CRC tissues and adjacent non-cancerous tissues were collected from 21 patients who had undergone surgical resection of CRC at the Cancer Hospital of Hunan from January to September of 2015. The normal tissue samples were 5 cm from the edge of the tumor and were identified by a pathologist. Prior to the surgical resections, no preoperative treatment had been administered to these patients. All tissue samples were immediately frozen in liquid nitrogen after being surgically resected and then stored at −80°C until required for the analyses. Prior to sample collection, written informed consent was obtained from all patients, and all of the experiments were approved by the Research Ethics Committee of the Shangyao Hospital of TCM.

Human normal colorectal cell line NCM460 and CRC cell lines COLO320, HCT116 and LoVo were purchased from Auragene Bioscience Co. (Changsha, China). NCM460 cells were cultured in McCoy's 5A medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). COLO320, HCT116 and LoVo cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.). All cells were cultured at 37°C in humidified air with 5% CO₂. All media were supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Shanghai, China).

Total RNA was extracted from tissues or cultured cells with TRIzol reagent (Invitrogen, Shanghai, China). For quantitative RT-PCR (qRT-PCR), cDNA was synthesized using random primers and a reverse transcription kit (Takara Biotechnology Co., Ltd., Dalian, China). qRT-PCR analyses were performed using SYBR-Green qPCR Mix (Toyobo Life Science, Osaka, Japan). All protocols were performed according to the manufacturer's instructions. The qRT-PCR assays and data collection were performed using an ABI 7300 instrument. Primers for qRT-PCR were synthesized by Invitrogen (Shanghai, China) and the sequences were as follows: ENST00000547547 sense, 5′-TTTTCTAAGGCACCAACT-3′ and antisense, 5′-CCAAATGCTCTAAGGGA-3′; ZEB1 sense, 5′-AAATGGAACACCAGATGC-3′ and antisense, 5′-TTACACCCAGACTGCGTC-3′; Sna1 sense, 5′-GCCTGTCTGCGTGGGTTT-3′ and antisense, 5′-GTGAGTCTGTCAGCCTTTGTCC-3′; vimentin sense, 5′-CCTGAACCTGAGGGAAACTAATC-3′ and antisense, 5′-GAAGTTTCGTTGATAACCTGTCC-3′; E-cadherin sense, 5′-AGGTCTCCTCTTGGCTCTG-3′ and antisense, 5′-AATAGGCTGTCCTTTGTCG-3′; N-cadherin sense, 5′-GCATCATCATCCTGCTTATCC-3′ and antisense, 5′-GTCCTGGTCTTCTTCTCCTCC-3′; β-actin sense, 5′-AGGGGCCGGACTCGTCATACT-3′ and antisense, 5′-GGCGGCACCACCATGTACCCT-3′. Thermocycling conditions were as follows: Initial DNA heat denaturation at 95°C for 3 min, followed by 39 cycles at 95°C for 10 sec and then, annealing and extending at 60°C for 30 sec in the qRT-qPCR experiment. The expression levels of the target genes were normalized to the transcription level of β-actin. Each sample was analyzed in triplicate.

Paraffin-embedded sections of CRC and adjacent non-cancerous tissues were used to detect ENST00000547547 expression using the RNAscope^® Assay-Brown (Advanced Cell Diagnostics, Advanced Cell Diagnostics, Inc., Newark, CA, USA) according to the manufacturer's instructions with the following modifications: Antigen retrieval for 15 min, protease digestion for 30 min at 37°C, probe incubation at 42°C overnight. The probe sequence for ENST00000547547 was 5′-GGTTTACTCTTCCCCTCTGTTC-3′. The expression level of ENST00000547547 in the nucleus was visualized with DAB and quantitated using an automatic imaging system (Leica DMLA; Leica Microsystems, Wetzlar, Germany).

For the overexpression of ENST00000547547, a pCDNA3.1-ENST00000547547 plasmid was constructed by Auragene Bioscience Co. The plasmid carrying pCDNA3.1-ENST00000547547 (1 µg/µl) was transfected into the CRC cell lines HCT116 and LoVo using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a Lipofectamine 2000:pCDNA3.1-ENST00000547547 ratio of 1:2.

After pCDNA3.1-ENST00000547547 transfection, the transfected and control cells were seeded into 96-well plates at an initial density of 5,000 cells/well. After culture for 24, 48 and 72 h, the cells were treated with 10 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sangon Biotech, Co., Ltd., Shanghai, China) by adding it into each well. The cells were incubated at 37°C for another 4 h, and then the medium was carefully removed and 150 µl dimethyl sulfoxide (DMSO) solution (MP Biomedicals, Santa Ana, CA, USA) was added to lyse the cells for 10 min. Finally, the absorbance was assessed at 570 nm using a Multiskan MK microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All experiments were performed in triplicate.

