Cell culture
The human cell lines (including HCT116 and HEK293T) originated from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in Roswell Park Memorial Institute-1640 medium (RPMI 1640) medium with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37 ℃.
Clinical Samples
CRC specimens were collected with informed consent from 80 cases of surgical patients in Suqian Hospital Affiliated to Xuzhou Medical University and Jiangning Hospital Affiliated to Nanjing Medical University between 2011 and 2014 following the protocols approved by the Ethics Committee of the Affiliated Suqian Hospital of Xuzhou Medical University. The samples had paired samples of adjacent normal CRC tissue. The definitive histological diagnosis of each CRC patient was confirmed after surgery, and no patients received radiotherapy or chemotherapy. All participants signed informed consent forms.
Immunohistochemistry (Ihc) Analysis
Paraffin embedded tumor tissues or peritumor tissues were cut into 4-µm-thick sections. The slide was heated by microwave in 0.01 M citrate buffer (pH = 6.0) for 10 minutes to recover the antigen. Subsequently, the sections were incubated with primary antibodies [anti-TCN1 rabbit antibody (ab202121, Abcam), anti-ITGB4 rabbit antibody (ab182120, Abcam), anti-Ki-67 antibody (ab16667, Abcam), and anti-PCNA antibody (ab92552, Abcam)] in a humidified chamber overnight at 4°C. Horseradish peroxidase (HRP)-labeled secondary antibodies were incubated at room temperature for 1 h. All colorectal cancer sections were examined by two experienced pathologists. TCN1 or ITGB4 staining was independently scored by two pathologists who did not know the clinical data using the H-score system.27 The intensity of immunostaining was 0–3:0, negative; 1. Weak; 2. Medium; 3. Strong. H score was the product of different staining intensities in 0-3 and the percentage of positive cells.
Q-PCR
Total RNA was extracted from snap-frozen CRC tumor tissues and paired noncancerous tissues using TRIzol Reagent (B5704-1, Takara), followed by treatment with DNase I (2212, Takara). After the RNA concentration was determined by spectrophotometer (NanoDrop 2000c, Thermo Scientific), the cDNA was synthesized using PrimeScriptTM RT reagent Kit (RR037A, Takara). Q-PCR was performed using the Light Cycler PCR QC Kit (6746381001, Roche) and a 7300 Real-Time PCR System (LC96, Roche). Human TCN1- and GAPDH-specific primers (TCN1-F, 5’- AGA TCA AAG CAG ATG AAG GCA GTT T -3’ and TCN1-R, 5’- TCC GTG AGC ACT GTA TTC AGA GTT -3’ for amplifying 208 bp; GAPDH-F, 5’-ACG GAT TTG GTC GTA TTG GG-3’ and GAPDH-R, 5’-CGC TCC TGG AAG ATG GTG AT-3’ for amplifying 214 bp) (Genesprit Biotechnology) were used. The relative expression level of the target genes was normalized to that of the GAPDH and calculated by the ΔCT method [ΔCT = (mean CT−mean CT GAPDH)] [28]. Data analysis was performed using GraphPad Prism 8 software.
Survival Analysis
Survival analysis was performed on 80 patients, and the survival curves were drawn according to the Kaplan-Meier method. Patients were divided into TCN1 and ITGB4 high expression group (ΔCt ≤4.25) and low expression group (ΔCt > 4.25) according to the median levels of TCN1 and ITGB4 in cancer tissues. Overall survival (OS) was the time from surgery to patient’s death. Follow-up was for 60 months, although patients who survived beyond 5 years were still followed up.
Generation Of Stable Cell Lines Using Lentivirus Infection
To generate a lentivirus expressing short hairpin RNA (shRNA) targeting human TCN1 (GenBank Accession No. NM_001062.4), the shTCN1 sequences listed in Supplemental Table 1 were designed and synthesized. HEK293T cells were co-transfected with lentiviral expression constructs (4 µg), viral envelope plasmid (pMD2.G, 4 µg), and viral packaging plasmid (psPAX2, 4 µg) using Lipofectamine 2000 (Invitrogen). The empty vector was used as a shRNA control (TCN1-KDC). The expression constructs (TCN1-KD1, TCN1-KD2, and TCN1-KDC) plasmid maps are listed in Supplemental Figure 1A, B. After 72 h of transfection, the virus supernatant was collected and purified by 0.45 µm filters. The mCherry-expressing lentiviruses, including LV-TCN1-KD1, LV-TCN1-KD2, and LV-TCN1-KDC, were then concentrated by ultracentrifugation (20,000 rpm) for 2 h.
The full-length coding sequence (CDS) of human TCN1 was cloned into pLenti-CMV-PuroR-mCherry lentiviral plasmid (CAVR Gene, Zhenjiang, China) to construct the recombinant TCN1-overexpression lentivirus (TCN1-OE). The empty vector was used as a shRNA control (TCN1-OEC). HCT116 cells were infected with LV-TCN1-KD1, LV-TCN1-KD2, LV-TCN1-KDC, LV-TCN1-OEC and LV-TCN1-OE plus 8 µg/ml polybrene. 24 h after lentiviral infection, the cells were selected using puromycin (2 µg/ml, Sigma) prior to use in experiments. The primer sequences for TCN1 cloning are listed in Supplemental Table 1. The TCN1-OE plasmid maps are listed in Supplemental Figure 1C, D.
