Long non-coding RNA colon cancer-associated transcript 2 may promote esophageal cancer growth and metastasis by regulating the Wnt signaling pathway

The aim of this study was to investigate how long non-coding (lnc)RNA colon cancer-associated transcript 2 (CCAT2) regulates the proliferation, invasion and metastasis of esophageal cancer cells via the Wnt signaling pathway. The expression of lncRNA CCAT2 was quantified by reverse transcription-quantitative PCR in four esophageal cancer cell lines (Eca-109, EC9706, KYSE150 and TE-1) and normal human esophageal epithelial cells (HEECs). The effect of silencing CCAT2 (si-CCAT2) and inhibiting Wnt signaling (using the inhibitor FH535) on the proliferation, migration and invasion of Eca-109 cells was measured by MTT, wound-healing and Transwell invasion assays. Flow cytometry was used to evaluate apoptosis in si-CCAT2 Eca-109 cells. The expression of β-catenin and proliferating cell nuclear antigen (PCNA) proteins was detected by immunohistochemistry. The pro-apoptotic protein Bax, cyclin D1 and Wnt target proteins, including c-Myc and adenomatous polyposis coli (APC), were detected by western blotting. LncRNA CCAT2 was highly expressed in the four esophageal cancer cell lines compared with the HEEC cells. The expression of CCAT2 was significantly decreased in si-CCAT2 Eca-109 cells. Treatment with si-CCAT2 and FH535 alone or in combination significantly inhibited the proliferation, migration and invasion of Eca-109 cells. The treatments also promoted apoptosis, upregulated the expression of Bax and APC proteins, and downregulated β-catenin, PCNA, cyclin D1 and c-Myc proteins. In summary, lncRNA CCAT2 is upregulated in esophageal cancer cells and the knockdown of lncRNA CCAT2 inhibits their proliferation, migration and invasion via the Wnt signaling pathway.


Introduction
Esophageal cancer, including squamous cell carcinoma and adenocarcinoma, affected half a million people worldwide in 2014, and causes a fatal outcome in the majority of cases (1,2). The incidence rate of esophageal cancer has increased dramatically over the past 40 years (2). Alcohol consumption and tobacco use are major risk factors (3)(4)(5). Untreated gastroesophageal reflux disease usually progresses to Barrett's esophagus, which is 50-100 times more likely to develop into cancer (1). Obesity can also increase the risk of developing esophageal cancer, via low-grade inflammation (2). Due to the lack of serosa in the esophageal wall and extensive lymphatic drainage to intrathorax and intra-abdominal lymph nodes, esophageal cancer can spread rapidly into the neck and thorax (2). Advanced stages of esophageal cancer can also spread hematogenously (2).
LncRNA CCAT2 was found to be associated with ESCC by in silico analysis and subsequent clinical studies (4,19). These studies showed that CCAT2 was highly expressed in cancer tissue and was associated with smoking (4,19). Lymph node metastasis, advanced lymph node metastasis and Myc amplification were also associated with high lncRNA CCAT2 expression (19). Furthermore, the expression of this lncRNA was positively correlated with Myc amplification and progression of cancer (19).
To investigate the molecular mechanism of lncRNA CCAT2 function in esophageal cancer, the expression of lncRNA CCAT2 in esophageal cancer cells and its association with proliferation and metastasis were investigated in the present study. The results from the current study may provide a theoretical basis for new treatment options for esophageal cancer. Grouping. Based on the expression level of lncRNA CCAT2 in the four esophageal cancer cell lines, the Eca-109 cell line was selected for further experiments because it exhibited a higher expression level of lncRNA CCAT2 than the other three cell lines. The cells were divided into 5 groups: i) Control, no treatment; ii) negative control group (si-NC), scramble sequence transfected; iii) lncRNA CCAT2-silenced group (si-CCAT2), transfection with siRNA sequences targeting CCAT2; iv) Wnt pathway inhibitor (FH535) group, 10 µM FH535 treatment (cat. no. HY-15721; MedChemExpress); and v) CCAT2 silencing and inhibitor group (si-CCAT2 + FH535), transfection with siRNA sequence targeting CCAT2 and 10 µM FH535 treatment. The esophageal cancer Eca-109 cells were digested and passaged with 0.25% trypsin (Invitrogen; Thermo Fisher Scientific, Inc.). Cells (2x10 5 ) were inoculated on a 6-well plate. After 24 h, when the cells reached 30-50% confluence, 10 µl/250 µl siRNA was transfected with Lipofectamine ® 2000 Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A further ~24 h later, the medium containing the mixture was preplaced with fresh medium containing 10% FBS. Cell transfection was assessed by reverse transcription-quantitative PCR (RT-qPCR). The sequences of the control and lncRNA CCAT2 siRNAs were as follows: si-CCAT2 sense strand, 5'-GCC UGU AGG AAG AGU CAA ATT-3'; si-CCAT2 antisense strand, 5'-UUU GAC UCU UCC UAC AGG CTT-3'; si-NC sense strand, 5'-UUC UCC GA A CGU GUC ACG UT T-3'; and si-NC antisense strand, 5'-ACG UGA CAC GUU CGG AGA ATT-3'.

