Specimens and cell culture
Serum samples from 100 pancreatic cancer patients who underwent surgical resection at the Fudan University Shanghai Cancer Center (FUSCC) from July 2012 to April 2016 were collected for the detection of serum LRG1 levels. Serum samples from 87 normal human subjects collected within the same period were used as controls. Forty pairs of frozen pancreatic cancer and adjacent normal pancreas tissue samples collected between March 2015 and June 2015 were retracted for tissue RNA or tissue protein extraction. Formalin-fixed, paraffin-embedded (FFPE) tissue specimens, including tissue microarrays (TMAs), were obtained for immunohistochemical (IHC) analysis. The study was approved by the Institutional Review Board of FUSCC, and written informed consent was obtained from the patients.
Human pancreatic cancer cell lines (MIA PaCa-2, PANC-1, BxPC-3, Capan-1, CFPAC-1, SW 1990, and HPAF-II) were purchased from the American Type Culture Collection (ATCC) and cultured at 37°C in a humidified atmosphere containing 5% CO2. A human pancreatic ductal epithelial cell line (HPDE) was a kind gift from Professor Ming-Sound Tsao (University Health Network, Toronto, Canada).
Enzyme-linked Immunosorbent Assay (Elisa)
Serum LRG1 levels were determined using a human LRG assay kit (IBL, Fujioka, Japan) according to the manufacturer's instructions. Each sample was assayed in triplicate. The absorbance at a wavelength of 450 nm was measured with an ELx800 absorbance microplate reader (BioTek Instruments, Winooski, VT, USA), and the serum LRG1 concentration was determined from a standard curve generated using internal control samples.
Rna Isolation And Quantitative Rt-pcr (Qrt-pcr) Analysis
Total RNA was isolated from frozen pancreatic tissues and cultured cells using RNAiso Plus reagent (Takara Bio Inc., Otsu, Shiga, Japan) according to the manufacturer’s instructions. Reverse transcription was conducted using the PrimeScript RT reagent kit (Takara Bio Inc.). qRT-PCR was performed using the QuantiTect SYBR Green RT-PCR kit (QIAGEN, Hilden, NRW, Germany) on a QuantStudio 7 Flex system (Applied Biosystems, Foster City, MA, USA) in triplicate. The following primers were used:
LRG1
forward, 5’-CAGACAGCGACCAAAAAGC-3’
reverse, 5’-GGAACACCTGGCAGTCTTTG-3’
DTX3L
forward, 5’-CCAGGTTATGAGTCCTTTGGCAC-3’
reverse, 5’-TGCAGTTCGCTGTATTCCAGGG-3’
β-actin: forward, 5’-CTACGTCGCCCTGGACTTCGAGC-3’
reverse, 5’-GATGGAGCCGCCGATCCACACGG-3’
Relative LRG1 or DTX3L expression was determined in accordance with the 2−∆∆Ct method using β-actin as the reference gene.
Western Blot Analysis
Total protein extraction from frozen tissues and cultured cells and western blotting were performed as previously described [15]. The primary antibodies used were mouse anti-β-actin (66009-1-Ig; Proteintech Group Inc, Rosemont, IL, USA), rabbit anti-LRG1 (HPA001888; Sigma-Aldrich, St. Louis, MO, USA) and rabbit anti-DTX3L (HPA010570; Sigma-Aldrich). β-Actin was used as a loading control. The immune complexes were detected by chemiluminescence (Immobilon; Millipore, Billerica, MA, USA). The intensity of the protein bands was quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to the β-actin protein levels.
Ihc Assay
FFPE specimens and TMAs were obtained from patients who underwent surgical resection at the FUSCC between March 2010 and April 2016 (last follow-up: July 2017). Briefly, heat-mediated antigen retrieval was performed using sodium citrate buffer (pH 6.0) in a pressure cooker for 10 min, and endogenous peroxidase activity was suppressed by incubation with a 3% solution of H2O2 for 15 min. Slides were incubated with anti-LRG1 (1:200) or anti-DTX3L (1:200) antibodies overnight at 4°C, followed by incubation with horseradish peroxidase polymer-conjugated secondary antibody. Based on staining intensity (H-scores: 0, no staining [no coloration]; 1, weak staining [pale yellow]; 2, moderate staining [yellow]; 3, strong staining [brown]) the samples were divided into two groups (low expression: 0 or 1; high expression: 2 or 3).