Cell Apoptosis Detection
An annexin V-fluorescein isothiocyanate (FITC) detection kit (556547; BD Biosciences) was used to detect the apoptosis rate of HCT116 cells. Briefly, the cells were washed twice with cold phosphate-buffered saline (PBS) and then resuspended in 500 µL of binding buffer containing 5 µL Annexin V-FITC antibody and 5 µL Propidium Iodide and incubated for 15 min at room temperature in the dark. Samples were analyzed using flow cytometry (BD FACSCalibur; BD Biosciences).
Transwell Invasion Assays
For cell migration assays, 1 × 105 cells were seeded in the upper chamber of Transwell apparatus (Corning Costa) in serum-free medium, and 10% FBS medium was added in the lower chamber. After 24 h, the cells on the upper surface that did not pass through the 8-µm pore-size polycarbonate filter were removed using a moistened cotton swab; the cells migrating to the lower membrane surface were fixed in 100% methanol for 10 min, stained with 0.4% crystal violet for 15 min, and counted under a microscope (IX-71, Olympus).
Cell Viability
Cell viability was detected at 0 h, 12 h, 24 h, 36 h, 48 h, and 60 h using Cell Counting Kit-8 (CCK-8) reagent (HY-K0301, MedChemExpress). Briefly, the cells were washed twice with PBS. Then, 100 µL RPMI 1640 medium and 10 µL CCK-8 solution were added to each well. The cells were incubated for 1.5 h at 37°C, and the absorption value was detected at 450 nm. All experiments were repeated three times.
Immunofluorescence Staining
HCT116 cells were fixed with 4% formaldehyde for 15 min at room temperature. After washing the cells 3 times in PBS with 0.1% Triton X-100, cells were blocked with 4% BSA for 30 min. Cells were incubated with primary ITGB4 antibody (ab182858, Abcam), FLNA antibody (4762, Cell Signaling Technology), FITC-phalloidin antibody (P5282, Sigma) and PLEC antibody (ab11220, Abcam) for 1 h at room temperature. Following washes with PBS, 0.1% Triton X-100, cells were incubated with FITC/TRITC-conjugated secondary antibodies for 1 h at room temperature. Following washes with PBS, 0.1% Triton X-100, cells were stained with DAPI (Sigma) and visualized by confocal scanning microscopy (710, Zeiss).
Transmission Electron Microscopy Assays
The cells were fixed in 2.5% glutaraldehyde (pH 7.4) at 4 ℃ for 48 h, and then in 0.5% osmium tetroxide for 24 h. After dehydration, the samples were embedded in epoxy resin to make ultrathin sections (70-nm-thick) and examined by transmission electron microscope (Tecnai G2 Spirit Bio TWIN; FEI).
Rna-sequence Analysis
Total RNA was extracted from TCN1-KD1, TCN1-KD2, TCN1-KDC, TCN1-OE and TCN1-OEC HCT116 cell lines. RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 2 µg RNA per sample was used as input material for the RNA sample preparations. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 4000 platform and paired-end 150 bp reads were generated. The genes with a fold change value greater than 2, and a p-value < 0.01 were considered differentially expressed. Relationships of differently expressed genes were determined by GO and GSEA analysis.
Chromatin Immunoprecipitation (Chip) Assays
ChIP assays were performed as described previously [29]. The protein–DNA complexes were immunoprecipitated using anti-TCN1 antibody (ab202121, Abcam), anti-ITGB4 antibody (ab182120, Abcam) or immunoglobulin G (IgG, ab172730, Abcam), which served as a control. ChIP samples were analyzed by quantitative real-time PCR. The primer sequences for Chips are listed in Table S1.
Establishment Of Tumor Xenografts In Nude Mice
All animal experimental procedures were approved by the Animal Care and Use Committee of Nanjing Medical University. Thirty BALB/c nude mice (4 weeks old, male) were randomly divided into five groups (TCN1-KDC, TCN1-KD1, TCN1-KD2, TCN1-OEC and TCN1-OE) and housed at 22°C on a 12/12 h light/dark cycle and freely received standard mouse chow and tap water. A total of 2.5 × 106 cells were inoculated into the mammary fat pads of mice. The growth of the primary tumor was measured with a caliper every week for 6 weeks. The tumor volume was calculated by multiplying the length by the square of the width.
Western Blot Analysis
The cells and cancer tissues were lysed in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Bioteke Company). Protein samples (50 µg) were separated on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes. TBST buffer (100 mM NaCl, 10 mm Tris HCl, and 0.1% Tween 20) containing 5% skim milk was used to block the membrane at room temperature for 1 h. Then, the membrane was incubated with primary antibodies against TCN1 (ab202121, Abcam), ITGB4 (ab182120, Abcam), FLNA (4762, Cell Signaling Technology) and GAPDH (sc‑166574, Santa Cruz Biotechnology) at 4°C overnight. On the second day, an HRP-conjugated goat anti-rabbit IgG secondary antibody (sc‑2004, Santa Cruz Biotechnology, Inc.) or goat anti-mouse IgG HRP binding secondary antibody (sc‑2005, Santa Cruz Biotechnology, Inc.) was incubated with the membrane for 1 h at room temperature. Then, the membrane was rinsed three times with TBST. Western blot analyses were performed with Pierce ECL Western Blotting Substrate (32209, Thermo Fisher Scientific) and a ChemiDoc XRS+ molecular imager (Bio-Rad), and quantitative analysis was performed with ImageJ software.
Statistical analysis
Data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad 8.0 statistical software (GraphPad Software). One way ANOVA was used to compare the differences among the groups. Univariate survival analysis was performed using the Kaplan-Meier method. Survival curves were compared by log-rank test. P<0.05 was defined as statistically significant difference.