The present study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (Nanjing, China; Ethics no. 2010-SR-091.A1). In total, 80 pairs of human CRC tissues and adjacent normal tissues were collected from patients with CRC, who had signed an informed consent form, between 2010 and 2012 at the First Affiliated Hospital of Nanjing Medical University. All the patients were aged between 27 and 88 years old (average, 61.6 years old), and none of the patients had a history of radiotherapy or chemotherapy prior to surgery. All samples were immediately preserved in liquid nitrogen within 5 min following resection and then placed at −70°C for long-term preservation. The tumor-node-metastasis stage was determined based on the National Comprehensive Cancer Network (23).

All CRC cell lines (LoVo, HCT116, SW480, DLD-1 and HT29) and normal epithelial colon cells (NCM460) were purchased from the American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (both from Winsent, Inc., St. Bruno, QC, Canada), 100 U/ml of penicillin and 100 µg/ml of streptomycin in a humid incubator (stabilized at 5% CO₂ and 37°C)

Total RNA was extracted from CRC tissues and cells using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. The cDNA was produced from the RNA by performing reverse transcription using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The RT-qPCR experiment was performed in a 20 µl volume consisting of 2 µl cDNA, 1.2 µl primers, 6.8 µl dH₂O and 10 µl SYBR, using a SYBR-Green PCR kit (Roche Diagnostics, Indianapolis, IN, USA). The final reaction was performed in a StepOnePlus Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and comprised: Hot-start DNA polymerase activation to 95°C for 10 min; 40 cycles of 95°C for 15 sec and 60°C for 1 min; followed by one cycle of melt curve analysis at 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec. The specific primers were as follows: TRIM27, forward, 5′-AGCCCATGATGCTCGACTG-3′ and reverse, 5′-GGGCACGACACGTTAGTCT-3′; GAPDH, forward, 5′-AGAAGGCTG GGGCTCATTTG-3′ and reverse, 5′-AGGGGCCATCCACAGTCTTC-3′. TRIM27 expression was normalized to GAPDH and relative expression levels were calculated using the 2^−ΔΔCq method (24).

To detect the level of TRIM27 in CRC, 50 CRC tissues and 25 adjacent normal tissues were evaluated using IHC. The formalin-fixed and paraffin-embedded tumor and normal tissue sections (4-µm) were deparaffinized in xylene and rehydrated in different concentrations of alcohol and distilled water, followed by microwave antigen retrieval. The sections were washed in phosphate-buffered saline (PBS) three times and were placed in 3% H₂O₂ for 20 min in the dark, followed by three washes with PBS. The tissues were then soaked in 5% BSA (Servicebio, Wuhan, China) for 30 min. Finally, the tissues were reacted with anti-TRIM27 antibodies (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393; Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with anti-rabbit antibodies (horseradish peroxidase-tagged, diluted 1:1,000, polyclonal, cat. no. ab6721; Abcam) at room temperature for 50 min, and with the color agent diaminobenzidine. The nuclei were counterstained with hematoxylin, and the sections were dehydrated using different grades of ethyl alcohol and xylene. The sections were then viewed under an inverted microscope (NIKON ECLIPSE TI-SR; Nikon Corporation, Tokyo, Japan). Based on the staining intensity, the level of TRIM27 was graded as 0 (no staining), 1 (+), 2 (++), and 3 (+++). According to the proportion of TRIM27-positive cells, the scores were as follows: 0 (negative), 1 (<30%), 2 (31–60%), 3 (>60%). The total score was calculated as the intensity score plus the positive rate score. Scores ≥4 were regarded as a high level of TRIM27, and scores <4 were regarded as a low level of TRIM27.

Small interference RNA (siRNA) targeting TRIM27 and a negative control sequence (NC) were designed by GenePharma Corporation (Shanghai, China). The target sequences are shown in Table I (13). The TRIM27 inhibitor lentivirus [short hairpin (sh)TRIM27] in vivo experiment was designed based on the sequence of siRNA918, with the specific sequence: 5′-GCAGCTGATATCACTCCTTA-3′. For the overexpression of TRIM27, a plasmid expressing TRIM27 was designed by GeneCopoeia, Inc. (Rockville, ME, USA). The above siRNAs and plasmid were transfected into cells using Lipofectamine^® 3000, according to the manufacturer’s protocol (Invitrogen; Thermo Fisher Scientific, Inc.).