Establishment Of Panc-1-lrg1-overexpressing And Mia Paca-2-lrg1-knockdown Cell Lines
shRNA sequences were cloned into the hU6-MCS-Ubiquitin-EGFP-IRES-puromycin vector (GV248; GeneChem, Shanghai, China) according to the manufacturer’s instructions. The scramble shRNA and two LRG1-targeting shRNA sequences were used in this study: scramble shRNA, 5’-TTCTCCGAACGTGTCACGT-3’; shLRG1-A, 5’-AGGCAACAAATTGCAAGTA-3’; shLRG1-B, 5’-ATGTTTCTAGAACTCTGTT-3’. Lentiviral shRNA particles were generated by cotransfection of the lentiviral constructs with psPAX2 and pMD2.G into HEK-293T cells using Lipofectamine 3000 (Thermo Fisher Scientific, Rochester, NY, USA). To establish a stable LRG1-knockdown cell line, MIA PaCa-2 cells were infected by exposure to the lentiviral supernatant and polybrene (2 µg/ml) for 2 days. Infected cells were selected in culture medium containing puromycin (1 µg/ml).
The Flag-tagged coding sequences of human LRG1 were cloned into the BamHI and AgeI sites of the expression vector GV 492 (GeneChem). The preparation of lentiviral overexpression particles and cell infection were carried out as described above, and the empty GV 492 vector was used as a control.
Rna Sequencing
Total RNA was isolated from scrambled-MIA PaCa-2, shLRG1-A-MIA PaCa-2, shLRG1-B-MIA PaCa-2, LRG1-OE-PANC-1, and GV492-OE-PANC-1 cells using RNAiso Plus reagent (Takara Bio Inc.). RNA sequencing was performed using the Illumina HiSeq 4000 sequencing system.
Colony Formation Assay
Cells were seeded at an initial density of 1,000 cells/well in six-well plates and cultured for 2 weeks. Then, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min and stained with 0.1% crystal violet (Sigma-Aldrich) for 20 min. Colonies containing more than 50 cells were counted under a light microscope.
Cell Proliferation Assay
Cells were seeded in 96-well plates at a density of 2×103 cells/well. Cell viability was quantified every 24 h using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay.
Wound Healing Assay
A wound healing assay was performed as previously described [7]. Briefly, when the confluency of the cells was ideal, a scratch wound was made across the center of the well with a pipette tip. The cells were then cultured in medium supplemented with 0.5% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) for 24 h to allow wound closure. The degree of cell migration was analyzed as a percentage of wound area with ImageJ software.
Transwell Migration Assay
Twenty-four-well plates with 8-µm pore filters (Corning Incorporated, Corning, NY, USA) were used for the cell migration assay. A total of 4×104 cells were seeded in the upper chamber of a Transwell plate and cultured in serum-free medium (200 µl). The cells were allowed to migrate into the lower chamber, which was filled with culture medium (800 µl) containing 10% FBS. After 48 h of incubation, the noninvaded cells that remained in the upper chamber were removed with cotton swabs. The migrated cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde (Sigma-Aldrich), stained with 0.1% crystal violet (Sigma-Aldrich), and counted in five random microscopic visual fields.
In vivo tumorigenicity assay
Four- to six-week-old female BALB/c nude mice were purchased from the Shanghai Laboratory Animal Company (SLAC, Shanghai, China). Approximately 3×106 cells (in 50% Matrigel) were subcutaneously implanted into the second mammary fat pads of the mice (n = 5 for each cell line). After 4 weeks, the mice were euthanized, and the tumor specimens were surgically dissected and fixed with 4% paraformaldehyde. Xenograft tumor experiments were performed at the Laboratory Animal Center of Fudan University (Shanghai, China) according to an animal protocol approved by the Committee on the Ethics of Animal Experiments of Fudan University.
Immunofluorescence Assay
Immunofluorescence assays of cultured cells were performed on glass-bottom dishes (Thermo Fisher Scientific). Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with 0.5% Triton X-100 (Sigma-Aldrich), blocked with bovine serum albumin (BSA; Sigma-Aldrich), and incubated with anti-LRG1 or anti-DTX3L overnight at 4°C. The cells were then incubated with fluorochrome-conjugated secondary antibody in the dark and photographed using a Leica TCS SP5 confocal laser scanning microscope. Double immunofluorescent staining of FFPE tissue specimens was performed as previously described [16].
Statistical Analyses
Statistical analyses were carried out with SPSS software (IBM, Chicago, IL, USA). Quantitative data were compared between every two groups using Student’s t test or the paired t test. The Kaplan-Meier method was used to estimate overall survival, and comparisons between curves were performed using the log-rank test. The Cox proportional hazard regression model was used to estimate the hazard ratio with a 95% confidence interval for variables associated with overall survival. Correlation analyses were performed using Spearman’s correlation test. P < 0.05 was used to indicate statistical significance.