A total of 88 tumor tissues were obtained from patients with CRC who underwent surgery between January 2013 and December 2014 at Jinan Central Hospital Affiliated to Shandong University (Jinan, China). Prior to the surgery, the patients had not received chemotherapy, radiotherapy or immunotherapy. Among the patients, 77.27% (68/88) were male and 22.73% (20/88) were female. Patients were aged from 20-90 years old with a mean age of 67.17 years. Follow up data were summarized on April 14th 2018, with a median follow-up time of 60 months. Tumor tissues were classified according to the American Joint Committee on Cancer Cancer Staging Manual (21). The present study was supported by the Institutional Review Boards of Jinan Central Hospital Affiliated to Shandong University, and written informed consent was obtained from all patients.

Human CRC tissues and adjacent normal tissues were fixed in 10% formalin (Phygene, Fuzhou, China; cat. no. PH0996) for 24 h at room temperature. Subsequently, it was dehydrated with different concentrations of ethanol (70, 85, 95 and 100%) and xylene, embedded in paraffin and cut to a thickness of 4 μm. When the sections had been de-paraffinized by xylene and rehydrated by different concentrations of ethanol (100, 95 and 75%), antigen retrieval was performed by heating the specimens in Tris EDTA buffer (Wuhan Sanying Biotechnology, Wuhan, China; cat. no. B600011) for 10 min in a microwave oven (Guangdong Galanz Group Co., Ltd., Guangdong, China; P70D20TL D4) at medium heat. Following naturally cooling the sections, they were incubated in 3% H₂O₂ at room temperature for 10 min and blocked in 10% goat serum (OriGene Technologies, Inc., Rockville, MD, USA; cat. no. ZLI 9021) at room temperature for 1 h. The sections were incubated with rabbit polyclonal anti ILT4 anti body (1:100; OriGene Technologies, Inc.; cat. no. TA349368) and mouse monoclonal anti HLA-G antibody (1:200; Abcam, Cambridge, UK; cat. no. ab52455) at 4°C overnight, followed by detection with an Elivision Plus Polymer

Horseradish Peroxidase (mouse/rabbit) IHC kit (25 min) and streptavidin conjugated peroxidase (25 min). The sections were visualized using 3,3′ diaminobenzidine solution (Fuzhou Maixin Biotech Co., Ltd., Fuzhou, China) and counterstained with hematoxylin at room temperature. Normal mouse IgG (1:20,000; Abcam; cat. no. ab205719) and rabbit IgG (1:10,000; OriGene Technologies, Inc.; cat. no. 17502-0.2mG) were used instead of the primary antibody as a negative control.

A total of two independent researchers analyzed the immunohistochemistry results. The stained cell percentages in at least five fields were recorded at ×400 magnification under a light microscope (NIKON ECLIPSE TI SR; Nikon Corporation, Tokyo, Japan). The sections were scored as positive or negative by a previously described method (11).

The human colon epithelial cell line FHC was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The HCT1116, HT29, SW480 and SW620 cell lines were obtained from the Institutes of Biochemistry and Cell Biology (Shanghai, China), and originated from the ATCC. All cells were cultured in RPMI 1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences). All cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C.

Downregulation of ILT4 expression was performed by transfecting plasmid pGPU6/GFP/Neo short hairpin (sh)ILT4-1 (shILT4; Shanghai GenePharma Co., Ltd., Shanghai, China) in to HCT116 and SW620 cells. Non targeting plasmid [sh negative control (NC); Shanghai GenePharma Co., Ltd.] was used as a negative control. Plasmid Pez lv105 ILT4 (ILT4) (GeneCopoeia, Inc., Rockville, MD, USA) was added to HT29 and SW480 cells to upregulate the level of ILT4, plasmid Pez lv105 (NC; GeneCopoeia, Inc.) was used as negative control. When the cells had reached 40-60% confluence in 6-well plates, plasmids and X-treme GENE HP reagents (Roche Diagnostics, Basel, Switzerland) were added to the cells at a ratio of 2 μg/6 μg according to the manufacturer’s protocols. The level of mRNA were detected by reverse transcription quantitative polymerase chain reaction (RT qPCR) after 24 h and the level of protein was detected by western blot assays after 48 h following transfection. The interference sequences of ILT4 are listed in Table I. The overexpression sequences of ILT4 are listed in Fig. S1.