To investigate the effect of TRIM27 on the proliferation of CRC cells, 2×10³ cells per well were cultured in 96-well plates with each well containing 100 µl of medium. Cell viability was detected using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kummamoto, Japan) assay according to the manufacturer’s protocol. After 24, 48, 72 and 96 h, 10 µl of CCK-8 assay reagent was added to each well mixed with 90 µl of serum-free medium. The absorbance was measured 2 h later using a microplate reader at a test wavelength of 450 nm and a reference wavelength of 630 nm.

The cells (500 per well) were cultured in 6-well plates to investigate the effect of TRIM27 on the efficiency of colony formation in CRC cells. After 7 days, each well was washed with PBS three times at room temperature. The cells were then fixed in each well using ethyl alcohol for 30 sec and stained for 20 min using crystal violet dye. Following washing with PBS, colonies (≥50 cells/colony) in each well were manually counted and images were captured using a digital camera (Canon DS126211; Canon, Tokyo, Japan).

The CRC cells (4×10⁵) were seeded in 6-well plates and cultured until they reached a density of 90%. A 200-µl micropipette tip was then used make a wound in the cells and the medium was replaced with serum-free medium. Electron microscopy was used to observe the shape of wound at 0 and 24 h.

Cell migration and invasion were assayed in a 24-well plate with polycarbonate sterile chambers (8-µm filters; BD Biosciences, Franklin Lakes, NJ, USA) with or without Matrigel coating. The CRC cells (2×10⁴) were cultured with 100 µl of serum-free DMEM in the upper chamber and 600 µl of DMEM + 10% serum in the lower chamber. After 24 h, the lower chamber was washed twice with PBS and crystal violet dye was added to the lower chamber incubated for 20 min. The lower chamber was washed with PBS three times, following which a cotton bud was used to remove cells and medium from the upper chambers. The migrated or invaded cells in the lower chambers were observed under an electron microscope.

Following 48 h of the siRNA or plasmid transfection, the cells were collected and washed with cold PBS. The cells were then mixed with an Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) in flow cytometry tubes, following the manufacturer’s protocol. The number of apoptotic cells was analyzed using flow cytometry (BD Biosciences).

At 48 h following siRNA or plasmid transfection, the transfected CRC cells were collected in PBS. Following washing twice with PBS, the cells were fixed with 75% ethyl alcohol at 4°C overnight. The following day, the separated ethyl alcohol was removed, and the cells were fixed with 500 µl of propidium iodide (PI) staining solution and incubated for 30 min in the dark at room temperature. The analysis of the cell cycle was performed using fluorescence-activated cell sorting with a FACSCalibur flow cytometer with CellQuest software (version 3.0; BD Biosciences).

Protein was extracted from the CRC cells using a Radioimmunoprecipitation Assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s protocol. The protein concentration, which determined the quantity loaded for SDS-PAGE, was determined using the Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). The proteins (40 µg) were separated using 10% SDS-PAGE in running buffer and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Bedford, MA, USA) in transfer buffer. The membranes were then blocked in 5% non-fat milk for >2 h and incubated with specific primary antibodies at 4°C overnight. Following washing with TBST three times (10 min each time), the membranes were incubated with secondary antibodies (anti-rabbit or anti-mouse) at room temperature for 2 h. The immunoreactive protein bands were visualized using ECL Plus (EMD Millipore) with a bio-imaging system. The specific primary and secondary antibodies were as follows: TRIM27 (diluted 1:1,000, polyclonal, rabbit, cat. no. ab78393), E-cadherin (diluted 1:500, monoclonal, mouse, cat. no. ab1416), N-cadherin (diluted 1:1,000, poly-clonal, rabbit, cat. no. ab18203), vimentin (diluted 1:2,000, monoclonal, rabbit, cat. no. ab92547), AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab8805), p-AKT (diluted 1:500, polyclonal, rabbit, cat. no. ab38449) (all from Abcam), anti-rabbit secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAB007), anti-mouse secondary antibodies (diluted 1:5,000, polyclonal, cat. no. GAM007) (both from Hangzhou Multi Sciences Biotech Co., Ltd., Hangzhou, China). GAPDH (diluted 1:5,000, monoclonal, mouse; Abcam) was used as an internal control.