Six 4-week-old male BALB/c nude mice (original weight, 25 g) were obtained from the Hunan Branch of the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Changsha, China). The mice were housed on a 12 h light/dark cycle under pathogen-free conditions and were fed an autoclaved diet ad libitum. To assess tumor formation, the mice were subcutaneously injected with 5×10⁶/ml HCT116 cells stably expressing Lv-NC or Lv-ENST00000547547. For the tumor formation assay, the tumor volume was calculated as follows: Tumor volume = (width² × length)/2. At 28 days after the Lv-NC and Lv-ENST00000547547 inoculation, the mice were sacrificed and the tumors derived from each group were used for comparisons. The protocol was approved by the Ethics Committee on the Animal Experiments of the Third Xiangya Hospital of Central South University.

Cell migration and invasion assays were performed using Transwell chambers (micropore size, 8 µm, 24-well; Corning^®; Thermo Fisher Scientific, Inc.) without Matrigel (for migration assays) or with Matrigel (for invasion assays). Both assays were performed according to the manufacturer's instructions. Briefly, the treated cells were plated in the upper chamber at a concentration of 5×10⁴ in 500 µl FBS-free media. The bottom chamber contained 500 µl media with 10% FBS. The plates were incubated for 24 h, and then the chamber was fixed and stained with crystal violet staining solution (Beyotime Institute of Biotechnology, Haimen, China). The stained cells were imaged under a microscope at ×100 magnification and the optical density (OD) values were detected using a microplate reader (Thermo Multiskan MK3; Thermo Fisher Scientific, Inc.).

The CRC cell lines HCT116 and LoVo were transfected with pCDNA3.1-ENST00000547547 or pCDNA3.1-NC for 48 h and harvested. Then, the cells were washed three times with cold phosphate-buffered saline (PBS) and fixed with cold 70% ethanol overnight. For the cell cycle analysis, the cells were stained with propidium iodide (PI) (Keygentec, Inc., Nanjing, China) for 30 min at 4°C in the dark. The cell cycle profiles were assayed using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). For the apoptosis analysis, the cells were harvested 48 h after transfection and were stained with Annexin V-fluorescein isothiocyanate (FITC) and PI (Keygentec, Inc.) for 10 min in the dark at room temperature. The cells were then examined by flow cytometry (BD Biosciences). All of the samples were assayed in triplicate.

Cells were lysed using RIPA lysis buffer (Auragene Bioscience Co.), supplemented with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) and phenylmethylsulfonyl fluoride (PMSF) (Auragene Bioscience Co.). Equal amounts (25 µg) of protein were loaded on a 10–12% SDS-polyacrylamide separating gel. The proteins were then transferred to a Immobilon-P polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 3% bovine serum albumin-Tris-buffered saline with Tween-20 (BSA-TBST) with gentle shaking at room temperature for 90 min. Then, the membrane was probed with the indicated primary antibodies with gentle shaking at 4°C overnight. The membranes were washed with TBST (5×3 min) and incubated with specific secondary antibodies at room temperature for 1 h. A β-actin antibody (cat. no. LCA01; Auragene, Changsha, China) was used as a control and Bcl-2 (1:1,000; cat. no. AM2211; Abzoom, Changsha, China), Bax (1:500; cat. no. AM2198; Abzoom), PCNA (1:200; cat. no. AM3547; Abzoom), P21 (1:200; cat. no. B7175; Assay Biotechnology, California's San Francisco Bay Area, CA, USA), cyclin D1 (1:1,000; cat. no. ab134175; Abcam, Dallas, TX, USA), cyclin E1 (1:1,000; cat. no. ab33911; Abcam), P27 (1:200; cat. no. YM0496; ImmunoWay, Changsha, China), ZEB1 (1:500; cat. no. 21544-1-AP; ProteinTech, Wuhan, China), Sna1 (1:500; cat. no. ab82846; Abcam), N-cadherin (1:1,000; cat. no. YM0465; ImmunoWay), vimentin (1:800; cat. no. BM0147; Abzoom) and E-cadherin (1:800; cat. no. BM0530; Abzoom) antibodies were used for each group. The second antibody was goat anti-rabbit IgG (H+L)-HRP (1:18,000; cat. no. SA009; Auragene Bioscience Co.) or goat anti-mouse IgG (H+L)-HRP(1:18,000; cat. no. SA001; Auragene Bioscience Co.) incubation for 1 h in room temperature.

All statistical analyses were performed using SPSS 20.0 (IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analyses were determined with Student's t-test and AVONA as appropriate. All P-values were two-sided and P<0.05 was considered to indicate a statistically significant result.