Total proteins were extracted from CRC and FHC cells. Following washing in ice cold PBS for 1 min, cell lysates were lysed in radioimmunoprecipitation assay lysis buffer containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Proteins were quantified using a Pierce bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). A mass of 30 μg protein/lane was separated via 10% SDS PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) using semidry transfer apparatus. Following blocking in TBS with 0.1% Tween 20 and 5% non fat milk for 1 h at room temperature, the PVDF membranes were incubated with mouse monoclonal anti HLA-G antibody (1:1,000; Abcam; cat. no. ab52455), rabbit polyclonal anti ILT4 antibody (1:500; Abcam; cat. no. ab128349), rabbit monoclonal anti phospho AKT antibody (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 4060), rabbit monoclonal anti AKT antibody (1:1,000; Epitomics; Abcam; cat. no. 2957-1), rabbit monoclonal anti phospho ERK antibody (1:1,000; Abcam; cat. no. ab201015) and rabbit monoclonal anti ERK antibody (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4695) at 4°C overnight. The blots were incubated in horseradish peroxidase conjugated secondary goat anti mouse (1:10,000; Wuhan Sanying Biotechnology; cat. no. SA00001-1) or goat anti rabbit antibodies (1:10,000; Wuhan Sanying Biotechnology; cat. no. SA00001-2) for 1 h at room temperature. Finally, the blots were developed in Enhanced Chemiluminescence reagent (EMD Millipore) and exposed using a ChemiDoc™ XRS+ system (Bio Rad Laboratories, Inc., Hercules, CA, USA). GAPDH (1:10,000; Wuhan Sanying Biotechnology; cat. no. 10494-1 AP) antibody was used as the loading control. Image J 1.8.0 (National Institutes of Health, Bethesda, MD, USA) was employed to quantify protein levels.

Total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The cDNAs were synthesized from 2 μg total RNA using a Thermo Scientific RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). RT-qPCR was performed using a Fast Start Universal SYBR^® Green RT PCR kit (Roche Diagnostics) and ABI 7500 Fast Real Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.; 50°C for 2 min, 95°C for 10 min and 95°C for 15 sec for 40 cycles). The forward and reverse primer sequences are listed in Table II. The mRNAs were normalized to GAPDH. The 2^-∆∆Cq method was used to evaluate the relative expression levels of the genes (22).

HT29 cells were plated at a concentration of 1×10⁵ cells/ml in 6 well plates, each well containing 2 ml RPMI 1640 medium with 10% FBS, and cultured at 37°C. When the cells had reached 60-70% confluence in 6 well plates, HLA-G fusion protein (Wuhan Sanying Biotechnology; cat. no. Ag10839) at concentrations of 10, 20, 50, 100, 200 and 500 ng/ml was added according to the manufacturer’s protocols. Each well contained 2 ml RPMI 1640 medium with 10% FBS. The cells were incu bated at 37°C, and then analyzed at 24 h with an RT-qPCR assay and at 48 h by western blotting according to the afore mentioned protocols.

HT29 cells were treated with 0.5 μg/ml anti ILT4 blocking antibody (R&D Systems, Inc., Minneapolis, MN, USA; cat. no. MAB2078) in 6-well plates at 37°C, using 0.5 μg/ml IgG (R&D Systems, Inc.; cat. no. 1-001 A) and PBS as negative controls. After 12 h, cells were treated with 200 ng/ml HLA-G fusion protein at 37°C, and after 48 h the total protein levels were detected by western blotting, according to the aforementioned protocol. The peptide sequence of the HLA-G fusion protein was as follows: SHSMRYFSAAVSRPS RGE PRFIAMGYVD DTQFVRFDSDSACPRMEPRAPWVEREGPEYWEEETRN TKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGC DLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAAD TAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLEN GKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYP AEIILTWQRDGEDQTQDVELVETKPAGDGTFQKWAAV VVPSGEEQRYTCHVQHEG LPEPLMLRWKQSSLPTIPI (26-308aa encoded by BC021708).

