We obtained paired CRC tumor samples (bulk samples) and adjacent non-tumor colorectal tissues from 120 patients who had undergone gastrointestinal surgery between 2008 and 2013 at the First Affiliated Hospital of Zhengzhou University, China. All specimens were immediately frozen in liquid nitrogen and stored at 80 °C until total RNA extraction. Written informed consent was obtained from all patients. No patient received chemotherapy or radiotherapy before surgery. The follow-up periods ranged from 2 months to 5 years, with a mean of 3 years. Our study was approved by the Research Ethics Committee of Zhengzhou University.
Cell lines and cultivation
Human colorectal cancer cell lines including SW480, HCT116, HT29, SW620, and LOVO, were obtained from the Key Laboratory of Cancer Prevention and Intervention, Cancer Institute, The Affiliated Hospital, Zhengzhou University School of Medicine, Zhengzhou, China. The SW480, SW620, and LOVO cell lines were cultured in L-15 (with 10 % FBS and 1 % streptomycin/penicillin); HCT-116 cell lines were cultured in McCoy’s 5A (with 10 % FBS and 1 % streptomycin/penicillin); and HT-29 cell lines were cultured in RPMI-1640 (with 10 % FBS and 1 % streptomycin/penicillin). Normal human colorectal cells were purchased from the American Type Culture Collection and cultured in RPMI1640 supplemented with 10 % FBS and 2 mM l-Glutamine (Gibco). All cell lines were maintained at 37 °C and 5 % CO2 in an incubator, and passaged with 0.25 % trypsin (Sigma, St Louis, MO, USA) in 0.2 mol/l phosphate-buffered saline (PBS; Sigma). The study was approved by the ethics committee of the Cancer Institute, The Affiliated Hospital, Zhengzhou University School of Medicine, Zhengzhou, China.
Real-time quantitative PCR
Total RNA was extracted from colorectal tumor samples and CRC cell lines using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. Complementary DNA was synthesized from total RNA with the Revert Aid™ First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The primer sequences were as follows: TUG1 forward primer: 5c-TAGCAGTTCCCCAATCCTTG-3′, reverse primer: 5′-CACAAATTCCCATCATTCCC-3′; GAPDH forward primer: 5′-CGCTCTCTGCTCCTCCTGTTC-3′, GAPDH reverse primer: 5′-ATCCGTTGACTCCGACC-TTCAC-3′. The PCR was performed in a total reaction volume of 20 ml and was completed in the ABI PRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA, USA). GAPDH was used as an internal control. The PCR cycling parameters were: one denaturation step of 10 min at 95 °C; 40 cycles, with one cycle consisting of 15 s at 95 °C, 20 s at 55 °C, and 30 s at 70 °C. The median in each triplicate was used to calculate the relative TUG1 expression level using the comparative DCt method (value of 2−DCt(TUG1-GAPDH)). Expression fold changes were calculated using 2−DDCt methods.
Protein isolation and western blotting
For the protein expression analyses, standard western blot assay was carried out. Cultured or transfected cells were washed twice with cold phosphate-buffered saline (PBS) and were lysed with iced RIPA buffer containing 1 % PMSF (KeyGen, Nanjin, China). After total protein detection using a BCA kit (Beyotime, Shanghai, China), protein lysates were separated on 10 % SDS polyacrylamide gel, transferred to PVDF membranes, and blocked in 0.1 % Tween 20 and 5 % skim milk protein in Tris Buffer Saline at room temperature for 2 h. Target proteins were probed with rabbit anti-HDAC1 antibody (1:800; Proteintech Corporation, USA), rabbit anti-HDAC2 antibody and rabbit anti-HDAC3 monoclonal antibody (1:1000; Bioworld Corporation, USA), and mouse anti-E-cadherin, N-cadherin, fibronectin, vimentin (1:1000; Abcam, USA), and rabbit anti-β-Actin antibody (1:2000; acted as an internal control, Zhongshan, Inc., Beijing, China) overnight at 4 °C. The membranes were washed twice and visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. Tagged proteins were detected by enhanced chemiluminescence (KeyGen, Nanjing, China).
Plasmid and lentivirus vector
Ectopic expression of TUG1 in cells was realized through pcDNA-TUG1 transfection. The TUG1 sequence was synthesized and subcloned into the pCDNA3.1 (Invitrogen, Shanghai, China) vector. The empty pcDNA3.1 vector was used as a control. To assess the effect of TUG1 on liver metastasis in vivo, TUG1 was overexpressed by SW480 transfecting with Lenti-GIII-CMV-Human-TUG1 Lentivirus [Applied Biological Materials (ABM) Inc, Canada]. Following this step, the expression levels of TUG1 were detected by quantity real-time PCR.
