Four human CRC cell lines: SW480, SW620, HT-29 and HCT-116, and one normal colon epithelial cell line (CCD-18Co), were purchased from American Type Culture Collection (Manassas, VA, USA). The HEK-293T normal human embryonic kidney cell line was obtained from Shanghai Cell Collection (Shanghai, China). The above cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 4 mM glutamine at 37°C in an atmosphere containing 5% CO₂.

CRC tissue and adjacent normal colonic mucosa tissue samples were obtained by surgical removal from patients diagnosed with primary CRC (eight men and seven women; mean age, 53.4 years) at the Department of Gastrointestinal Surgery, China-Japan Union Hospital, Jilin University (Changchun, China). The patients provided written informed consent and the present study was approved by the ethics committee of the China-Japan Union Hospital, Jilin University. The tumor samples (average size, 2.23 cm) and adjacent non-cancerous tissue samples (average size, 1.1 cm) were placed in DMEM supplemented with 20% FBS, minced with scissors, and digested in 1% Collagenase I (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 1 h at 42°C. Subsequently, the cells were re-suspended in DMEM supplemented with 20% FBS and cultured at 37°C in a humidified atmosphere containing 5% CO₂. After 24 h, the media was replaced with fresh DMEM containing 10% FBS.

RT-qPCR was performed according to the procedures described in a previous study (12). Briefly, total RNA was isolated from the digested CRC and paired normal colon tissue samples and CRC and normal colon cell lines using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer's instructions. To detect miRNA expression in paraffin-fixed CRC sections, a paraffin-fixed tissue RNA extraction kit was used (Beinuo Life Science, Dusseldorf, Germany), according to the manufacturer's instructions. Briefly, sections of the tissue samples were placed in liquid nitrogen and then stored at 280 uC for RNA and protein extraction, for RT-qPCR and western blotting analysis, respectively. The tissue samples were fixed in 10% buffered formalin and embedded in paraffin. Formalin-fixed and paraffin-embedded tissues were cut into 4 µm sections. For the detection of miR-155, an RT reaction was performed using an All-in-One™ First-Strand cDNA Synthesis kit (cat. no. AORT-0020; GeneCopoeia, Rockville, MD, USA), according to the manufacturer's instructions. qPCR was performed using an All-in-One™ miRNA qRT-PCR Detection kit (AOMD-Q020; GeneCopoeia) and a CFX96™ Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). U6 was selected as an endogenous control. The primers and probes for miR-155 and U6 were obtained from GeneCopoeia.

In order to detect the mRNA expression levels of Axin2, CD44 and Lgr5, the isolated RNA was transcribed into cDNA using a ReverTra Ace^® qPCR RT kit (Toyobo Co., Ltd., Osaka, Japan), according to the manufacturer's instructions, followed by a qPCR assay. Total RNA was polyadenylated using a Poly(A) Polymerase Tailing kit (Epicentre, Chicago, IL, USA). The reaction mixture included 1 mg RNA, 1 ml of 10 mM ATP, 1 ml of 10X reaction buffer, and 1 U Poly(A) polymerase. The reaction was conducted by incubation at 37°C for 25 min followed by enzyme inactivation at 65°C for 6 min. Reverse transcription was subsequently performed with a reaction mixture containing 1 ml polyadenylation reaction product, 1 ml AMV 10X reaction buffer, 1 ml of 0.5 mM RT primer, 0.5 ml of 10 mM dNTP, and 50 U of AMV High Performance Reverse Transcriptase. The reaction was conducted with incubation at 42°C for 45 min, followed by 70°C for 10 min. The PCR reaction was performed at 95°C for 30 sec, followed by 36 cycles of 95°C for 10 sec and 60°C for 30 sec. cDNA was mixed with primers and SYBR^® Green PCR Master Mix according to the manufacturer's protocol. Quantification was performed using a Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA). The primer sequences were as follows: Axin2, forward 5′-CCGGTGGACCAAGTCCTTAC-3′ and reverse 5′-TCCATTGCAGGCAAACCAGA-3′; CD44, forward 5′-AGTCCCTGGATCACCGACAG-3′ and reverse 5′-GTTTCTTGCCTCTTGGTTGCT-3′; Lgr5, forward 5′-TGAACACCTGCTTGATGGCT-3′ and reverse 5′-TGCTGCGATGACCCCAATTA-3′; HMG-box transcription factor 1 (HBP-1), forward 5′-CTTGCCTTATCCGTGCAGGT-3′ and reverse 5′-GCCTGAGATTTCGACTTGCC-3′; and GAPDH, forward 5′-TCAGTGGTGGACCTGACCTG-3′ and reverse 5′-TGCTGTAGCCAAATTCGTTG-3′. RT-qPCR was conducted using an ABI7900-HT Sequence Detection system (Applied Biosystems Life Technologies, Foster City, CA, USA). The RT-qPCR products were separated on a 1% agarose gel. For relative quantification of mRNA expression levels, and the mRNA expression levels in methionine-exposed cells were plotted as the fold increase compared with untreated samples. RNA (100–300 ng/µl) was also quantified with a Nanodrop 2000. GAPDH was used for normalization and the Ct values for each triplicate sample were averaged.

