A total of 31 pairs of tumor tissues and adjacent tissues, which were at least 2-cm distal to tumor margins, were collected from 31 patients with CRC who were admitted to the Affiliated Hospital of Shandong University and underwent surgery between January 2014 and January 2015. There were 15 male patients and 16 female patients with the age ranged from 47 to 73 years old. Following collection, tissues were quickly frozen and stored in −196°C liquid nitrogen. None of the patients received neoadjuvant therapy. The present study was approved by the Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Jinan, China). Informed consent was obtained from each patient. Tissues were used for the detection of mRNA expression levels.

The human CRC cell line, SW480, was purchased from Nanjing KGI Biotechnology (Nanjing, China) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin, and incubated in a humidified chamber at 37°C with an atmosphere of 5% CO₂ and 95% air.

SW480 cells were seeded into 24-well plates at a density of 1×10⁵ cells/well. Cells were randomly divided into two groups: miR-766-5p inhibitor group and miR-negative control (NC) inhibitor group. Transfection of SW480 cells with 30 µM miR-766-5p inhibitor or miR-NC inhibitor (GeneCopoeia, Rockville, MD, USA) was conducted by Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. SW480 cells were collected 48 h after transfection for subsequent experiments.

Suppressor of cancer cell invasion (SCAI) was predicted to be recognized by miR-766-5p using TargetScan (http://www.targetscan.org/), and the recombinant plasmids of pmir-SCAIwt-3′UTR and pmir-SCAImut-3′UTR (Promega Corporation, Madison, WI, USA) were constructed. SW480 cells were incubated for 24 h at 37°C, followed by co-transfection of miR-766-5p inhibitor and pmir-SCAIwt-3′UTR or pmir-SCAImut-3′UTR using Lipofectamine 2000 reagents. After 24 h, luciferase activities were assessed using the Dual-Luciferase Reporter Assay System (Promega Corporation). Renilla luciferase was used as an internal control.

SW480 cells (2×10³ cells/well) were seeded onto 96-well plates. Cell viability was evaluated with a cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Rockville, MD, USA), according to the manufacturer's protocol, at 24, 48 or 72 h after transfection with miR-766-5p inhibitor or miR-NC inhibitor. Absorbance was read at 490 nm with an iMARK plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Transfected SW480 cells were seeded into 12-well plates at the density of 2×10⁵/well, cultured for 48 h at 37°C and collected via centrifugation at 23,200 × g for 5 min at 4°C. Following three washes with PBS, SW480 cells were re-suspended in 100 µl binding buffer, stained with annexin V-fluorescein isothiocyanate and propidium iodide (Roche Diagnostics, Basel, Switzerland) for 15 min at room temperature in the dark. The rate of cell apoptosis was determined by flow cytometry (Beckman Coulter, Inc., Miami, FL, USA) in 1 h. The cell number at each phase was analyzed suing FloJo software (Version 7.6.3, Treestar, Inc., Ashland, OR, USA).

Cell invasiveness of SW480 cells was evaluated by Transwell chambers, which were coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Transfected SW480 cells were placed in Transwell upper chambers that were coated with gelatin. Functioning as a chemoattractant, 0.2% BSA containing fibronectin (Bio-Techne, Shanghai, China) (10 µg/ml) was added to the bottom chamber. For the invasion assay, 100 µl transfected SW480 cells at a concentration of 1×10⁵ cells were cultured in the upper chamber in serum-free Dulbecco's modified Eagle's medium at 37°C in 5% CO₂ humidified air. A total of 16 h later, non-invading cells on the upper chambers were removed with a cotton swab, and invading cells that reached the bottom chambers were fixed with 70% ethanol for 15 min at room temperature and stained by Hemacolor^® Rapid staining solution (Merck KGaA, Darmstadt, Germany) at room temperature for 30 min. The number of invasive cells was counted under a light microscope (magnification, ×400).

Cell migration was evaluated using a wound healing assay. Transfected SW480 cells were cultured in an incubator at 37°C with 5% CO₂. After incubation for 24 h, a wound gap was created using a 20-µl pipette tip. Transfected SW480 cells were further cultured for 24 h. Thereafter, wounded monolayers were photographed with an inverted microscope (magnification, ×400).

Protein lysates were obtained with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China). Concentration of protein samples were determined using the BCA method (Beyotime Institute of Biotechnology). Protein samples (15 µg/lane) were first separated by 10% SDS-PAGE and transferred, at 4°C, onto polyvinylidene difluoride membranes followed by blocking by 5% skim milk at room temperature for 1 h. Membranes were first incubated overnight with primary antibodies (SCAI, 12892, 1:1,000, Cell Signaling Technology, Inc., Danvers, MA, USA; MMP2, ab37150, 1:1,000, Abcam, Cambridge, UK; p-PI3K, ab151549, 1:1,000, Abcam; p-AKT, 4060, 1:1,000, Cell Signaling Technology, Inc.; GAPDH, 5174, 1:1,000, Cell Signaling Technology, Inc.) at 4°C, then treated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature. Blots were visualized with an enhanced chemiluminescent kit (Beyotime Institute of Biotechnology).