The animal experiment was approved by Animal Ethics Committee of Nanjing Medical University. A total of 20 male mice (age, 3–4 weeks; weight, 13–15 g) were purchased from the Animal Center of Nanjing Medical University after signing the animal-raising agreement. The mice were maintained under the following conditions: Room temperature, 20–26°C and 12-h light/dark cycle. The mice received 5 g food and 100 ml water per 100 g body weight per day. To detect whether TRIM27 can affect tumor growth in mice, 2×10⁶ of differently treated LoVo cells mixed with 200 µl of PBS were subcutaneously injected into the anesthetized mice. Each mouse was randomly injected with cells treated with shTRIM27 or NC in their right and left armpits. At 4 weeks post-injection, all mice were sacrificed and the tumor tissues were surgically removed. To further investigate the role of TRIM27 in tumor metastasis in vivo, 2×10⁶ LoVo cells suspended in 200 µl of PBS were injected into the mouse tail vein. The liver tissues were removed after 7 weeks and stained with hematoxylin and eosin.

The results are expressed as the mean ± standard deviation. The mRNA expression was analyzed using unpaired t-tests. IHC and the clinical features were analyzed using Pearson χ² tests. One-way analysis of variance and the least-significant difference post hoc test were the main statistical methods used to compare datasets containing multiple groups. Cumulative survival analysis was assessed using the Kaplan-Meier method. Independent prognostic factors were identified using univariate and multivariate Cox proportional hazard regression models. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS (version 15.0; SPSS, Inc., Chicago, IL, USA) and GraphPad Prism5 (GraphPad Software, Inc., La Jolla, CA, USA).

To reveal the role of TRIM27 in the progression of CRC, the mRNA expression of TRIM27 was first detected in 80 pairs of CRC tissues and adjacent normal tissues using RT-qPCR analysis. It was found that the expression of TRIM27 was significantly upregulated in CRC tissues (Fig. 1A). Then we detected the presence of TRIM27 using IHC in 50 CRC tissues and 25 adjacent normal tissues. IHC also showed that the protein level of TRIM27 was significantly upregulated in CRC tissues (Fig. 1B and C; Table II). The patients were next divided into two groups based on the average TRIM27 levels, and the association between levels of TRIM27 and the clinicopathological features was analyzed. It was found that a higher expression of TRIM27 was associated with deeper invasion (P=0.005), increased lymph node metastasis (P=0.014), more advanced tumor stage (P=0.004), and increased liver metastasis (P=0.043) (Table III). However, no significant association was found for sex, age, CEA, or location. Kaplan-Meier curves were used to analyze the effect of TRIM27 on overall survival (OS) in patients with CRC. The median follow-up time was 60 months, and the result showed that higher mRNA expression of TRIM27 predicted poorer prognosis in patients diagnosed with CRC (Fig. 1D). Multivariate analysis further suggested that TRIM27 was an independent prognostic factor for CRC (Table IV).

To evaluate the expression level of TRIM27 in CRC cells, five CRC cell lines (SW480, LoVo, HT29, HCT116 and DLD-1) and the NCM460 normal colon epithelial cell line were detected by RT-qPCR and western blot analyses. It was found that the mRNA and protein levels of TRIM27 were upregulated in the CRC cell lines compared with those in the NCM460 cells (Fig. 2A and B). To knock down or overexpress TRIM27, the LoVo and HCT116 cells were transfected with a TRIM27 inhibitor and TRIM27-overexpressing plasmid, respectively. According to the results of RT-qPCR and western blot analysis, the expression of TRIM27 was significantly inhibited by siRNA-918, siRNA-1251 and siRNA-1578 in LoVo cells (Fig. 2C and D), and was overexpressed by trans-fection with the TRIM27-overexpressing plasmid in HCT116 cells (Fig. 2E and F). To further investigate the role of TRIM27 in CRC, the TRIM27-overexpressing plasmid and siRNA-918 were selected to perform further in vitro experiments.