Cells were plated in 96 well culture plates at a concentration of 3×10³ cells/well, each well containing 100 μl RPMI-1640 medium with 10% FBS. After incubation for 6, 24, 48, 72 and 96 h at 37°C, cells were treated with 10 μl Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) solution and incubated at 37°C for 4 h, and subsequently measured at 450 nm. All assays were performed at least three times.

CRC cells (HCT1116, HT29, SW480 and SW620) were plated at a concentration of 1×10⁵ cells/ml in 6 well plates, each well containing 2 ml RPMI 1640 medium with 10% FBS, and cultured at 37°C for 24 h. Subsequently, the cells were scratched with a sterile 200 μl pipette tip. PBS was used to wash the cell debris. Images were acquired at 0 and 24 h in the same location under a light microscope (Carl Zeiss AG, Oberkochen, Germany; magnification, ×40). The scratch wound widths were calculated in three random fields of view.

Cell migration was assessed using Transwell insert chambers with a pore size of 8 μm (Corning Inc., Corning, NY, USA). Approximately 1×10⁵ cells in RPMI 1640 without FBS were cultured in the upper chamber. RPMI 1640 medium supplemented with 10% FBS was added to the bottom chamber as a chemoattractant. Subsequently, the cells were incubated at 37°C for 24 h. The migrated cells were fixed with 20% methanol for 20 min at 37°C and stained with 0.1% crystal violet (Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min at 37°C. The number of cells in three fields of each sample was counted under a light microscope (Carl Zeiss AG; magnification, ×100). Cell numbers were calculated from five random fields.

Cell invasion was determined using Matrigel invasion chambers (BD Biosciences; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The upper chambers were covered with Matrigel, which was diluted in cold PBS and incubated at 37°C for 1 h. Approximately 1×10⁵ cells were suspended in RPMI 1640 medium without FBS and transferred into the upper chambers. RPMI 1640 medium supplemented with 10% FBS was added to the bottom chamber as a chemoattractant. Subsequently, the cells were incubated at 37°C for 24 h. The migrated cells were fixed with 20% methanol for 20 min at 37°C and stained with 0.1% crystal violet for 15 min at 37°C. The number of cells in three fields of each sample was counted under a light microscope (Carl Zeiss AG; magnification, ×100). Cell numbers were calculated from five random fields.

All data are presented as the mean ± standard deviation. SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) were selected to perform the statistical analysis. OS curves were generated via the by Kaplan Meier method and compared by means of the log rank test. The associations between ILT4, HLA-G and clinicopathological characteristics of patients with CRC were analyzed with the χ² test. The correlation between ILT4 and HLA-G expression in CRC tissues was analyzed by Spearman’s correlation coefficient. The differences in characteristics between two groups were examined by Student’s t test. The differences in characteristics between three or more groups were examined by one way analysis of variance followed by Tukey’s post hoc test. P<0.05 was considered to indicate a statistically significant difference.

ILT4 and HLA-G positive expression was detected in the cell cytoplasm and in some cancer stromal cells (Fig. 1A). In adjacent normal colorectal tissues, ILT4 and HLA-G expression was too weak to be observed (Fig. 1B). In total, 68.18% (60/88) of the tissue samples exhibited overexpression of ILT4 and 59.09% (52/88) of the tissue samples exhibited overexpression of HLA-G. There was a notable positive correlation between ILT4 and HLA-G expression, and positive ILT4 expression had an increased probability of being observed in HLA-G positive tissues, compared with HLA-G negative tissues (P<0.001; Fig. 1C).

Firstly, the correlation of ILT4 and HLA-G expression was analyzed with various clinicopathological characteristics. ILT4 expression was positively correlated with males and increased lymph node metastasis (Table III; P=0.002 and P=0.002, respectively). Additionally, increased expression of HLA-G was correlated with increased CRC Tumor Node Metastasis (TNM) staging (P=0.030).

