Between February 2016 and May 2018, 20 patients with colon cancer who underwent surgical resection at The Yantai Yeda Hospital (Yantai, China) were selected. Patients with a confirmed cancer diagnosis followed by postoperative pathological examination were enrolled in the study. Patients exhibiting additional types of tumors and patients who underwent preoperative radiotherapy or chemotherapy were excluded from the study. A total of 12 males and 8 females were included in the present study, with an average age of 45.1±6.9 years. In total, 4 patients exhibited T1 primary tumor stage, 7 patients presented T2 and nine patients T3. According to the tumor, node and metastasis staging system, 4 patients exhibited colon cancer at stage I, 4 at stage II, 7 at stage III and 5 at stage IV (26). A total of 12 patients exhibited low and middle degrees of differentiation (27) and 8 patients presented high differentiation. Lymph node metastasis was observed in 13 patients. The present study was approved by The Ethics Committee of Yantai Yeda Hospital and informed consent was obtained from all patients. The tumor tissues and adjacent tissues were collected and stored at −80°C. Healthy tissues, as confirmed by histopathological assays, at 2 cm away from the tumor tissue were considered as adjacent normal tissue controls.

Colorectal cancer cell lines (HCT116, SW480 and HT29) and a normal human intestinal epithelial cell line (FHC) were purchased from The Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Science, Shanghai, China; http://www.sibcb.ac.cn/). The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified water-jacketed incubator with 5% CO₂ at 37°C. The cells were subcultured at 90% confluence.

Total RNA was isolated from colorectal carcinoma tissues, adjacent normal tissues and cell lines, and the expression levels of miR-101 and CREB1 were examined. The experiments were conducted as previously described (28). Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. DNA was synthesized using the TransScript miRNA RT Enzyme Mix (TransGen Biotech Co., Ltd., Beijing, China), according to the manufacturer's protocol, as follows: RT at 50°C for 60 min and inactivation of reverse transcriptase at 70°C for 15 min. The primer sequences used were: miR-101 forward, 5′-GCGGCGTACAGTACTGTGATAA-3′, reverse, 5′-GTGCAGGGTCCGAGGT-3′; CREB1 forward, 5′-AACAATGGTACGGATGGGGT-3′, reverse, 5′-GCCATAACAACTCCAGGGGC-3′; GAPDH forward, 5′-AGAAGGCTGGGGCTCArTTG-3′, reverse, 5′-AGGGGCCATCCACAGTCTTC-3′. PCR amplification was conducted using SYBR Premix Ex Taq™ II (Takara Biotechnology Co., Ltd., Dalian, China) with a 20-µl reaction system under the conditions of 95°C for 30 sec, 95°C for 30 sec and 60°C for 30 sec for 40 cycles, following the manufacturer's instructions. RT-qPCR analysis was conducted using the LightCycler^® 480 Instrument (Roche Applied Science, Penzberg, Germany) GAPDH small nuclear RNA was used as internal reference gene. The 2^−ΔΔCq method (29) was used to quantify expression.

HT29 cells transfected with negative control mimics (miR-NC; Thermo Fisher Scientific, Inc.) or miR-101 mimics (Thermo Fisher Scientific, Inc.) were seeded into 96-well plates, using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The following sequences were used: miR-101 mimics 5′-UACAGUACUGUGAUAACUGAA-3′. The negative control for the agomir was sense 5′-UUCUCCGAACGUGUCACGUTT-3′. The final concentration of miRNA mimics was 50 nM. Following a 48-h incubation, viability was evaluated using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. Cell proliferation was analyzed by measuring the absorbance at 450 nm using a microplate reader. HT29 cells were seeded at a density of 5×10⁵ into 6-well plates and cultured under standard conditions. When the confluence reached 100%, a scratch was made using a 200 µl pipette tip. Cell migration was determined by counting the cells migrating into the scraped area. The process of wound closure was monitored and images were captured at 0 and 12 h under an inverted microscope at ×400 magnification and counted.

The targets and binding sites of miR-101 were predicted using a number of online tools and databases, including EIMMO (version2; http://www.mirz.unibas.ch/EIMMo2/) (30), miRBase (version21; http://www.mirbase.org/) (31) and TargetScan (version 7.2; http://www.targetscan.org/) (32). CREB1 was predicted to be a target of miR-101 in silico and was validated in vitro using a dual luciferase assay.

HT29 cells were plated in a 24-well plate and cotransfected with 200 ng pMIR-CREB1-wild-type (WT) or pMIR-CREB1-mutant (Mut)plasmid (Promega Corporation, Madison, WI, USA), together with miR-101 or NC mimics using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) together with miR-101 mimics or miR-NC at a concentration of 100 nM. A total of 10 ng of the pRL-TK vector containing the Renilla luciferase gene (Promega Corporation) was transfected using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Reporter activity was determined at 48 h after transfection. The luciferase activity was analyzed using a Dual-Luciferase Reporter Assay system (Promega Corporation) according to the manufacturer's protocols.