CCK-8 and colony formation assays were used to detect the effect of TRIM27 on the proliferation of CRC cells. It was found that the knockdown of TRIM27 significantly inhibited cell proliferation in LoVo cells (Fig. 3A). By contrast, cell proliferation was promoted in HCT116 cells overexpressing TRIM27 (Fig. 3B). Consistently, in the colony formation assay, it was found that LoVo cells transfected with the siRNA showed reduced formation of colonies (Fig. 3C), whereas HCT116 cells transfected with the TRIM27-overexpressing plasmid showed the opposite result (Fig. 3D).

Flow cytometry was used to investigate whether TRIM27 regulates apoptosis and the cell cycle of CRC cells. It was found that the LoVo cells transfected with the siRNA showed a significant increase in the percentage of cells in the G0-G1 phase and a reduced percentage of cells in the S phase, whereas HCT116 cells transfected with the TRIM27-overexpressing plasmid showed the reverse result (Fig. 4A). Similarly, LoVo cells transfected with the siRNA showed a significant increase in the percentage of apoptotic cells, whereas HCT116 cells transfected with the TRIM27-overexpressing plasmid showed fewer apoptotic cells (Fig. 4B). Taken together, these results suggested that TRIM27 promoted the proliferation of CRC cells by promoting apoptosis resistance and cell cycle transition in the G0-G1/S phase.

Wound-healing and Transwell assays were used to detect the metastasis and invasiveness in LoVo and HCT116 cells. In the wound-healing assay, it was found that the wound area was wider in LoVo cells transfected with siRNA (Fig. 5A), but narrower in HCT116 cells transfected with the TRIM27-overexpressing plasmid (Fig. 5B). In the Transwell assay, the numbers of cells that traversed the Transwell and Matrigel were significantly reduced by TRIM27 knockdown in the LoVo cells, whereas the overexpression of TRIM27 in HCT116 cells increased the number of cells that traversed the Transwell and Matrigel (Fig. 5C–F). These results suggested that TRIM27 promoted the invasion and metastasis of CRC cells.

To determine whether TRIM27 affects tumor growth in vivo, tumor xenografting was performed in nude mice. At 4 weeks post-implantation, the tumor size (Fig. 6A and B) and weight (Fig. 6C) was significantly decreased in the group treated with shTRIM27 compared with the untreated control. To further investigate whether TRIM27 affects tumor metastasis in vivo, a tail vein metastatic assay was performed in nude mice using LoVo cells transfected with shTRIM27. At 7 weeks post-injection, the results of hematoxylin and eosin staining showed that the knockdown of TRIM27 correlated closely with reduced liver metastasis (Fig. 6D and E). These in vivo results further confirmed that TRIM27 promoted proliferation and metastasis in CRC.

To further detect whether TRIM27 was associated with the EMT process, the levels of EMT-associated proteins in LoVo and HCT116 cells were evaluated using western blot analysis. Following TRIM27 knockdown, it was found that the level of the epithelial marker E-cadherin increased, and the level of the mesenchymal marker N-cadherin decreased. The opposite effect was observed in HCT116 cells transfected with the TRIM27-overexpressing plasmid (Fig. 7). Further investigation revealed that the knockdown of TRIM27 decreased the level of vimentin in LoVo cells; however, the level of vimentin increased in HCT116 cells following the overexpression of TRIM27 (Fig. 7). These results suggested that TRIM27 promoted EMT in CRC cells. Additionally, the levels of p-AKT and AKT in LoVo and HCT116 cells were detected. It was found that the level of p-AKT increased when TRIM27 was overexpressed, but decreased when TRIM27 was knocked down. However, no significant changes occurred in the expression of AKT, suggesting that TRIM27 promoted the activation of p-AKT (Fig. 7).