According to ILT4 and HLA-G expression levels, patients were divided into 4 groups to further examine the association between ILT4/HLA-G co expression and various clinicopathological factors. As presented in Table IV, compared with the ILT4 /HLA-G group, ILT4/HLA-G co expression in the ILT4+/HLA-G+ group was significantly associated with males (P=0.025) and increased lymph node metastasis (P=0.041). Additionally, compared with the ILT4+/HLA-G group, ILT4/HLA-G co expression was associated with advanced TNM staging (P=0.001). Older age (P=0.042), males (P=0.001), increased lymph node metastasis (P=0.038) and advanced TNM staging (P=0.030) were associated with ILT4/HLA-G co expression, compared with the ILT4 /HLA-G+ group.

As depicted in Fig. 1D, patients with ILT4+/HLA-G+ had a decreased survival time, compared with the ILT4 /HLA-G (P=0.032) and ILT4+/HLA-G (P=0.043) groups. However, no significant difference in OS was observed, compared with the ILT4 /HLA-G+ group (P=0.395).

Co expression of ILT4 and HLA-G is also detected in CRC cells. The expression levels of ILT4 and HLA-G were examined in FHC and CRC cells (HCT1116, HT29, SW480 and SW620 cells) by western blotting (Fig. 2A) and RT qPCR (Fig. 2B and 2C). CRC cells exhibited increased levels of ILT4 and HLA-G expression, compared with normal cells. Additionally, increased levels of ILT4 and HLA-G were detected in HCT116 cells, and reduced levels in HT29 cells, indicating that the proteins ILT4 and HLA-G may be co expressed in CRC cells.

To investigate the regulatory effects of ILT4 on the expression of HLA-G in CRC cells, ILT4 was overexpressed in HT29 and SW480 cells via an ILT4 plasmid (ILT4 vector), and ILT4 expression was inhibited in HCT116 and SW620 cells via ILT4 shRNA (shILT 4). Western blotting and RT qPCR assays were performed to examine HLA-G expression. The overexpression of ILT4 in HT29 (Fig. 3A and B) and SW480 cells (Fig. 3C and D) upregulated the expression of HLA-G.

Simultaneously, the present study assessed whether ILT4 influenced CRC cell proliferation, migration and invasion. The results demonstrated that ILT4 overexpressing HT29 and SW480 cells had a notably increased capacity for proliferation, migration and invasion, compared with the negative control (Fig. 4).

Similarly, HLA-G expression was notably downregulated when ILT4 expression was reduced in HCT116 (Fig. 5A and B) and SW620 cells (Fig. 5C and D). Additionally, downregulation of ILT4 notably reduced the proliferation, migration and invasion of HCT116 and SW620 cells (Fig. 6). These results indicated that ILT4 promotes an increased proliferative, migratory and invasive phenotype in CRC cells.

The effects of HLA-G expression on ILT4 expression were assessed in CRC cell lines. HT29 cells, which express a low level of ILT4/HLA-G, were treated with different concentrations (10, 20, 50, 100, 200 and 500 ng/ml) of HLA-G fusion protein. In the range of 10-200 ng/ml concentration, HLA-G fusion protein notably upregulated the expression of ILT4 at the mRNA and protein levels in a dose dependent manner (Fig. 7).

To identify the underlying molecular mechanisms of ILT4/HLA-G in CRC progression, HT29 cells were treated with 200 ng/ml HLA-G fusion protein, and the expression levels of ERK1/2 and AKT were analyzed. The results demonstrated that the levels of phospho ERK1/2 and phospho AKT were notably enhanced, while the total ERK and AKT levels did not change significantly (Fig. 8A). Simultaneously, following stimulation by HLA-G, HT29 cells exhibited an increased capacity for proliferation, migration and invasion (Fig. 8B E).

The present study sought to block the ILT4 expression in HT29 cells via anti ILT4 antibody to identify its function in the cellular malignant behaviors promoted by HLA-G. Notably, the upregulation of phospho ERK1/2 and phospho AKT expression induced by HLA-G was no longer significantly altered (Fig. 9A), and the cell proliferation, invasion and migration of HT29 were notably reduced (Fig. 9B E).