Materials and methods
RT-qPCR. Total RNA was extracted from each cell line using the Total RNA Extraction kit (Invitrogen; Thermo Fisher Scientific, Inc.), and RNA was reverse-transcribed into cDNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Inc.). SYBR Green PCR kit (Qiagen, Inc.) and Mastercycler ® Ep Realplex2 (Eppendorf) were used for RT-qPCR. The RT-qPCR was performed using 2 µl cDNA as a template under the following conditions: 95˚C for 10 min, 95˚C for 15 sec and 60˚C for 1 min for 40 cycles. GAPDH was used as the internal reference and the relative expression of lncRNA CCAT2 was calculated by the 2 -ΔΔCq method (20). The following primer pairs were used: CCAT2 forward, 5'-AGA CAG TGC CAG CCA ACC-3' and reverse, 5'-TGC CAA ACC CTT CCC TTA-3'; and GAPDH forward, 5'-AAT GGA CAA CTG GTC GTG GAC-3' and reverse, 5'-CCC TCC AGG GGA TCT GTT TG-3'.
MTT assay. Cells (~100 µl; 1x10 4 cells/ml) were inoculated on a 96-well flat-bottomed culture plate and cultured for 24, 48, 72 and 96 h. MTT (100 µg) was added and incubated at 37˚C for 4 h. The supernatant was dissolved with 200 µl DMSO (Sigma-Aldrich; Merck KGaA). Optical density (OD) of each well was measured at a wavelength of 490 nm using a plate reader (Bio-Rad Laboratories). The relative absorbance of each group was calculated by the following equation: Wound-healing assay. Cells (~1.0 ml; 3x10 5 cells/ml) were plated in each well (6-well plate). When cells reached 100% confluence within 24 h, a 10-µl pipette tip was used to scratch the monolayer of cells. The cells were washed with PBS twice to remove suspended cells and serum-free medium was gently added. The cell-free wound area was photographed with an inverted microscope (Olympus Corporation) and the results were analyzed by ImageJ 1.46r software (National Institutes of Health). The percentage of the wound size at 24 h relative to the 0 h time point of each treatment group was calculated.
Transwell assay. Matrigel mixture (~50 µl) was added to the upper chamber of Transwell inserts (EMD Millipore) and incubated at 37˚C for 30 min. Cells (2x10 5 cells/ml) were resuspended in serum-free DMEM (Gibco; Thermo Fisher Scientific, Inc.). A total of ~100 µl cell suspension was added into the upper compartment of the chamber, and 600 µl of 10% FBS-DMEM culture media was added to the lower chamber. Membranes were collected after 24 h incubation, fixed in 5% glutaraldehyde at 4˚C for 30 min, stained with 0.5% crystal violet at room temperature for 20 min and observed under a light microscope (Olympus Corporation) at x200 magnification. Images of nine random fields were captured for analysis of the number of invading cells.
Flow cytometry. Cells were digested by trypsin and collected by centrifugation at 11,180 x g for 5 min at 4˚C. The cells were washed twice with pre-cooled sterile PBS solution at 4˚C. Cells [~195 µl; 1x10 6 cells in 250 µl 1X binding buffer (Beyotime Institute of Biotechnology)] were incubated with 5 µl of FITC-labeled annexin-V (Beyotime Institute of Biotechnology) for 3 min. Then, 10 µl propidium iodide solution (20 µg/ml) was added and incubated in the dark for 10 min at room temperature. Binding buffer (~400 µl 1X solution) was added prior to analysis using a flow cytometer (Gallios; Beckman Coulter, Inc.) and Cell Quest 5.1 software (BD Biosciences). The cell numbers at different stages were recorded and the ratio of apoptotic cells was defined as the ratio of cells in quadrants 2 and 4 to total cells.