Northern blot analysis was performed to determine the levels of miR-155 in the indicated samples and cell lines, according to previously described procedures (13). Briefly, total RNA was isolated from the tissues and cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), which was then separated using 15% urea-PAGE (8 M; 480 g urea/liter; Sigma-Aldrich). The blots were subsequently transferred onto nylon membranes (GE Healthcare Life Sciences, Little Chalfont, UK), and subjected to ultraviolet cross-linking. The blots were hybridized with digoxigenin (DIG)-labeled miR-155 or U6 probes (Exiqon, Vedbæk, Denmark) overnight at 4°C. The membranes were then washed using a low-stringency buffer containing Tris-Hcl 20 mM, Nacl 150 mM, CaCl2 10 mM, 1% Triton x-100 (Sigma-Aldrich). A DIG Luminescent Detection kit (Roche Diagnostics, Basel, Switzerland) was used to examine the abundance of miR-155, according to the manufacturer's protocol.

To alter the expression levels of miR-155 in the cells, mirVana™ miRNA inhibitors or mimics (30 nM) of the miR-155 and control molecules (30 nM) (Invitrogen Life Technologies) were transfected into the SW620, HT-29, CCD-18Co and HEK-293T cells using Lipofectamine^® 2000, according to the manufacturer's instructions, 48 h prior to subsequent experiments.

The SW620 and HT-29 cells were plated at a density of 5×10³ cells into 96-well plates. After 6 h at 37°C, the cells were treated with the indicated mimics/inhibitor. After 0, 24, 48 or 72 h, the cells were treated with 10 µl MTT (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37°C. Following treatment, the medium containing the MTT solution was removed and 150 µl dimethyl sulfoxide was added. Absorbance was then spectrophotometrically determined at a wavelength of 570 nm using a Bio-Rad model 550 microplate reader (Bio-Rad Laboratories, Inc.). Cell viability was calculated as follows: Viability (%) = absorbance value of adenovirus-infected cells / absorbance value of control cells.

Cell cycle analysis was performed according to previously described methods (14). Briefly, 2×10⁵ cells were fixed with cold 70% ethanol for 1 h at 48 h following the treatments described above. RNase A (1%; (Sigma-Aldrich) was then used to remove the contaminated RNA. Propidium iodide (50 mg/ml; Sigma-Aldrich) was added to the fixed cells, which were then subjected to flow cytometry (FACSAria™ II; BD Biosciences, Franklin Lakes, NJ, USA). The proportion of cells in the G₀/G₁, S and G₂/M phases were determined using Modfit software 2.0 (BD Biosciences).

