Study population and data collection: A total of 96 CRC patients (including 22 with distant metastasis) were recruited from Shengjing Hospital of China Medical University between 2014 and 2015 based on the criteria of the National Comprehensive Cancer Network (NCCN) classification system. Rechecks and follow-ups were performed every three to six months after surgery. Follow-up was terminated in January 2020. The patient follow-up period ranged from 49–70 months (median duration: 60.0 months). The overall survival (OS) and disease-free survival (DFS) periods were determined. Written informed consent was obtained from study participants.
Tumor specimens and adjacent normal colorectal tissues (5 cm from the primary tumor) were collected prior to radiotherapy or chemotherapy. The NCCN guidelines were used to assign the histological types.
Main reagents
Anti-FKBP51 polyclonal antibody (Abcam, Cambridge, UK). Anti-GAPDH monoclonal antibody, horseradish peroxidase-labeled secondary antibody, SP kit and DAB working solution (Zsgb-Bio, Beijing, P.R. China). NF-κB, p-NF-κB, and p-IKKα polyclonal antibodies and IKKα and IκBα monoclonal antibodies (Cell Signaling Technology, MA, US). MMP-2, MMP-9, BAX, and BCL-2 polyclonal antibodies (Proteintech, Wuhan, CN). PrimeScript RT and SYBR Premix Ex Taq II (Takara, Shiga, Japan).
Immunohistochemistry (IHC): Formalin-fixed, paraffin-embedded sections (5 µm) were used for IHC as previously reported[14]. After paraffin removal, dehydration, and antigen repair, endogenous peroxidase activity was inhibited, and nonspecific binding was blocked. Then, the sections were incubated with the FKBP51 antibody (1:800) at 4 ℃ overnight. The secondary antibody and horseradish enzyme-labeled streptavidin were added. DAB working solution was used to develop staining on positive cells. The results were observed and recorded by optical microscopy. According to the IHC scoring system, five random fields of each section were scored at 400× magnification. Image-Pro Plus software 6.0 (Media Cybernetics, Rockville, USA) was used to analyze the expression intensity.
Western blotting: Protein samples were collected from tissues and cells, and the protein concentration was determined using the BCA method. The proteins were separated by 10% SDS–PAGE and then transferred to PVDF membranes. After blocking with 5% skim milk for 3 h, the membranes were incubated at 4°C overnight with the following antibodies: FKBP51 (1:3000), NF-κB (1:3000), p-NF-κB (1:3000), IκB-α (1:3000), p-IκB-α (1:3000), MMP-2 (1:2000), MMP-9 (1:2000), BAX (1:5000), BCL-2 (1:5000) and GAPDH (1:5000). The blots were incubated with horseradish peroxidase-labeled secondary antibody (1:5000) for 2 h at RT. The blots were observed using a Bio–Rad chemiluminometer. The density of each band was measured by ImageJ (v1.8). GAPDH was used as an internal control.
RT–qPCR
Briefly, mRNA was extracted from samples using TRIzol and converted into cDNA by PrimeScript RT. The prepared samples were mixed at the correct dilution with SYBR Premix Ex Taq II and analyzed by real-time PCR (Roche 480; Roche Diagnostics GmbH, Mannheim, Germany) under thermocycling conditions. RNA expression was quantified with GAPDH as an internal reference.
Cell culture
The normal colon cell line NCM460 (NCM) and CRC cell lines SW480 and SW620 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in complete RPMI 1640 culture medium (Gibco) containing 10% fetal calf serum, 2 mM glutamine (Gibco), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C with 5% CO2 in a humidified atmosphere.
Lentivirus transfection: The lentivirus plasmids to silence FKBP51 (target sequence: 5'-GCCTAAATTTGGCATTGAACC-3') or overexpress FKBP51 (GenBank: NM_001145775.3) were synthesized by Beijing Syngentech Co., Ltd, China. Colon cancer cells were plated at a density of 4×105 cells/well in a 6-well plate for 24 h. Next, the cells were cultured in lentivirus-containing medium for 12 h and then in medium containing 10% fetal bovine serum for 24 h. Stable knockdown was ensured by selection with puromycin (2 µg/ml). Transfection efficiency was determined by RT–qPCR. Empty vectors were used to measure the change in FKBP51 levels.
MTS assay
A total of 5×103 cells were seeded in 96-well dishes and cultured for 24, 48, and 72 h. The cells were washed twice with PBS before analysis. Then, 100 µl of MTS working solution (Promega) was added to each well, and the plates were incubated for 1 h. The absorbance was recorded at 490 nm using a 96-well plate reader.
Scratch assay
A total of 1×106 cells were cultured in 6-well dishes overnight. A sterile pipette tip was used to generate a scratch in the center of the well. After washing with PBS, the cells were cultured in serum-free culture medium for 24 h and 48 h. Cell migration was assessed based on photos obtained under an inverted microscope. The cell migration rate was measured as [Distance (0 h)-Distance (24 or 48 h)]/Distance (0 h) × 100%.
Migration and invasion assays
A total of 100 µl of gel working fluid was added in an even layer to the upper chamber and allowed to solidify (this step was omitted for migration assays). CRC cells (1×105) in 200 µl of serum-free culture medium were added to the upper chamber, and 600 µl of culture medium containing 10% FBS was added to the lower chamber. After incubation for 24 h, the migrated cells present on the underside of the transwell membrane were stained with crystal violet and counted under a microscope.
Flow cytometric analysis
The procedure has been previously published [16]. Briefly, 300 µl of SW620 cells in suspension (1×106 cells/ml) were mixed with FITC-Annexin V and PI in a 1.5 ml tube. Apoptosis was examined by flow cytometry (Beckman Coulter, USA), and data were analyzed using CytExpert 2.1 software (Beckman Coulter, USA).
Animal experiments
Twenty-four male BALB/c nude mice (6 weeks old, 20 ± 2 g) were purchased from the Experimental Animal Center of China Medical University (CMU). The animal experiment protocol was approved by the Ethics Committee for Animal Experiments of CMU. A 100 µl suspension of SW620 cells (5×106/ml) was injected into the right lower abdomen of nude mice. The mice were divided into 4 groups (n = 6) according to the FK506 dose [0 (CON), 1, 3, and 5 mg/kg]. FK506 solution (or normal saline in the CON group ) was injected every day after the tumors reached the predetermined size. Tumor size was measured by a Vernier caliper, and tumor volume was calculated as Volume=(length × width2)/2. On the 21st day of FK506 treatment, the mice were sacrificed, and subcutaneous tumor tissues were rapidly resected.
Statistical analysis
The results were analyzed statistically and plotted using GraphPad Prism 8.0 (GraphPad Software Inc., California, USA). The results were shown as the mean ± standard deviation (SD). For comparisons among groups, one-way ANOVA was applied. Statistically significant differences were determined by Student’s t test. The significance of differences in Kaplan–Meier curves for the indicated populations was determined by the log-rank test. The chi-square test was used to identify statistically significant differences in the relationship between FKBP51 expression and the clinicopathologic features of patients. Univariate and multivariate Cox regression analyses of CRC prognosis were performed.