Immunocytochemistry. Cells, cultured for 24 h, were washed with PBS two to three times and fixed with paraformaldehyde for 30 min. PBS was used to wash the cells two to three times prior to adding 0.5% Triton X-100 in PBS and incubated for 20 min. This was followed by two to three PBS washes before 3% H 2 O 2 was added for 15-20 min. Cells were then washed with PBS two to three times and 5% bovine serum albumin (Beyotime Institute of Biotechnology) was added and incubated at 37˚C for 30 min. The primary antibodies, including rabbit anti-human anti-β-catenin (1:200; cat. no. ab6302) or anti-PCNA (1:100; cat. no. ab15497) (both Abcam), were added and incubated at 37˚C for 30 min. After two to three PBS washes, horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000; cat. no. ABIN101988; antibodies-online GmBH) was added at 37˚C for 30 min. Cells were washed with PBS two to three times before streptavidin-biotin complex (Beyotime Institute of Biotechnology) was added and incubated at 37˚C for 30 min. Following three washes with PBS, 3,3'-diaminobenzidine was added, followed by incubation with hematoxylin at room temperature for 2 min and washing with ethanol at increasing concentrations (70% alcohol for 1 min, 80% alcohol for 1 min, 95% alcohol for 2 min and absolute ethanol for 4 min). Observations were performed at x400 magnification under a light microscope (Olympus Corporation) and three to five fields of each section were randomly selected for analysis.
Western blotting. Cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology) and the protein concentration was measured by the bicinchoninic acid (Pierce™ BCA Protein Assay kit; Thermo Fisher Scientific, Inc.). Each protein sample (~40 µg) was separated on a SDS-PAGE 10% gel (Mini-Protean-3 type; Bio-Rad Laboratories) and then transferred to a PVDF membrane (Merck KGaA) for 30 min. The membrane was blocked with 5% non-fat dry milk in TBST solution for 1 h. TBST with 3% bovine serum albumin was used to dilute each of the antibodies, including rabbit anti-human anti-Bax (cat. no. ab53154), anti-APC (ab15270, Abcam), anti-cyclin D1 (cat. no. ab226977), anti-c-Myc (cat. no. ab39688) and β-actin (cat. no. ab8227) (all 1:1,000) polyclonal antibodies, and goat anti-rabbit IgG (1:2,000; cat. no. ab6721) (all Abcam). The membrane  was incubated with primary antibody overnight at 4˚C. Following incubation with horseradish peroxidase-labeled goat anti-rabbit secondar y antibody (1:1,000; cat. no. ABIN101988; Antibodies Online) at room temperature for 1 h, the ECL system (Thermo Fisher Scientific, Inc.) was used to detect the signals. The expression levels were quantified by ImageJ 1.46r software, and β-actin was used as an internal control.
Statistical analysis. Each experiment was repeated at least three times. SPSS 19.0 (IBM Corp.) was used to analyze all the data, which are expressed as the mean ± SD. One-way analysis of variance was used for analysis of data among groups. Least-Significant Difference was used for post hoc analysis. P<0.05 was considered to indicate s statistically significant difference.

