN BRC Cell Bank, Tsukuba, Ibaraki, Japan) which are high expression of HABP1 in our possession. An immortalized cell line derived from human pancreatic duct, HPDE, was a kind gift from Dr. M.S. Tsao (Univ. of Toronto, Canada). PDAC cell lines were maintained in RPMI1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, NY, USA) and 1% streptomycin and penicillin (Life Technologies, Grand Island, NY, USA). HPDE was maintained in HuMedia-KG2 (KURABO, Kyutaro-machi, Chuo-ku, Osaka, Japan), in a 5% CO2 incubator at 37 °C.
siRNA targeting for HABP1
The siRNA targeting for HABP1 (ON-TARGETplus Smart Pool Human HABP1 L-011225-01-0005) and negative control siRNA (ON-TARGETplus Control siRNA Non-Targeting siRNA #1 D-001810-01-05) were purchased from GE healthcare (Chicago, IL, USA). NOR-P1 and PANC-1 were transfected with 100 nM siRNA using DhermaFECT 1 Transfection Reagent (GE healthcare, Chicago, IL, USA) according to manufacturer’s instructions. After 48 hours treatment, the cells were immediately used for further experiments.
Quantitative real-time RT-PCR
Total RNA was isolated from cell lines using RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacture’s protocol. First strand cDNA was synthesized from 1.0 µg of total RNA using SuperScript® VILO cDNA synthesis Kit and Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA). Real-time mRNA expression analysis of HABP1 and a housekeeping gene (GAPDH) for control was performed using TaqMan@ Gene Expression Assays and Step One Plus real-time PCR system (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacture’s instruction. The assay numbers for these genes were as follows: Hs00241825_m1 (HABP1); and Hs02758991_g1 (GAPDH).
The relative quantification was given by the Ct values, determining the reactions for target genes and an internal control gene in all samples.
Cell Proliferation Assay
PDAC cells treated with siRNA targeting for HABP1 and negative control siRNA were incubated for 1, 3, and 5 days and counted using trypan blue staining.
Colony Formation Assay
After treatment with siRNA, PDAC cells were harvested, counted, and the same number of cells in each group were seeded in dishes. Cells were grown for 14 days and colonies were fixed and stained with hematoxylin and eosin. We counted the number of colonies on each dish.
Migration assay
The migratory ability of cells was determined by transwell cell migration assay using cell culture inserts equipped with a filter membrane containing 8 µm pores (BD Biosciences, Franklin Lakes NJ). The lower chamber was filled with RPMI1640 containing 10% FBS. The upper chamber was filled with 2.0 × 104 cells (for PANC-1) or 5.0 × 104 cells (for NOR-P1) in the RPMI1640. After 24 h incubation, the cells remaining on the upper side of the filters were removed. The cells on the bottom surface of the membrane were stained with hematoxylin and eosin and the number of cells that had migrated to the bottom surface of the membrane were counted in five randomly selected microscopic fields in each sample.
Western blot analysis
The cells were harvested and total protein was extracted with PRO-PREP protein extraction solution (iNtRON Biotechnology Inc., Seongnam-si, Gyeonggi-do, Republic of Korea). Equal amount of protein was subjected to 12% Mini-PROTEAN Precast Gel (Bio-Rad Laboratories, Inc., Philadelphia, PA, USA) and transferred on to PVDF membranes (ATTO, Daito-ku, Tokyo, JAPAN). Membranes were blocked for 1 h with 3% BSA (Sigma-Aldrich, St. Louis, MO, USA) in TBST buffer at room temperature, then were incubated with antibodies against HABP1 (Monoclonal Anti-C1QBP antibody produced in mouse sc-23885, Sigma-Aldrich, St. Louis, MO, USA) and ß-Actin (Proteintech Group, Rosemont, IL, USA) for overnight at 4 °C, followed by incubation with secondary antibodies (Proteintech Group, Rosemont, IL, USA) for 1 h at room temperature. The proteins were visualized using an ECL Western Blotting Detection System (GE healthcare, Chicago, IL, USA).
Statistical analysis
Statistical analyses were performed using SPSS statistical software version 25.0 (SPSS, Chicago, Illinois, USA). Chi-square tests, Student’s t test and Mann-Whitney U test were used for group comparison. The Kaplan–Meier survival curves and log rank tests were used for survival analysis. Prognostic factors were evaluated by univariate and multivariate analyses using Cox proportional hazard regression models. A p-value of < 0.05 was considered statistically significant.