An immunoblot assay was performed, according to routine laboratory procedures. Briefly, all of the cells were lysed using radioimmunoprecipitation assay buffer containing 1 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mmol/l phenylmethylsulfonyl fluoride (Sigma-Aldrich) for 45 min. Subsequently, 45 µg of each total protein was separated by 10% SDS-PAGE prior to being transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated in 1% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature. The membranes were then washed three times with Tris-buffered saline with Tween 20 (TBST; Sigma-Aldrich). The membranes were incubated with the following primary antibodies at 4°C overnight at a dilution of 1:1,000: Monoclonal rabbit anti-human Ki-67 (cat. no. 9129), total β-catenin (cat. no. 9582), phosphorylated β-catenin (cat. no. 2009) and GAPDH (cat. no. 5174) (Cell Signaling Technology, Inc.; Danvers, MA, USA) and HBP1 (cat. no. ab83402; Abcam, Cambridge, UK). Then the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h following three washes with TBST. The expression levels were quantified using a Tanon GIS Gel Imager system (Tiangen, Beijing, China).

The animal experiments performed in the present study were approved by the Committee on the Use and Care of Animals of the China-Japan Union Hospital, Jilin University. A SW620 CRC xenograft was established by injecting 4×10⁶ cells into the flanks of 4–7 week-old male BALB/c nude mice (n=14) obtained from the Institute of Zoology, Chinese Academy of Sciences (Beijing, China). The mice were house at 26°C with 10 h light each day, and provided access to sterile food and water with separate feeding. The 14 mice were randomly divided into two groups (n=7), and six of those were chosen for subsequent experimentation. Once the diameter of the tumor reached 5–9 mm, miR-155 inhibitors or control molecules were intratumorally administered into the xenografts (30 nM in 100 µl). The tumor diameters were periodically measured using vernier calipers, and the tumor volume was calculated as follows: Tumor volume (mm³) = [maximal length (mm) × perpendicular width (mm)]²/2.

A TOPflash assay (Genomeditech) was performed to determine the activation of Wnt/β-catenin signaling, in which a TCF-responsive luciferase-expressing plasmid (Genomeditech, Shanghai, China) was transfected into the SW620 and HT-29 cell lines at a cell density of 70% in 10 cm culture dishes. The transfection was performed at 37°C for 24 h. A luciferase assay was performed, as previously described (15). Briefly, the growth medium was removed from the cells to be assayed. The cells were then washed twice with phosphate-buffered saline, with care so as not to dislodge any of the cells. A minimal volume of 1X Cell Culture Lysis reagent (Promega Corporation, Madison, WI, USA) was added to cover the cells (250 µl for a 60 mm dish), and the cells were incubated for 15–30 min at room temperature. The attached cells were removed from the culture dish and transferred to a microcentrifuge tube. The solution was centrifuged for 5 sec at 10,000 × g to pellet the cell debris. The supernatant (cell extract) was then transferred to a new tube and the pelleted cell debris were discarded. A total of 20 µl cell extract was added to 100 µl luciferase assay reagent (Promega Corporation) at room temperature. The solution was placed in an Lmax II Luminometer (Molecular Devices, Sunnyvale, CA, USA). The light produced was measured for 10 sec and the data recorded.

The TargetScan (http://www.targetscan.org/) online database was used to identify potential miR-155 targets, and HBP1 was identified as a predicted miR-155 target. In order to verify HBP1 as an authentic miR-155 target, a pMIR-REPORT vector was reconstructed by inserting a luciferase reporter vector (Genomeditech) containing a 205-bp long DNA sequence of HBP1 3′-UTR with a putative miR-155 binding site (AGCATTAA), in order to generate pMIR-REPORT-HBP1-3UTR-wt (Genomeditech). A luciferase vector harboring a mutant miR-155 binding site (AGC AAA TT) was also constructed and used as a control (pMIR-REPORT-HBP1-3UTR-mut). miR-155 mimics were transfected into the HEK-293T cells using Lipofectamine^® 2000, and luciferase activity was detected using a Dual-Luciferase^® Reporter Assay kit (Promega Corporation).

HBP1-specific small interfering RNA (siHBP1) was used to reduce the expression levels of HBP1 in the cells. The HBP1 siRNA had the following sequence: forward, 5′-GCUUGACUGUGGUACAGCATT-3′, and reverse, 5′-UGCUGUACCACAGUCAAGCTT-3′. siHBP1 was obtained from Shanghai Genepharma Co., Ltd. (Shanghai, China), and 10 ng/ml was transfected into the SW-620 cells, together with the miR-155 inhibitors or controls. Following 48 h culture at 37°C, the previously described immunoblot assay was performed.