The lncRNA microarray data revealed that lncRNA 3837 was downregulated and lncRNA 1181 was upregulated in CRC tissues. In addition, the ENST00000547547 level was decreased in the microarray data in CRC tissues compared to normal tissues (Fig. 1A). Subsequently, its expression was analyzed by qRT-PCR and in situ hybridization assays in CRC and adjacent non-cancerous tissues. The qRT-PCR results revealed that the expression of ENST00000547547 in CRC tissues was significantly decreased compared to adjacent non-tumor tissues (Fig. 1B). The in situ hybridization assay results revealed that the CRC tissues displayed lower ENST00000547547 staining than the adjacent non-cancerous tissues (Fig. 1C). The ENST00000547547 level in the human CRC cell lines COLO320, HCT116 and LoVo, and the normal human colonic epithelial cell line NCM460 was examined by qRT-PCR. As displayed in Fig. 1D, the expression of ENST00000547547 was significantly decreased in CRC cell lines compared to the expression in normal human colorectal cell lines. These results indicated that ENST00000547547 was significantly downregulated in CRC.

To investigate the potential biological function of ENST00000547547 in CRC cells, an ENST00000547547-overexpressing plasmid was constructed and used to transfect HCT116 and LoVo cells (16). The overexpression efficiency was verified by qRT-PCR analysis (Fig. 2A).

To determine the effect of ENST00000547547 on CRC cell proliferation, an MTT assay and flow cytometric analysis were performed. The MTT results revealed that the overexpression of ENST00000547547 significantly inhibited cell proliferation in the HCT116 and LoVo cells compared to the control group (Fig. 2B). As displayed in Fig. 2C, the overexpression of ENST00000547547 significantly increased cell apoptosis in the HCT116 and LoVo cells. Various mechanisms have been studied to explain lncRNA-mediated cell apoptosis and the most important factor identified is the activation of anti- or pro-apoptotic regulators. Bcl-2 and Bax proteins are one of the many ways to induce apoptosis (17), and proliferating cell nuclear antigen (PCNA) is known as a molecular marker for proliferation (18,19). To confirm the role of ENST00000547547-induced apoptosis, the expression levels of Bcl-2, PCNA and Bax were examined. Western blot analysis revealed that the protein levels of Bax were significantly increased in ENST00000547547-overexpressing cells, whereas the protein levels of Bcl-2 and PCNA were decreased (Fig. 2D). Collectively, these data indicated that the overexpression of ENST00000547547 could inhibit cell growth and promote apoptosis in CRC cells.

To further determine the effect of ENST00000547547 on tumorigenesis, cells stably expressing Lv-NC and Lv-ENST00000547547 were subcutaneously injected into nude mice. Nine days after the injection, a palpable tumor could be observed in both groups. There was a dramatic decrease in tumor volume in the Lv-ENST00000547547 group compared with the Lv-NC group (Fig. 2E). In addition, at 28 days, tumors derived from the Lv-ENST00000547547 group were significantly smaller than those in the control group (Fig. 2F). These results indicated that the overexpression of ENST00000547547 significantly inhibited CRC growth in vitro and in vivo.

To further assess whether the antiproliferative effects of ENST00000547547 on CRC cells were mediated by inhibiting cell cycle progression, the cell cycle was evaluated by flow cytometry and western blotting. The flow cytometric results demonstrated that overexpression of ENST00000547547 induced a significant G1-phase increase and caused an obvious reduction in the number of cells in the S phase in the HCT116 and LoVo cells (Fig. 3A and B). Furthermore, western blotting results revealed that the cell cycle-related proteins P21 and P27 were significantly increased in the ENST00000547547-overexpressing cells, whereas the protein levels of cyclin D1 and E1 were decreased in the HCT116 and LoVo cells (Fig. 3C). These observations indicated that ENST00000547547 may induce cell cycle arrest by affecting the expression levels of cyclins. Collectively, these data demonstrated that ENST00000547547 induced G0/G1 phase arrest in CRC cells.

To analyze the role of ENST00000547547 in cell invasion and migration, Transwell assays were performed in HCT116 and LoVo cells and the results revealed that ENST00000547547 overexpression greatly decreased cell invasion and migration compared with the control groups (Fig. 4). These data demonstrated that the overexpression of ENST00000547547 inhibited the invasion and migration of CRC cells in vitro.

To investigate whether the expression of ENST00000547547 regulated the progress of the epithelial-mesenchymal transition (EMT), we examined the expression levels of ZEB1, Sna1, N-cadherin, vimentin and E-cadherin in ENST00000547547-overexpressing HCT116 and LoVo cells. qRT-PCR analysis indicated that overexpression of ENST00000547547 resulted in an obvious downregulation in the transcription levels of EMT-inducing genes (ZEB1, Sna1) and the biomarkers of mesenchymal cells (N-cadherin and vimentin), as well as a clear upregulation in the transcription level of E-cadherin, which is a hallmark of epithelial cells (Fig. 5A). These results were reconfirmed by western blot analysis (Fig. 5B). These data indicated that ENST00000547547 inhibited the metastasis of CRC cells by suppressing the expression of EMT-inducing genes.