Results
Expression of lncRNA CCAT2 is higher in esophageal cancer cell lines. The expression of lncRNA CCAT2 in four esophageal cancer cell lines (Eca-109, EC9706, KYSE150 and TE-1) and one normal esophageal epithelial cell (HEEC) was examined by RT-qPCR. The expression was significantly higher in all esophageal cancer cell lines compared with that in HEEC (P<0.05; Fig. 1A). Subsequently, siRNA was used to silence lncRNA CCAT2 in Eca-109 cells. The RT-qPCR data showed no off-target effect occurring, and the knockdown of lncRNA CCAT2 was successfully performed (Fig. 1B).

Inhibition of lncRNA CCAT2 attenuates proliferation, migration and invasion of esophageal cancer cells.
After 24, 48, 72, and 96 h of culture following transfection with si-CCAT2, proliferation decreased significantly in the si-CCAT2 group compared with the control and si-NC groups (P<0.05; Fig. 2). To further investigate the effect of lncRNA CCAT2 on the metastasis of esophageal cancer cells, the wound-healing and Transwell assays were performed. As shown in Fig. 3, the cell migration and invasion in the si-CCAT2 group were significantly suppressed compared with the control and si-NC groups (P<0.05).

Inhibition of lncRNA CCAT2 promotes apoptosis in esophageal cancer cells.
Flow cytometry was used to analyze apoptosis in esophageal cancer cells, following annexin V staining. The number of apoptotic cells in the si-CCAT2 group was significantly higher compared with the control and si-NC groups (P<0.05; Fig. 4). Similarly, the immunohistochemistry staining showed significantly decreased expression of β-catenin and PCNA in the si-CCAT2 group (P<0.05; Fig. 5). Protein expression, determined by western blotting, showed significant upregulation of Bax and APC in the si-CCAT2 group compared with the control and si-NC groups, whereas cyclin D1 and c-Myc proteins were significantly downregulated (P<0.05; Fig. 6).
LncRNA CCAT2 and Wnt signaling pathway. To further analyze how lncRNA CCAT2 functions in esophageal cancer cells, the effect of the Wnt signaling pathway on the cell proliferation, invasion and metastasis was investigated, using a Wnt signaling inhibitor (FH535). No differences in the effects on apoptosis, proliferation, migration and invasion of Eca-109 cells were observed between the si-CCAT2 + FH535 group and the si-CCAT2 or FH535 groups (Figs. 7-9). Expression of β-catenin, PCNA, Bax, APC, cyclin D1 and c-Myc were also similar among these groups (Figs. 10 and 11).

Discussion
β-catenin can bind T-cell factor (TCF) family DNA-binding proteins and translocate to the nucleus. This complex regulates cellular differentiation and proliferation (21)(22)(23)(24). Wnt signaling molecules can activate the β-catenin/TCF complex and induce gene expression (21)(22)(23). This signaling pathway has been identified to serve a major role in in certain cancer types including highly malignant carcinoma such as esophageal cancer (21,(25)(26)(27). It is believed that this pathway plays a critical role in the maintenance and self-renewal of cancer stem cells, which leads to malignant tumors and poor prognosis (22,25). Wnt signaling is positively correlated with higher ratio of cancer stem cells and advanced clinical stages of esophageal carcinoma (25). Other genes, such as Dapper homolog 2, naked cuticle homolog 2, microRNA (miR)-942, transcription factor Sox17 and miR-141, can modulate the  Wnt signaling pathway and affect tumorigenesis in esophageal cells (22,23,26,28). Human papillomavirus can lead to esophageal cancer via the miR-125b and Wnt/β-catenin signaling pathway (27).
The present study demonstrated that esophageal cancer cell lines had higher expression levels of lncRNA CCAT2 compared with normal esophageal cells. To further explore the function of lncRNA CCAT2, si-CCAT2 was constructed and tested in esophageal cancer Eca-109 cells. The results showed that si-CCAT2 decreased the cell proliferation. Furthermore, the population of apoptotic cells, quantified by annexin V staining, increased significantly following knockdown of lncRNA CCAT2. This downregulation also significantly decreased cell migration and invasion. To elucidate the molecular mechanism of lncRNA CCAT2, a Wnt inhibitor (FH535) was used to study the associated signaling pathway. FH535 and lncRNA CCAT2 equally elicited suppressive effects on proliferation, migration and invasion, suggesting that lncRNA may function via the Wnt pathway.
LncRNA CCAT2 has been demonstrated to be involved in the Wnt pathway in a number of cancer types, and one of its functions is to modulate the Wnt pathway (35)(36)(37)(38). For example, downregulating CCAT2 suppresses the transcriptional activity of Wnt/β-catenin signaling pathway and the combination of siCCAT2 and FH535 synergistically inhibits Wnt signaling in breast cancer (39). The present study demonstrates an association between lncRNA CCAT2 and Wnt signaling pathway in esophageal cancer.
In the present study, only one esophageal cancer cell line (Eca-109) was used to study the effects and mechanism of lncRNA CCAT2. Therefore, a limitation in the present findings is that they may not apply to other esophageal cancer cell lines or different esophageal cancer types. In addition, the present results showed that the expression level of lncRNA CCAT2 was different in the four esophageal cancer cell lines, which was not investigated any further in this study. Furthermore, more detailed studies of the regulatory effect of CCAT2 on the Wnt pathway should be conducted.  In the future, the correlation between the expression level of lncRNA CCAT2 and the proliferation and metastasis of the four esophageal cancer cell lines will be explored. In addition, the hypothesis tested in the present study will be applied in an  in vivo model. These studies will provide more critical insights into the mechanism, for the development of novel treatments for esophageal cancer in the future.
In summary, the present study demonstrated that lncRNA CCAT2 was highly expressed in esophageal cancer cells, and its downregulation led to the inhibition of cell proliferation,