The long non-coding RNA TUG1 indicates a poor prognosis for colorectal cancer and promotes metastasis by affecting epithelial-mesenchymal transition

Clinical samples

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.

Cell transfection

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

SW480^pcDNA-TUG1, LOVO^si-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 (SW480^pcDNA-TUG1 or LOVO^si-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 SW480^pcDNA3.1 or SW480^pcDNA-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

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.

Upregulation of TUG1 is correlated with CRC progression

To investigate the clinical relevance of abnormal TUG1 expression in CRC, we collected 120 pre-frozen primary CRC tissue samples (Table 1), and determined TUG1 expression levels in all of these samples using quantitative real-time PCR. By data statistics and analysis, we observed that TUG1 expression was significantly elevated in tumor tissues compared with para-tumor tissue (Fig. 1a), and univariate analysis revealed a close association between high TUG1 expression and patient survival (Fig. 1b). These data suggest that TUG1 upregulation is strongly associated with the clinical progression of human CRC.

Characteristics	All cases	TUG1 expression levels
		Tumor high expression	Tumor low expression	P value
Age				0.512
<60	61	38	23
≥60	59	33	26
Gender				0.761
Male	80	60	20
Female	40	29	11
Grade				0.027*
Well and moderately	86	14	72
Poorly differentiated	34	20	14
Depth of tumor				0.034*
m, sm, mp	30	27	3
ss, se, si	90	65	25
Lymph node-metastasis				0.013*
Negative	70	23	47
Positive	50	35	15
Liver metastasis
Negative	95	25	70	0.01*
Positive	25	18	7
Dukes’ stage
A and B	69	50	19	0.234
C and D	51	36	15

Tumor high expression, the relative expression level of TUG1 is fivefold than para-carcinoma tissue. Tumor low expression, the relative expression level of TUG1 is less than fivefold than para-carcinoma tissue

*Difference is statistically significant

Possible negative regulating TUG1 expression by histone deacetylase

Next, we attempted to investigate the possible mechanism underlying aberrant TUG1 expression in CRC. The five representative CRC lines of SW480, HCT116, HT29, SW620, and LOVO were used for following analysis. We observed that TUG1 expression was significantly elevated in all CRC lines compared to normal colorectal cells (Fig. 2a). Interestingly, TUG1 expression was found to be 4–6 times higher than in corresponding cell lines (marked as control with no TSA treatment in presence of TSA, an inhibitor for histone deacetylase (Fig. 2b). We then evaluated HDAC1, HDAC2, and HDAC3 expression in these CRC lines in comparison to control and we found that HDAC1 expression was upregulated, while the expression levels of HDAC2 and HDAC3 protein remained unchanged in CRC cells (Fig. 2c). These data suggested that TUG1 expression was possibly regulated by abnormal expression HDAC1 expression. Thus, the CRC lines were used to create sub-lines with HDAC1 stably knocked down by shRNA focusing on analysis of TUG1 expression. From the result, we observed that HDAC1 silencing induced promotion (3.4–4.0 times the level of the si-control) of TUG1 expression (Fig. 2d).

TUG1 promotes the aggressiveness of CRC cells in vitro

Considering that TUG1 has a strongly correlation with CRC pathological courses, we transfected the SW480 cell line with pcDNA-TUG1 and detected the effect of overexpressed TUG1 on CRC cell metastasis capacity. The results showed that TUG1 overexpression induced an increase in colony formation (Fig. 3a) and caused a significant facilitation of cell migration in a wound-healing assay (Fig. 3b). We also utilized the classical Transwell assay to assess the contribution of TUG1 to cell migration and invasion. As shown in Fig. 3c and d, overexpressed TUG1 increased migration of SW480 cells and enhanced cell invasion ability.

Knock-down TUG1 suppresses CRC cell metastasis in vitro

Another CRC cell line- LOVO was used to establish cell subline with TUG1 stably knocked down by TUG1-shRNA transfection with the purpose of evaluating CRC cell metastasis ability. The results showed that, compared with the si-control, TUG1 silencing reduced the number of colony formations (Fig. 4a). TUG1 silencing also promotes cell migration showed by a wound-healing assay (Fig. 4a). Additionally, by classical Transwell assay, we observed that TUG1-shRNA interference caused significant suppression in ability of cell migration and invasion as shown in Fig. 4c and d.

Inhibition of mice CRC progression by enhancing tumor-expressing TUG1

Liver metastasis of colon cancer is a common clinical phenomenon. Considering the identification of the key regulator role of TUG1 on CRC cell metastasis in vitro, we next established an in vivo experimental liver metastasis model by injecting human SW480 CRC cells into the spleens of BALB/c nude mice and followed their ability to invade-via the portal vein-the liver to form metastases and then investigated whether TUG1 plays an important role in the liver metastasis of CRC. To define the relationship between TUG1 and CRC liver metastasis in vivo, six, 7-week-old female BALB/c nude mice in each group were injected with SW480^pcDNA-TUG1 or SW480^pcDNA cells into the spleen before splenectomy. After 5 weeks, the mice were sacrificed, and the metastatic tumor nodules that formed in the liver were numbered. The data showed that metastatic tumor nodules were more frequently found in the livers of the SW480^pcDNA-TUG1 group than in the SW480^pcDNA group (Fig. 5).

TUG1 facilitates epithelial-mesenchymal transition (EMT) in CRC cell lines

EMT is an important factor in cell invasion. Thus, we next determined whether EMT markers were altered in our model. The expression of E-cadherin, N-cadherin, vimentin, and Fibronectin protein level was analyzed by Western blot. By data, we found that the expression of N-cadherin, vimentin and Fibronectin was increased while E-cadherin expression was decreased, in SW480-TUG1 cells (Fig. 6a), whereas the opposite results were obtained when TUG1 was knocked down in LOVO cells (Fig. 6b).