Cellular proteins were extracted by RIPA buffer (ab156034; Abcam, Cambridge, UK). Total protein concentration was quantified using BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc). The proteins (50 µg/well) were transferred onto a polyvinylidene difluoride membrane following electrophoresis with 12% SDS-PAGE. The membrane was incubated for 1 h at 37°C in 5% non-fat dry milk powder. A rabbit polyclonal antibody against CREB1 (ab31387, 1:1,000, Abcam), and a mouse monoclonal antibody for GAPDH (ab8245, 1:5,000, Abcam) were used as the primary antibodies. The membranes were subsequently probed with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (ab150077, 1:20,000, Abcam) at room temperature for 2 h. Signals were detected following incubation with a SuperSignal West Pico chemiluminescent substrate (Pierce; Thermo Fisher Scientific, Inc.), and imaged using a ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and quantified using Quantity One software (version 4.6.8; Bio-Rad Laboratories, Inc.).

HT29 cells were transfected with 150 nM miR-101 mimics or miR-NC using Amaxa-Nucleofector II (Amaxa Biosystems, Inc, Gaithers- burg, MD). Following transfection, cells were incubated in fresh medium for 24 h at 37°C. Subsequently, the cells were collected and washed with ice-cold PBS three times and subsequently suspended in PBS at a concentration of 5×10⁶ cells/ml. Nude mice (n=12, male; age, 06–08 weeks; weight, 18–22 g) were purchased from the Shanghai Laboratory Animal Center Laboratory Animal Co., Ltd. (Shanghai, China). These mice were housed five per cage under the following conditions: Constant temperature, 25°C; humidity, 40–75%; 12 h light/dark cycle and free access to food and water. These mice were divided into two groups; one group (n=6) was inoculated with cells transfected with miR-101 mimics, and the other group (n=6) with cells transfected with miR-NC. Mice were xenografted with transfected tumor cells by subcutaneous injection of 5×10⁵ cells in 100 µl. Tumor growth was measured every 3 days from the 7th day. Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated with the following formula: V=(L × W²) ×0.5. The tumors were isolated from the mice with a surgical scissor, weighed on the 22nd day, and subsequently preserved in 4% paraformaldehyde at 4°C for subsequent immunohistochemical analysis. All experiments involving animals were approved by The Ethics Committee of Yantai Yeda Hospital.

Tumor tissues were fixed overnight in 4% paraformaldehyde at room temperature, dehydrated, permeabilized and embedded in paraffin using a Leica embedding machine (Leica Microsystems, Inc., Buffalo Grove, IL, USA). Using an RM2016 slicing machine (Leica, Wetzlar, Germany), the paraffin blocks were sliced into 5-µm serial paraffin sections, which were placed in an oven overnight. Hydrated tissue sections were treated with 3% hydrogen peroxide solution to block endogenous peroxidase; the sections were subsequently acid-fixed using a pre-configured citrate buffer and the sections were placed in liquid using a microwave heating method. Following repair, blocking was performed with 5% normal goat serum albumin (Thermo Fisher Scientific, Inc.) at room temperature for 20 min. Sections were incubated overnight at 4°C with primary antibodies (rabbit polyclonal antibodies against CREB1, ab31387, 1:100; Abcam) and proliferating cell nuclear antigen (PCNA, ab18197, 1:50, Abcam). Then incubated with the secondary goat anti-rabbit IgG (horseradish peroxidase) antibody (ab150077, 1:200; Abcam) at 37°C for 30 min. Following 3,3′-diaminobenzidine staining at room temperature for 10 min, hematoxylin staining was performed at room temperature for 2 min, followed by dehydration and neutral resin mounting. Images were observed with a routine microscope at ×400 magnification and counted. The intensity of immunohistochemical staining was analyzed and quantified in three randomly selected fields per section using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).

All quantitative data for statistical analyses were from at least three independent experiments. Data are presented as the mean ± standard deviation. Comparisons between two groups were performed using Student's t-test, and paired Student's t-test was used to analyze paired data. Comparisons among three or more groups were performed using analysis of variance followed by Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The expression of miR-101 was determined in colon cancer tissues from 20 patients. The expression level of miR-101 in colorectal carcinoma tissues and the adjacent normal tissues was measured using RT-qPCR. Significant downregulation of the expression level of miR-101 was identified in all 20 colorectal carcinoma tissues compared with adjacent normal tissue (Fig. 1A). Furthermore, the expression level of miR-101 was significantly upregulated in the normal human intestinal epithelial cell line (FHC) compared with colorectal cancer cell lines (SW480, HT29 and HCT116; Fig. 1B). Collectively, the RT-qPCR results suggested that miR-101 may be involved in the development and progression of colon cancer.