Plasmid vector (pcDNA-TUG1 and pCDNA3.1) or lentivirus vector (Lenti-GIII-CMV-TUG1 and Lenti-GIII-CMV) were prepared using DNA Midiprep or Midiprep kits (Qiagen, Hilden, Germany), and were transfected into SW480 cells. Two individual siRNAs (TUD1-siRNA and HDAC1-siRNA) and negative control siRNA (silencer negative control siRNA) were purchased from Ambion. siRNA oligonucleotides (10 nmol/L) in Opti-MEM (Invitrogen) were transfected into LOVO cells or SW480 cells, respectively, using Lipofectamine RNAiMAX (Invitrogen) and following the manufacturer’s protocol. Forty-eight hours post-transfection, TUD1 and HDAC1 expression levels were measured, and/or cell metastasis ability was assessed. Target sequences for siRNAs were as follows: TUG1-siRNA1 (sense 5′-GGGAUAUAGCCAGAGAACAAUUCU-A-3′, antisense 5′-UAGAAUUGUUCUCUGGCUAUAUCCC-3′) and HDAC1-siRNA, sense 5′-CCAAGTACCACAGCGATGAC-3′, and antisense 5′-TGGACAGTCCTCACCAACG-3′.
Plate clone formation assay
Each well in a 6-well culture plate was seeded with 102 cells and each group contained three wells. The cells were incubated at 37 °C with 5 % CO2 for 2–3 weeks and the medium was replaced every 3 days. The incubation was stopped when colony formation was visible and the cells were then washed twice with PBS and stained with Giemsa solution for 20 min. The number of colonies containing 50 cells was counted under a microscope using the formula: plate clone formation efficiency = (number of colonies/number of cells inoculated) × 100 %.
Wound healing assay
SW480pcDNA-TUG1, LOVOsi-TUG1 and their corresponding control cells were separately seeded into 6-well plates, and were cultured until they were nearly 80 % confluent. Artificial wounds were created by scraping the monolayers with a sterile 10 ml tip, and the cells were washed with PBS several times to remove floating cells. Representative images of cells migrating into the wounds were captured after 0 and 24 h under a microscope (200×).
Cell migration assays
Cell migration assays were performed using 24-well Transwell plates (8.0-μm pore membranes; Costar). About 1 × 104 cells (SW480pcDNA-TUG1 or LOVOsi-TUG1) were loaded into the upper chambers. The lower chambers were filled with a medium (plus 1 % FBS) in the absence (DMSO 0.2 %) or presence of SecinH3 (10, 20, or 40 mM). The Transwell plates were then incubated in a 37 °C, 5 % CO2 incubator for 48 h. After cleaning the cells from the upper side of the polycarbonate membrane and following hematoxylin–eosin staining, the polycarbonate membrane was cut and placed on a microscope slide, cover slipped, and examined under the microscope. The migrated cell numbers and percentages were then counted.
Matrigel invasion assay
The Matrigel invasion assay was done using the BD Biocoat Matrigel Invasion Chamber (pore size: 8 mm, 24-well; BD Biosciences, San Jose, CA, USA) following the manufacturer’s protocol. Cells (5 × 104) were plated in the upper chamber in a serum-free medium. The bottom chamber contained a medium with 10 % FBS. After the 48 h incubation, the bottom of the chamber insert was stained with Calcein AM (Invitrogen). The cells that had invaded through the membrane to the lower surface were evaluated in a fluorescence plate reader at excitation/emission.
Experimental in vivo liver metastasis model
Female athymic BALB/c nude mice (aged 6 weeks) were purchased from the Shanghai Laboratory Animal Center Co. Ltd. (Shanghai, China) and maintained in a pathogen-free animal facility at the Laboratory Animal Research Centre of Zhengzhou University. For liver metastatic capacity, the spleen of BALB/c nude mice was injected with 1 × 106 cells per mouse. Briefly, BALB/c nude mice were anesthetized by i.p. injection of Pelltobarbitalum Natricum, and 1 × 106 SW480pcDNA3.1 or SW480pcDNA-TUG1 tumor cells in 25 ml were injected into the exteriorized spleen using an insulin syringe and following abdominal incision. Five minutes after injection, spleen blood vessels were ligated, and the spleen was removed. Finally, the abdominal wound was closed with staples. After 5 weeks, mice were sacrificed and their livers were removed and tumor nodules were numbered.
Statistical analysis was performed using GraphPad Prism 5.01 software. Statistical tests for data analysis included the log-rank test, the Chi square test. Multivariate statistical analysis was performed using a Cox regression model. The quantitative data were presented as the mean ± standard deviations (SD). Differences were considered to be statistically significant at values of P < 0.05.