Patients and tissue specimens
A total of 236 CRC patient samples were included at the time of diagnosis from patients treated at departments of general surgery, Zhejiang Provincial People’s Hospital. Informed consent was obtained from all individual participants included in the study and the research was carried out in accordance with the World Medical Association Declaration of Helsinki. All patient-derived specimens were collected and archived under protocols approved by the institutional review boards of Zhejiang Provincial People’s Hospital. Procedures were carried out in accordance with approved guidelines. Cancer tissues and their paired adjacent normal mucosa were collected immediately after surgical resection, then frozen in liquid nitrogen for subsequent RNA extraction or formalin-fixed and paraffin-embedded for immunohistochemical staining.
Quantitative PCR (qPCR)
The qPCR assays were performed as described previously [13-15]. The primers used for qPCR were: AGGF1, sense 5′-GGA CTA GTT TGC GAA AAC ATG GGT AGT G-3′ and antisense 5′-CCC AAG CTT TGC CTG CAA TGG TCT TTA TC-3′; GAPDH, sense 5′-GGG AAG GTG AAG GTC GGA GT-3′ and antisense 5′-GGG GTC ATT GAT GCRC AAC A-3′.
Western blot (WB) analysis
The WB assays were performed as described previously [13-15]. Primary antibodies specific to AGGF1 (1:500, # ab203680) and GAPDH (1:1000, # ab8245) were purchased from Abcam (Cambridge, UK).
Tissue microarray (TMA) construction and immunohistochemical (IHC) staining
TMA construction was undertaken as reported previously [15]. The expression of AGGF1 in the TMA was tested using standard IHC staining methods. The staining intensity for AGGF1 was scored as follows: 0 (negative), 1 (weak), 2 (moderate), 3 (strong); and staining extent, based on the percentage of positively stained cells, was scored as follows: 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), 4 (76%–100%). The sum of intensity and extent score were used as the final staining score: 0–2, negative expression; 3–4, weak expression; and 5–7, strong expression [16, 17]. The corresponding primary antibodies were: AGGF1 (1:100, Abcam, Cambridge, UK).
Cell culture and transfection
Human CRC cell lines (SW480, SW620, HT-29, RKO, HCT-8, HCT-116, and LoVo) and a normal colon epithelial cell line (FHC) were purchased from the American Type Culture Collection. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, USA) supplemented with 10% fetal bovine serum, under a humidified atmosphere containing 5% CO2 at 37 °C.
For gene expression manipulation studies, both small interfering RNA (siRNA) specially targeting AGGF1 and pcDNA3.1-AGGF1 plasmid and their control sequences were obtained from Obio Technology Co., Ltd (Shanghai, China). CRC cells were transfected using Lipofectamine 2000 following the manufacturer’s instructions.
Colony formation assays
Colony formation assays were used to evaluate the colony formation abilities of CRC cells and colony formation was determined by preparing single cell suspension solutions and seeding in 6-well plate with 800 cells/well. After a 14-day incubation, the cell colonies were fixed in methyl alcohol for 15 min and dyed with crystal violet for 15 min. Colonies were then counted, and the plates were photographed.
Wound healing assays
Transfected cells were trypsinized and seeded into 6-well plates (1.0×105 cells/well). Upon reaching the exponential growth phase, cells were wounded with a sterile pipette tip and then washed with PBS. Images were captured at 0, 12, 24, and 36 h intervals, and wound widths were quantified and compared with baseline values. Experiments were carried out in triplicate.
Transwell assays
The transwell 24-well Boyden chamber (Corning, USA) with an 8.0 μm pore size polycarbonate membrane was used for the cell migration (without Matrigel) and invasion (with Matrigel) assays according to the manufacturer’s protocol. Briefly, each group of cells (5×104/chamber) was plated in the upper chambers in 200 µl serum-free media for 36 h, while the bottom chambers contained 600 µl media supplemented with 10% FBS as a chemoattractant. Cells that migrated and invaded to the reverse side of chamber inserts were fixed by methyl alcohol and stained with 0.1% crystal violet.
Animal assays
To further investigate the metastatic effect of AGGF1 in vivo, we utilized a CRC metastasis model in four-week-old male, specific pathogen-free BALB/C nude mice, which were purchased from Shanghai Research Center for Model Organisms and housed under pathogen-free conditions in the animal experiment center of Zhejiang Provincial People’s Hospital. A total of 1×106 (200 μl) HCT-116/Lv-AGGF1 and RKO/si-AGGF1 as well as their control cells were transfected with lenti-LUC virus and injected into the tail veins of grouped nude mice (n=5), respectively. At weekly intervals, after ether inhalation, mice were subjected to i.p. injection with D-luciferin (150 mg/kg), then imaged 10 min later using the IVIS Illumina System (Caliper Life Sciences). After six weeks, the mice were euthanized by cervical dislocation. The lung and liver tissue samples were obtained, weighed, and embedded in paraffin. The in vivo assay using nude mice was approved by the Institutional Animal Care and Use Committee of Zhejiang Provincial People’s Hospital. All animal protocols were approved by Zhejiang Provincial People’s Hospital Animal Care.
Statistical analysis
The Student’s t-test was used to determine the differences in AGGF1 mRNA expression between CRC tissues and adjacent normal mucosa. The statistical significance of differences between AGGF1 expression and clinicopathological variables was estimated by the χ2 test or Fisher’s exact test. Survival curves were calculated by the Kaplan–Meier method with the log-rank test. The hazard ratio with a 95% confidence interval in Cox proportional hazards regressions were applied to estimate the hazard risk of individual factors for overall survival (OS) and disease-free survival (DFS). p < 0.05 with a two-sided test was considered to be statistically significant. All statistical analyses were carried out using the SPSS 22.0 statistical software package (SPSS, Chicago, IL, USA).