The human CRC cell lines HCT-116 and HT-29 were obtained from American Type Culture Collection (Manassas, VA, USA). Both cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Genom Biotech Pvt., Ltd., Bhandup, Mumbai) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA, USA), 100 unit/ml penicillin, 100 µg/ml streptomycin with normal glucose (NG; 5.5 mmol/l) or HG (30 mmol/l). Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

A total of 6 CRCs with or without T2DM in this study were histologically and clinically diagnosed at Ningbo Urology and Nephrology Hospital between October 2015 to March 2016 and the tissues were collected immediately following surgical resection for diagnosis. The inclusion criteria was as follows: i) Patients had to be diagnosed with CRC by preoperative pathological biopsy using a colonoscope; ii) aged between 18 and 75 years; iii) exhibit no distant metastasis; and iv) with or without diabetes. Patients were excluded if they: i) Received radiotherapy and chemotherapy prior to surgery; ii) exhibited acute infection; or iii) had a history of abdominal surgery or other malignant tumors. The specimens were then stored at −80°C. The present study was approved by Ningbo Yinzhou Ethics Committee and signed informed consent was obtained from the patients or their family. Patient data is summarized in Table I.

CRC tissues were fixed in 4% formaldehyde solution for 2 h at 25°C and then sectioned into 5-µM-thick frozen sections. The sections were washed in cold PBS 3 times and subsequently blocked with 2% bovine serum albumin V at 25°C (BSA-V; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 1 h. Samples were incubated with primary antibodies against E-cadherin (1:20; sc-8426; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) diluted with 1% BSA-V overnight at 4°C. Following 3 washes with PBS, the sections were incubated with tetramethylrhodamine conjugated goat anti-rabbit secondary antibody (1:1,000; sc-362281; Santa Cruz Biotechnology, Inc.) diluted with 1% BSA-V in the dark for 1 h at 25°C and washed in PBS again for 3 times. DAPI diluted with PBS was used to stain the nuclei at 25°C. Images at a magnification of ×40 were captured using an inverted fluorescence microscope (Nikon Corp., Tokyo, Japan).

Tissues and cells were homogenized in a radioimmunoprecipitation assay lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.). A BCA protein assay kit (Cwbiotech, Beijing, China) was used to determine protein concentrations. Proteins (20 µg) were separated by 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes, which were blocked in 5% nonfat milk for 1 h at 25°C and probed with primary antibodies against E-cadherin (1:400; #AF0131; Affinity Biosciences, Jiangsu, China), vimentin (1:600; #AF0292; Affinity Biosciences), GAPDH (1:10,000; #T0004; Affinity Biosciences) and high-mobility group A protein 2 (HMGA2; 1:500; 5269s; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. The membranes were subsequently incubated with goat anti-rabbit antibody diluted with 0.3‰ TBST (1:1,000; #SC2004; Santa Cruz Biotechnology, Inc.) or goat anti-mouse antibody diluted with 0.3‰ TBST (1:1,000; #SC2005; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The membranes were scanned with the Tanon 5200 automated image analysis system (Tanon, Shanghai, China) and the ImageJ software (version 1.48U; National Institutes of Health, Bethesda, MD, USA) was used to evaluate the band intensity.

HCT-116 and HT-29 cells (3–5×10⁵ cells/well) were seeded in 6-well plates and cultured in DMEM containing NG or HG for 4 days at 37°C. Confluent cultures were scratched with sterile 200 µl pipette tips and washed gently with PBS to remove floating cells. Then the cells were cultured in DMEM containing NG or HG and 5% FBS. Cells were viewed under an inverted fluorescence microscope (magnification, ×40) and images were captured after 0, 24, 48 and 72 h.

HCT-116 and HT-29 cells were cultured in DMEM containing NG or HG for 4 days and then serum-starved for 12 h. The cells (1×10⁵ cells/well) were seeded into Boyden chambers (EMD Millipore, Billerica, MA, USA) with 8-µm pore size filter membranes. The inserts were coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) for invasion assays or not coated for migration assays. The chambers were then placed in 24-well plates containing DMEM and 10% FBS at 37°C. After 72 h, the non-invaded cells on the upper side of the filter were removed with a cotton swab and cells attached to the underside of the membrane were fixed in ethanol, stained with crystal violet and images were counted using a microscope (CKX41; Olympus Corporation, Tokyo, Japan; magnification, ×40).

The RNA interference technique was used to downregulate HMGA2 in HCT-116 and HT-29 cells (22). HMGA2 small interfering 400 ng (si)RNAs (siHMGA2-1, 5′-GAAAGCAGAGACCAUUGGATT-3′; siHMGA2-2, 5′-GAAAGCAGAGACCAUUGGATT-3′; Shanghai Genechem Co., Ltd., Shanghai, China) were synthesized and transfected into cells using RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Following 48 h transfection, HMGA2 expression was confirmed by western blotting. Western blotting was then performed as aforementioned.