Due to the downregulation of the expression level of miR-101 in colorectal cancer tissues and cell lines, it was hypothesized that miR-101 may inhibit tumor cell proliferation and migration. In order to investigate the role of miR-101 on the proliferation of HT29 cells, HT29 cells were transfected with miR-101 mimics or miR-NC. RT-qPCR was performed to examine the expression level of miR-101, and a significant upregulation of miR-101 was detected following miR-101 overexpression (Fig. 2A). CCK-8 cell viability assay suggested that overexpression of miR-101 significantly inhibited the proliferation of HT29 cells (Fig. 2B). Using a wound healing assay, it was identified that overexpression of miR-101 significantly repressed the migration of HT29 cells (Fig. 2C). Collectively, the present results suggested that overexpression of miR-101 levels may inhibit the malignant features of colorectal cancer, including cell proliferation and migration.

Potential targets of miR-101 were predicted using miRBase (http://www.mirbase.org/), and miR-101 binding sites were identified in the 3′-UTR of CREB1, one of the putative targets (Fig. 3A). To investigate whether miR-101 interacts with the CREB1 transcript in vitro, the miR-101 binding sites in the 3′-UTR of CREB1 were mutated, and a dual luciferase assay was conducted (Fig. 3A). The pMIR-CREB1-WT plasmid, containing the 3′-UTR of CREB1, or the pMIR-CREB1-Mut reporter, containing a mutated form of the 3′-UTR, was cotransfected into HT29 cells together with miR-101 mimics or miR-NC mimics and luciferase assays were performed. Transfection of miR-101 mimics significantly decreased the luciferase activity of pMIR-CREB1-WT reporter plasmid compared with the miR-NC mimics. However, miR-101 overexpression did not affect the luciferase activity of the pMIR-CREB1-Mut reporter plasmid compared with the miR-NC mimics (Fig. 3B). Collectively, the present results suggested that miR-101 may be able to bind directly to the 3′-UTR of CREB1 to regulate gene expression. To investigate whether miR-101 regulated the expression of CREB1, HT29 were transfected with miR-101 and the protein and mRNA expression levels of CREB1 were examined using western blot and RT-qPCR analysis, respectively. miR-101 overexpression significantly decreased the mRNA and protein expression levels of CREB1 (Fig. 4). The present results suggested that miR-101 was able to decrease the expression level of CREB1 in colon cancer cells by directly binding the 3′-UTR of CREB1.

To investigate the role of CREB1 in colon cancer progression, the expression level of CREB1 was assessed using RT-qPCR in three human colorectal cancer cell lines (SW480, HT29 and HTC116) and the FHC cell line. Furthermore, the expression level of CREB1 was investigated in colorectal carcinoma tissues and adjacent normal tissues. The expression level of CREB1 was significantly increased in the three tumor cell lines compared with the FHC cell line (Fig. 5A). Furthermore, the expression levels of CREB1 were significantly upregulated in the tumor tissues compared with the adjacent normal tissues (Fig. 5B). Notably, the immunochemistry results suggested that the protein expression of CREB1 was significantly upregulated in the tumor tissues compared with the normal tissues (Fig. 5C). Additionally, it was identified, by western blotting, that the protein expression level of CREB1 in colon cancer tumor tissues was significantly increased compared with adjacent normal tissues (Fig. 5D). Collectively, the present results suggested that miR-101 may serve an important role in colon cancer by inhibiting CREB1 expression.

HT29 cells were injected into mice to generate a murine xenograft model of colon carcinoma. Transfected cells were injected subcutaneously into the back of nude mice. The tumor growth rate was measured every 3 days and at the end of the experiment the tumors were surgically removed, weighed and imaged (Fig. 6). The present results suggested that overexpression of miR-101 suppressed tumor growth in vivo. Subsequently, immunohistochemical analysis was performed using anti-PCNA and anti-CREB1 antibodies to detect proliferating cells and CREB1 protein expression levels, respectively. The number of cells positive for PCNA and CREB1 staining was significantly decreased in xenograft tumor tissues produced from HT29 cells transfected with miR-101 compared with miR-NC xenografts (Fig. 7A). Additionally, the protein expression level of CREB1 was significantly increased compared with adjacent normal tissue in nude mice xenografted with colon cancer cells, as detected by western blotting (Fig. 7B), suggesting that CREB1 may be involved the development and progression of colon cancer. The present results suggested that overexpression of miR-101 led to downregulation of the expression level of CREB1 and significant inhibition of tumor growth.