Total RNA was extracted from SW480 cells at 48 h after transfection and from tissue samples from patients with CRC using TRIzol (Thermo Fisher Scientific, Inc.), and RT-qPCR for miRNA was executed using a one-step TaqMan miRNA reverse-transcription kit (Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used: 95°C for 40 sec at initial denaturation, followed by 45 cycles for 30 sec at 95°C for denaturation and 30 sec at 58°C, with the final extension at 72°C for 35 sec. The relative mRNA expression levels of miR-766 (normalized to U6) and SCAI (normalized to GAPDH), were calculated using the 2^−ΔΔCq method (14). The primer sequences were as followed: GAPDH, forward 5′-AGCCTCCCGCTTCGCTCTCTGC-3′ and reverse 5′-ACCAGGCGCCCAATACGACCAAA-3′; U6, forward 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse 5′-CGCTTCACGAATTTGCGT-3′; SCAI, forward 5′-CGGGAAACACGAAATTATCC-3′ and reverse 5′-GCTTCTGGAGATGAGGATTCTC-3′; and miR-766, forward 5′-TCGAGTACTTGAGATGGAGTTTT-3′ and reverse 5′-GGCCGCGTTGCAGTGAGCCGAG-3′.

All data were presented as the mean ± standard deviation. The analysis was performed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Differences between two groups were analyzed using a Student's t-test, and differences between three or more groups were analyzed using one-way analysis of variance followed by Newman-Keuls analysis. P<0.05 was considered to indicate a statistically significant difference.

The miR-766-5p expression level in patient tissues was first evaluated by RT-qPCR. Results demonstrated that the expression level of miR-766-5p was significantly higher in cancer tissue than the level in healthy tissue, which suggests that miR-766-5p may be a promoter during the progression of CRC (Fig. 1).

TargetScan was applied for the prediction of interaction between miR-766-5p and its potential targets. SCAI demonstrated a relative higher score when compared with other potential targets, thus, SCAI was selected in the present study. Binding sequences between miR-766-5p and SCAI 3′UTR, as well as the mutant sequences, are presented in Fig. 2A. Luciferase activity in SW480 cells that were transfected with pmir-SCAIwt-3′UTR and miR-766-5p inhibitor was decreased compared with cells transfected with pmir-SCAIwt-3′UTR and miR-NC inhibitor, while there was no significant difference in cells transfected with pmir-SCAImut-3′UTR and miR-766-5p inhibitor in comparison with cells transfected with pmir-SCAImut-3′UTR and miR-NC inhibitor (Fig. 2B). These results demonstrate that miR-766-5p targets and binds with the 3′UTR of SCAI.

Patients' age, sex, tumor size, tumor differentiation degree and metastasis are presented in Table I. There were no significant differences in patient age or gender; however, there were significant differences in tumor size, differentiation degree and metastasis.

SCAI expression level in patient tissues was determined by RT-qPCR. Results demonstrated that the SCAI expression level was significantly lower in cancer tissue than in healthy tissue (Fig. 3).

For in vitro experiments, SW480 cells were transfected with miR-NC inhibitor and miR-766-5p inhibitor. The cells in the two different groups were used for the detection of miR-766-5p and SCAI expression levels by RT-qPCR. Results demonstrated that, compared with the NC group, miR-766-5p expression was significantly decreased and SCAI expression was significantly increased in the miR-766-5p inhibitor group (Fig. 4A and B). These results suggest that SW480 cells were successfully transfected with miR-766-5p inhibitor. The effects of miR-766-5p on cell behaviors and the corresponding molecules were subsequently determined.

CCK-8 assay was used for the examination of SW480 cell viability in the different groups. Results demonstrated that miR-766-5p inhibitor significantly inhibited SW480 cell proliferation compared with the NC group at 24, 48 and 72 h after transfection (Fig. 5).

Flow cytometry was used for the examination of SW480 cell apoptosis in the different groups. Results demonstrated that miR-766-5p inhibitor significantly promoted SW480 cell apoptosis (35.98%) compared with the NC group (17.79%) (Fig. 6A and B).

A wound healing assay was used for the examination of SW480 cell migration, while Transwell assays were used for the examination of SW480 cell invasion in different groups. Results demonstrated that the miR-766-5p inhibitor markedly inhibited SW480 cell migration/invasion compared with that observed in the NC group (Fig. 7A and B).

In comparison with the NC group, protein expression levels of MMP2 and phosphorylated (p)-PI3K/AKT were decreased, while SCAI was increased following treatment with miR-766-5p inhibitor. GAPDH was used as an internal control (Fig. 8). Taken together, these results indicated that, miR-766-5p inhibitor repressed the activation of p-PI3K/p-AKT signaling by targeting SCAI.