The data were representative of three independent experiments performed in triplicate and were presented as the mean ± standard deviation. The data in the present study were analyzed using a two-tailed Student's t-test. Statistical analyses were conducted using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Initially, the expression levels of miR-155 were elevated in the CRC tissue samples, compared with the paired normal colon tissue samples, as determined by RT-qPCR (n=30; P<0.01; Fig. 1A). Furthermore, in the CRC cell lines, the expression levels of miR-155 were increased (P<0.05; Fig. 1B). Northern blot analysis demonstrated that the miR-155 bands were significantly denser in the CRC tissue samples and cell lines, compared with normal tissue samples and cell line (Fig. 1C).

An miR-155 inhibitor was used to suppress the endogenous expression of miR-155 in the CRC cells (Fig. 2A). Subsequent MTT assays revealed that the proliferation rates of the SW620 and HT-29 cells were suppressed following transfection with miR-155 inhibitor (Fig. 2B). The downregulation of miR-155 also induced an accumulation of cells in the G₀/G₁ phase (Fig. 2C). In addition, a biomarker of proliferation, Ki-67, was consistently underexpressed in the SW620 and HT-29 cells transfected with the miR-155 inhibitor (Fig. 2D).

In a CRC xenograft murine model, treatment with miR-155 inhibitor impaired the growth of established SW620 xenografts, as evidenced by a lower tumor volume in the miR-155-silenced group (Fig. 3A). Furthermore, the weights of the CRC tumors in the mice treated with miR-155 inhibitor were reduced, compared with those in the control group (Fig. 3B).

The downregulation of miR-155 suppressed the activation of the Wnt/β-catenin pathway, as evidenced by downregulation of Wnt/β-catenin pathway responsive target genes, including Axin2, CD44 and Lgr5 (Fig. 4A) in the cells transfected with a miR-155 inhibitor, and reduced activity of the TOPflash plasmid, which reflects activation of the Wnt/β-catenin signaling pathway (Fig. 4B). However, in the cells transfected with the miR-155 inhibitor, the phosphorylation of β-catenin was not affected (Fig. 4C).

It was suggested that a potential MRE of miR-155 exists within the 3′-UTR of HBP1 mRNA (Fig. 5A). Inhibition of miR-155 induced an elevation in the protein expression of HBP1, whereas forced overexpression of miR-155 decreased its expression levels (Fig. 5B and C). In the HEK-293T cells, luciferase expression by pMIR-REPORT-HBP1-3UTR-wt was significantly suppressed following transfection with exogenous miR-155, whereas those of pMIR-REPORT-HBP1-3UTR-mut or pMIR-REPORT were unaffected (Fig. 5D).

siHBP1 was used to suppress the expression of HBP1 following miR-155 silencing (Fig. 6A). siHBP1 increased the mRNA expression levels of Axin, CD44 and Lgr5, which were reduced following transfection with the miR-155 inhibitor (Fig. 6B). The TOPflash assay further confirmed that HBP1 suppression restored the activation of the Wnt/β-catenin pathway following transfection with the miR-155 inhibitor (Fig. 6C). Additionally, the MTT assays revealed that the suppression of HBP1 attenuated the effects of miR-155 silencing on CRC cell proliferation (Fig. 6D). In addition, flow cytometric analysis of cell cycle progression demonstrated that siHBP1 reversed G₀/G₁ arrest in the SW620 cells transfected with the miR-155 inhibitor (Fig. 6E).

These above data indicated that HBP1 was required for the effects of miR-155 on CRC cell proliferation and activation of the Wnt/β-catenin pathway.

Spearman's rank analysis demonstrated that patients with high levels of miR-155 exhibited a shorter survival rate following surgery, compared with patients exhibiting low levels of miR-155. These results suggested an inverse association between the expression of miR-155 and patient survival rate (Fig. 7).