Human HCT116 colon cancer cells were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in McCoy's 5A medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS; GE Healthcare Life Sciences HyClone Laboratories, Logan, UT, USA) at 37°C with 5% CO₂. The culture medium was changed every 2–3 days, and the cells were digested with 0.25% trypsin (Beyotime Institute of Biotechnology, Haimen, China) for passage when 80% confluence was reached.

The biglycan-specific shRNA plasmid was constructed using the pGCsi-H1 plasmid (Shanghai GeneChem Co. Ltd.,, Shanghai, China). The human biglycan mRNA sequence (NM_001711.4) was obtained from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/nuccore/NM_001711.4). A total of four biglycan shRNA interference sequences, in addition to a sequence of an identical length, which was not associated with the human genome sequences (serving as the negative control), were generated, according to the principles of shRNA design: shRNA-biglycan-1, 5′-GATCCCCGATCTCCAAGATCCATGAGTTCAAGAGACTCATGGATCTTGGAGATCTTTTT-3′ (forward); shRNA-biglycan-2, 5′-GATCCCCGCTCTACATCTCCAAGAACTTCAAGAGAGTTCTTGGAGATGTAGAGCTTTTT-3′ (forward); shRNA-biglycan-3, 5′-GATCCCCGCTCAACTACCTGCGCATCTTCAAGAGAGATGCGCAGGTAGTTGAGCTTTTT-3′ (forward); and shRNA-biglycan-4, 5′-GATCCCCATCCAGGCCATCGAACTGGTTCAAGAGACCAGTTCGATGGCCTGGATTTTTT-3′ (forward). The shRNA fragments were subsequently ligated into the pGCsi-H1 vector between the HindIII and BamHI sites. The constructs obtained were verified through double restriction enzyme digestion (Fermentas, Vilnius, Lithuania) and sequencing, and these were termed shRNA-biglycan 1, 2, 3 and 4, or shRNA-control.

The HCT116 cells were seeded into six-well plates at a density of 1.0×10⁵ cells/well, and were cultured overnight until 70–80% confluence was reached. The shRNA-biglycan 1, 2, 3 and 4 co nstructs or the shRNA control plasmid were subsequently transfected into the cells using Lipofectamine™ 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. G418 (Invitrogen Life Technologies) was added to the culture media at 24 h following transfection to screen for stably transfected cells. G418-resistant individual clones were retrieved at 4 weeks post-treatment. Following confirmation of the expression of biglycan, the selected stable clones were continuously cultured in G418-containing medium, and expanded for subsequent experiments.

The total RNA was extracted from the cells using TRIzol^® reagent (Invitrogen Life Technologies). The RNA was reverse-transcribed into cDNA using the first-strand cDNA synthesis kit (Takara Bio, Inc., Dalian, China), according to the manufacturer's instructions. The primer sequences were as follows: Biglycan, upstream: 5′-GGTCTCCAGCACCTCTACGCC-3′ and downstream: 5′-AACACTCCCTTGGGCACCTT-3′; β-actin, upstream: 5′-CTTAGTTGCGTTACACCCTTTCTTG-3′ and downstream: 5′-CTGTCACCTTCACCGTTCCAGTTT-3′. The RT-qPCR reactions were performed using an Exicycler™ 96 quantitative fluorescence analyzer (Bioneer Corporation, Daejeon, Korea). The PCR reactions consisted of an initial step at 95°C for 10 min; 40 cycles of 95°C for 10 sec, 58°C for 20 sec and 72°C for 30 sec; and a final step at 4°C for 5 min.

