Immunohistochemistry analysis on tissue microarrays
Tissue microarrays (TMA) were constructed using formalin-fixed, paraffin-embedded tissue samples. Tumor samples were collected from 82 patients with colorectal cancer and preserved at Zhongshan Hospital, Fudan University, Shanghai, China. No patient received any other therapy before surgery. The tumor stage was determined according to the 2010 American Joint Committee on Cancer and International Union against cancer tumor-node-metastasis (TNM) classification system. All tumor samples were acquired at the time of operation, fixed with paraformaldehyde (4 %). Immunohistochemical stains were performed as previously described [17]. Overall survival (OS) was defined as the intervals between the dates of surgery and death. Patients without death were censored at the last follow-up. Detailed clinicopathological characteristics of the patients are given in Table 1. Ethical approval was obtained from the research ethics committee of Zhongshan Hospital, and written informed consent was obtained from each patient.
Table 1 Correlation between FGL2 and clinicopathological characteristics
Immunohistochemistry was conducted with the specific primary antihuman antibodies against FGL2 (1:50, Cell Signaling Technology). FGL2 staining in TMAs was evaluated at 200× magnification using light microscopy by two investigators blinded to the clinicopathologic data of the patients. For the expression intensity of FGL2, the integrated absorbance and the area in each 1-mm-diameter cylinder were measured using Image-Pro Plus version 6.0 software (Media Cybernetics, Inc.). A uniform setting of color segmentation was loaded for counting the integrated absorbance of all the pictures, and the mean FGL2 density was calculated as the product of the integrated absorbance/total area.
Cell culture
Three CRC cell lines (HT29, SW620, and LOVO) were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in our study. The cells were cultured as recommended by the manufacturer in DMEM supplemented with 10 % fetal bovine serum.
Gene constructs, lentivirus production, and transfection
The FGL2-RNA interference (RNAi) lentiviral vector was constructed by GeneChem Co., Ltd (Shanghai, China). Three double-stranded oligonucleotides specifically targeted to FGL2 mRNA were annealed and inserted into the shRNA expression vector pGCSIL-GFP. The cDNA encoding FGL2 was amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pGC-FU-GFP vector. The lentivirus was generated and harvested as described previously [18].
Quantitative real-time PCR
Real-time PCR analyses were performed as previously described [19]. In brief, the cells were harvested and total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Total RNA was reverse transcribed with RevertAid™ first-strand cDNA synthesis kit (Fermentas). FGL2 mRNA levels were determined by qPCR using SYBR Premix Ex Taq (TaKaRa, Dalian, China) and normalized to GAPDH.
Western blot
Western blot analysis was performed as previously reported [20]. Briefly, total cell lysates were prepared, and proteins were separated by SDS-PAGE, followed by transfer to polyvinylidene difluoride membrane. The membranes were washed, blocked, and incubated with the specific primary antihuman antibodies against FGL2 (1:1,000, Cell Signaling Technology) or GAPDH (1:5,000, Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Proteins were detected by enhanced chemiluminescence assay (Pierce-Thermo Scientific).
CCK8 assay
CCK8 assay was performed to determine the effect of FGL2 on cell proliferation. In brief, 1 × 103 cells were seeded in 96-well culture plates. At the time of 24, 48, 72, and 96 h, the cells were incubated with CCK8 reagent for 2 h at 37 °C. The staining intensity in the medium was measured by the absorbance at 450 nm.
Immunocytochemistry
Cells were grown on glass coverslips. After an attachment period of 24 h, cells were fixed in 4 % formaldehyde at room temperature for 30 min and permeabilized with 0.1 % Triton-X in PBS for 10 min and blocked with 10 % goat serum in PBS for 1 h. Cells were then blocked for nonspecific binding with 5 % milk in PBS and Tween-20 (PBST) overnight and incubated with E-Cad or N-Cad antibody at 37 °C for 1 h and then incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:500, Invitrogen) at 37 °C for 1 h. Immunofluorescence images were acquired on a fluorescence microscope.
Flow cytometry analysis
Cells plated on 20-cm2 tissue culture flasks were collected and fixed in 70 % cold ethanol for 1 h. Then, the cells were resuspended in a hypotonic propidium iodide (PI) solution containing RNase. Flow cytometry was performed with the use of Coulter epic flow cytometer. DNA histograms were analyzed using Modfit computer program. The percentage of apoptotic cells in the sub-G1 fraction was calculated.
Xenograft model
Mice were manipulated and housed according to the protocols approved by the Shanghai Medical Experimental Animal Care Commission. For the in vivo assays, SW620 cells (1 × 107) stably silencing of FGL2 expression or relative mock cells were suspended in 100-µl serum-free DMEM and subcutaneously injected into the upper flank of each mouse (six per group, male BALB/c-nu/nu, 8 weeks old). Tumor growth was monitored each week. Then, the mice were killed on day 25 after cell injection. At necropsy, tumor was measured for largest (a) and smallest (b) diameters, and the tumor volume was calculated as V = a × b
2/2.
The metastasis assay in vivo was performed as previously described. For intravenous injection, mice were placed in a restrainer, and SW620 cells (1 × 106 cells in 100 μl PBS) were injected through the tail vein using a 1-ml syringe. Then, the mice were killed on day 28 after cell injection. The lungs were excised and embedded with paraffin. Lung metastasis was determined by examining serial sections of every lung tissue block by microscopy.
Statistical analysis
Statistical analysis was performed with SPSS 15.0 for Windows (SPSS, Chicago, IL, USA). Student’s t test, Fisher’s exact test, and χ
2 test were used as appropriate. The optimal cutoff for dichotomizing FGL2 expression data was determined using X-tile 3.6.1 software (Yale University of New Haven) [21]. The cumulative survival rates were performed by the Kaplan–Meier method (log-rank test). Two-tailed P < 0.05 was considered statistically significant.