HCT-116 and HT-29 proliferation was measured using an MTT assay. Cells were incubated with 0.35 mg/ml MTT solution at 37 °C for 4 h. The medium was removed, 100 µl dimethylsulfoxide (DMSO) was added and the mixture was vortexed at 112 × g for 10 min at 25°C. The optical density was read at 490 nm and all experiments were performed 3 times.

Cells were seeded in 6-well plates and treated with NG or HG, respectively, for 4 days. Cells were digested using 1 ml trypsin (#C0201; Beyotime Institute of Biotechnology, Beijing, China), washed twice with PBS and incubated in 100 µl fixation buffer (Biolegend, Inc., San Diego, CA, USA) at room temperature for 15 min. Cells were then washed with 100 µl permeabilisation buffer (Biolegend, Inc.). Following centrifugation at 1,500 × g for 3 min at 25°C, the cells were resuspended in 100 µl permeabilisation wash buffer containing Alexa Fluor 647 mouse anti-Ki-67 antibody (1:100; 561126; BD Biosciences) and incubated at room temperature in the dark for 30 min. A total of 400 µl permeabilisation wash buffer was added to resuspend the cells for flow cytometric analysis using a FACS flow cytometer (BD Biosciences).

Cell apoptosis was assayed using the Annexin V-phycoerythrin (PE) Apoptosis Detection kit (BD Biosciences). Cells were washed twice with cold PBS and resuspended in Annexin V Binding buffer at a concentration of 1.0×10⁶ cells/ml. Specifically, this suspension (100 µl) comprised 1 µl Annexin V-PE, 1 µl 7-aminoactinomycin D and 98 µl Binding buffer. The cells were vortexed gently and incubated for 15 min at room temperature in the dark. To each tube, 400 µl of Binding buffer added and cells were analyzed using a FACS flow cytometer (BD Biosciences) and FlowJo 7.6 software (FlowJo LLC, Ashland, OR, USA).

Data are expressed as the mean ± standard deviation. One-way analysis of variance was used to test the Homogeneity of variance, then a Mann-Whitney U was used to compare differences between groups. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

E-cadherin protein expression was measured in tumor tissues from 3 patients with CRC and DM and 3 patients with CRC without DM using immunofluorescence and western blotting. The area of tumor cells with positive E-cadherin staining was increased in patients without DM compared with those with DM (Fig. 1A). Furthermore, the results of western blotting confirmed that the expression of E-cadherin protein was lower in patients with DM compared with patients without DM (Fig. 1B); however, the expression of vimentin protein was significantly higher in patients with DM compared with those without DM (Fig. 1B). HCT-116 and HT-29 cells were exposed to HG for 4 days and it was demonstrated that HG reduced the expression of E-cadherin protein, whereas the expression of vimentin protein was increased (Fig. 1C). These results suggest that HG serves an important role in the EMT of CRC cells.

EMT is characterized by a loss of cell-to-cell adhesion and increased cell migration and invasion (23). As such, the effect of HG on the metastatic capability of CRC cells was investigated. Scratch assays revealed that wound healing was faster in HCT-116 and HT-29 cells grown in HG conditions compared with those grown in NG (Fig. 2A and B). Furthermore, HG accelerated the cells ability to invade and migrate compared with NG (Fig. 2C and D). These results suggest that HG is able to promote the invasion and migration of CRC cells.

HMGA2 is known to control the expression of a diverse set of transcription factors associated with the regulation of E-cadherin transcription (24,25). HMGA2 has been reported to regulate EMT in gastric cancer (26,27), tongue squamous cell carcinoma (28) and prostate cancer cells (29). As such, it was hypothesized that HMGA2 may regulate HG-induced EMT and the expression of HMGA2 in CRC cells exposed to HG or NG for 4 days was assessed. HMGA2 was significantly upregulated in HG-stimulated cells compared with those treated with NG (Fig. 3A and B). HMGA2 expression was knocked down in HCT-116 and HT-29 cells and confirmed used western blotting (Fig. 3C and D). The results revealed that HMGA2 knockdown significantly increased E-cadherin protein expression and decreased vimentin protein expression in HG-stimulated cell compared with those treated with NG (Fig. 3E and F).

To characterize the functional roles of HG in cell proliferation, MTT assays were performed and Ki-67 was measured. The results revealed that HG enhances the viability of HCT-116 and HT-29 cells in a time-dependent manner (Fig. 4A and B). Ki-67 is a nuclear antigen present only in proliferating cells and is one of the most widely used proliferation-associated markers in cancer cells (30). Ki-67 staining demonstrated that HG enhances the expression of Ki-67 and therefore the proliferation of HCT-116 and HT-29 cells compared with NG (Fig. 4C and D). The role of HG on apoptosis in HCT-116 and HT-29 cells was also assessed and it was revealed that HG significantly decreased apoptosis compared with HG (Fig. 4E and F).