The cells were lysed using radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology), and the protein concentration of each sample was measured using the bicinchoninic acid method. Equal quantities of protein (40 µg) were separated by 13% SDS-PAGE (Solarbio Science and Technology Co., Ltd, Beijing, China) for caspase-3, p21 and p27, or by 10% SDS-PAGE in all other cases and subsequently transferred onto polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). The membrane was subsequently blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with the following diluted primary antibodies: Rabbit polyclonal anti-biglycan antibody (cat no. sc-33788; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-caspase-3 antibody (cat no. wl01992a; 1:1,000), rabbit polyclonal anti-p27 antibody (cat no. wl01769; 1:1,000), rabbit polyclonal anti-cyclin A antibody (cat no. wl01753; 1:200), rabbit polyclonal anti-p21 antibody (cat no. wl0362; 1:100), rabbit polyclonal anti-cyclin D1 antibody (cat no. wl01435a; 1:100) (all from Wanleibio, Shenyang, China), rabbit polyclonal anti-p38 antibody (cat no. sc-7149; 1:100) and anti-phosphorylated-p38 antibody (cat no. sc-101758; 1:100) (both from Abcam, Cambridge, MA, USA) at 4°C overnight. The membrane was subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (cat no. A0208; 1:5,000; Beyotime Institute of Biotechnology) at 37°C for 1 h. β-actin was used as an internal loading control, for which HRP-conjugated monoclonal mouse anti-β-actin antibody (cat no. KC-5A08; 1:10,000; Kangchen Biotech, Shanghai, China) was used. An enhanced chemiluminescence substrate (7 Sea Pharmtech, Shanghai, China) was added to the membrane in the dark for image development, and the scanned images were subsequently analyzed using Image J software (version 1.35; National Institutes of Health, Bethesda, MD, USA).

The cells were counted and seeded into 96-well plates (2×10⁴ cells/well) and subsequently cultured as before. Aliquots of 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) were added to the cell cultures at different time points, and incubated at 37°C for 1 h. The absorbance at 450 nm was measured for each well using a microplate reader (EL×800; Bio-Tek Instruments, Inc., Winooski, VT, USA).

The cells of each group were seeded into 6-well plates (1×10⁵ cells/well). The cells, which had reached 80% confluence, were used for the assay. The culture medium was discarded and a 200 µl pipette tip was used to perpendicularly scratch a wound on the surface of the cell monolayer. The cells were subsequently washed twice with serum-free culture media and cultured in McCoy's 5A medium, containing 10% FBS. Images of the cells were captured at 0, 12 and 24 h following the scratching procedure using a phase-contrast microscope (Motic AE31; Motic China, Xiamen, China), in order to calculate the distances that the cells had migrated into the denuded area.

Transwell chambers (Corning Life Sciences, Tewksbury, MA, USA) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were used for the cell invasion assay. A total of 5×10⁴ cells were collected from each group and resuspended in 200 µl serum-free cell culture medium. The cells were subsequently seeded into the upper Transwell chamber, and 800 µl McCoy's 5A culture medium, containing 10% FBS, was added to the lower chamber. Following a 24 h incubation, the cells in the upper chamber were removed using a cotton bud, and the cells present on the lower surface of the insert were fixed and stained with 0.5% crystal violet dye (Amresco, Inc., Solon, OH, USA). Subsequently, the cells were washed, images were captured and the number of cells was quantified under an inverted microscope (Motic AE31; Motic China).

The effect of biglycan on the cell cycle progression and cell apoptosis associated with colon cancer was measured by flow cytometry. Briefly, the cells were trypsinized and fixed with 70% pre-cooled ethanol for 2 h, prior to the re-suspension of the collected cells using staining solution containing 50 mg/ml propidium iodide (PI) and 1 mg/ml RNAse A (Beyotime Institute of Biotechnology) in 1 ml sodium citrate buffer. The cells were subsequently analyzed for cell cycle distribution using a BD FACSCalibur flow cytometer (BD Biosciences). Cellular apoptosis was detected using the annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (Nanjing KeyGen Biotech., Co., Ltd., Nanjing, China), according to the manufacturer's instructions. Cells which had reached 80% confluence were treated with trypsin and collected. Following washing with phosphate-buffered saline, the cells were resuspended in 500 µl annexin V-binding buffer (Nanjing KeyGen Biotech). Subsequently, 5 µl FITC-labeled annexin V and 5 µl PI dye were added the cells, prior to an incubation at room temperature in the dark for 15 min. The cells were analyzed by flow cytometry to determine the degree of cellular apoptosis.

All experimental data are expressed as the mean ± standard deviation. One-way analysis of variance was used for paired comparisons between groups, and the Bonferroni post-hoc test was used for multiple comparisons. Graphpad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was used for the data analysis and for generating the graphs. P<0.05 was considered to indicate a statistically significant difference.

To assess the role of biglycan in colon cancer cells, biglycan-specific shRNA vectors were initially constructed, which were subsequently transfected into HCT116 colon cancer cells. The mRNA expression levels of biglycan in each group of cells was determined by RT-qPCR and western blot analysis. As shown in Fig. 1, all the biglycan-specific shRNA fragments downregulated the mRNA expression level of biglycan in the transfected cells, compared with the shRNA-control group (P<0.05 or P<0.01), among which shRNA-biglycan 4 exhibited the greatest efficiency of interference (the percentage efficiency values for the downregulation were 71.33±1.53% for biglycan mRNA, and 72.33±3.51% for biglycan protein levels). On the basis of these results, the cells which were stably transfected with shRNA-biglycan 4 were selected for subsequent experiments.

The effect of biglycan on the proliferation of colon cancer cells was assessed using a CCK-8 assay. As shown in Fig. 2A, a significant decrease in cell viability was observed between 2 and 3 days in the shRNA-biglycan-transfected HCT116 cells, compared with that in the shRNA-control-transfected or non-transfected cells (P<0.01). In addition, the effect of biglycan on the progression of the cell cycle was also detected by flow cytometry. The results revealed that the downregulation of biglycan markedly affected the cell cycle distribution (Fig. 2B and C). The proportion of the cells in the G0/G1 phase was significantly increased in the shRNA-biglycan group compared with the shRNA-control or non-transfected groups (P<0.01), and the proportion of cells in S phase was decreased in the shRNA-biglycan group (P<0.01), which indicated that the cell cycle was arrested in the G0/G1 phase following biglycan silencing. To further confirm these results, the expression levels of the cell cycle markers, cyclin A and D1, and the cyclin-dependent kinase inhibitors, p21 and p27, were subsequently determined (14). The results from western blot analysis revealed that the downregulation of biglycan decreased the protein expression levels of cyclins A and D1 compared with the control cells, whereas the expression of p21 and p27 was increased following biglycan inhibition (P<0.01; Fig. 2D and E). These results suggested that the inhibition of biglycan expression arrested colon cancer cells in the G0/G1 phase, and inhibited cell proliferation.

Previous studies demonstrated that biglycan exerts a distinct role in different tumor entities (15–17). To further investigate the underlying mechanism of biglycan in colon cancer, the effect of biglycan on colon cancer cell migration and invasion was determined. A scratch wound assay revealed that biglycan knockdown markedly reduced the spontaneous migration distance of shRNA-biglycan-transfected HCT116 cells compared with that of the shRNA-control or non-transfected cells at 24 h after the scratching of the cell surface (P<0.01; Fig. 3A and B). Additionally, the effect of biglycan downregulation on the invasive ability of the cells was determined using a Transwell invasion assay. As shown in Fig. 3C and D, the number of invasive cells in the shRNA-biglycan group was significantly lower compared with that in the control group (P<0.01). These results indicated that the downregulation of biglycan expression inhibited the migration and invasive capability of the colon cancer cells.

The effect of biglycan on the apoptosis of colon cancer cells was investigated by flow cytometry. As shown in Fig. 4A and B, the proportion of apoptotic cells in the shRNA-biglycan group (18.45±1.09%) was significantly higher compared with that in the shRNA-control-transfected group (3.51%±0.16) or the non-transfected group (4.24±0.61%), indicating that the downregulation of biglycan induced colon cancer cell apoptosis. Western blot analysis also revealed that the expression of the proapoptotic caspase-3 protein was markedly increased following the downregulation of biglycan (Fig. 4C and D), which was consistent with the flow cytometry results. Furthermore, a previous study demonstrated that the p38 signaling pathway has an important role in cell apoptosis (18). In the present study, we identified that the p38 signaling pathway was activated following biglycan knockdown; the addition of the p38 signal inhibitor, SB203580, effectively reversed the increased apoptotic cell number compared with the shRNA-biglycan group (7.56±0.18%; P<0.01; Fig. 4A and B). These results indicated that biglycan may exert a role in regulating apoptosis in the colon cancer cells, specifically by inhibiting apoptosis via the regulation of the p38 MAPK signaling pathway.