A total of 62 tumor tissues and adjacent counterparts (<2 cm from the tumor site) were extracted from patients (42 male, 20 female; age, 28–79 years; median age, 66 years; T1-T4 stage; no distant metastasis or preoperative chemotherapy) with colon cancer who received tumor resections between January 2010 and December 2012 at the Department of Anus and Intestine Surgery, Weifang People's Hospital (Weifang, China). Patients' basic information was recorded, including Tumor-Node-Metastasis (TNM) stage, the degree of differentiation, lymph node metastasis and distant metastasis, according to the 2002 International Cancer Alliance TNM staging criteria (17). All tissues were rapidly placed in liquid nitrogen within half an hour after tumor resection. The Ethics Committee of Weifang People's Hospital approved the present study and informed consent from all patients was received upon admission. The clinical features of all cases are indicated in Table I.

The human colorectal cancer cell lines SW480, HT-29, LOVO, HCT116 and CACO-2 were purchased from the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were cultured at 37°C in an atmosphere containing 5% CO₂ until they reached a 60–80% confluence for subsequent experiments.

miR-193b was transfected in 6-well plates for 24 h at 37°C with 6×10⁵ SW480 cell culture cells (70–80% confluence). An miR-193b mimic (transfected with miR-193b mimic; all obtained from Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), negative-scramble control (transfected with the negative mimic, NC) and blank control (untransfected cells, BC) were used for transfection. The sequences of the miRNAs used were as follows: miR-193b mimic, sense 5′-AACUGGCCCUCAAAGUCCCGCU-3′ and antisense 5′-AGCGGGACUUUGAGGGCCAGUU-3′; and miR-193b SC, sense 5′-UUCUCGGAACCUGUCCAGUTT-3′ and antisense 5′-ACGUCAGACGUUGCGACAATT-3′. The transfection procedure was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The following mixtures were prepared: A) 10 µl miR-193b mimic (50 pmol/µl) + 250 µl opti-MEM, as provided by the kit; and B) 10 µl Lipofectamine^® 2000 + 250 µl opti-MEM. The A+B liquid was mixed together and plated for 20 min at room temperature. Subsequently, 500 µl cell culture was added drop-wise into the liquid media and 1.5 ml opti-MEM was added in each 6-well plate. The transfected cells were incubated at 37°C for 6 h and were left to culture with 2 ml RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS for 36 h at 37°C prior to further experiments. Each group was established in three replicate wells.

Using the above-described transfection method, 1×10⁵ cells/well were counted manually using a hemocytometer at 2 days following transfection. Transfected SW480 cells, which exhibited the lowest relative expression of miR-193b, were seeded at 8×10³ cells/well in a 6-well plate in serum-free RPMI-1640 medium for 12 h at 37°C in the upper chamber of Transwell plates (Costar; Corning Incorporated, Corning, NY, USA). The lower chamber contained 500 µl 20% fetal bovine Dulbecco's modified Eagle's medium. Following incubation for 36 h at 37°C the cells in the upper chamber were wiped out, fixed with 0.1% ethanol at room temperature for 20 min, stained with crystal violet at room temperature for 20 min. All migrated cells from the upper chamber were observed under an optical microscope (magnification, ×400) with crystal violet staining. Cells were counted in 5–6 randomly visual fields. Experiments were performed in triplicate.

Following transfection, transfected cells were seeded in 12-well plates (4×10⁵ cells/well) and cultured at 37°C. On reaching 100% confluence, a wound was created by scratching the cell surface with a 1 ml pipette tip. Cells were then washed three times with PBS and incubated in RPMI-1640 with 2% FBS. Wounds were observed under a light microscope (magnification, ×400) at 0 and 48 h. Cell migration was determined by subtracting the final wound width (µm) from the initial wound width (µm).

RNA was extracted from tissues and all cell lines using TRIzol reagent extract (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA RT-qPCR TaqMan method was used to detect miR-193b expression level. Specific steps were followed using the TaqMan miRNA reverse transcription kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The following conditions were applied to conduct the RT reaction: 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. The PCR product was preserved at 4°C. qPCR reaction system operation steps were performed according to the TaqMan Small RNA Assay instructions (Takara Bio, Inc., Otsu, Japan). The following thermocycling conditions were applied for qPCR: 95°C for 10 min; 95°C for 15 sec and 60°C for 60 sec for 42 cycles; and 72°C for 10 min using a Stratagene Mx3005P PCR system. The results were calculated using the 2^−ΔΔCq method (18). The miR-193b and U6 Ct value in each sample was revised via U6 internal calibration, with 2^−ΔΔCt relative quantitative method to compare the miR-193b expressed in colon cancer and matched normal tissues: ΔΔCq=Cqcarcinoma miR-193b-Cqcarcinoma U6-(Cqadjacent miR-193b-Cqadjacent U6). The relative levels of gene expression were represented as ΔCq-Cq gene-Cq reference, and the fold change of the gene expression was calculated by the 2-ΔΔCt method. Experiments were repeated in triplicate. The following primers were used: RAB22A, forward 5′-TTGTAGTTGCCATTGCAGGA-3′ and reverse 5′-AGGCTGTCTTCGGAGTTTGA-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′.

Total SW480 cell protein was extracted using radioimmunoprecipitation assay lysis buffer (Pierce; Thermo Fisher Scientific, Inc.), centrifuged at 12,000 × g for 15 min at 4°C and quantified using the Bradford method (19). Protein (50 µg) was separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA), which were then blocked for 1 h with 5% skimmed milk in Tris-buffered saline with Tween-20 at room temperature. Primary antibodies against RAB22A (cat. no. ab137093; 1:5,000, Abcam, Cambridge, UK), extracellular signal-related kinase (ERK; cat. no. AF1576; 1:800; Novus Biologicals Canada ULC; Oakville, ON, Canada), GAPDH (cat. no. sc-25778; 1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Ras (cat. no. sc-68743; 1:1,000; Santa Cruz Biotechnology, Inc.) and phosphorylated (p-)ERK l/2 (cat. no. sc-101761; 1:800; Santa Cruz Biotechnology, Inc.) were added and incubated at 4°C overnight. Following elution, mouse anti-rabbit horseradish peroxidase conjugated Immunoglobulin G (cat. no. sc-2357; 1:5,000; Santa Cruz Biotechnology, Inc.) was added and incubated for 2 h at room temperature. GAPDH was used as the reference protein. Enhanced chemiluminescent color (EMD Millipore) was added, and the blots were developed. The experiment was repeated in triplicate.

Ras separation and detection was performed using the Active Ras Pull-Down and Detection kit (cat. no. 16117; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. A total of 2×10⁶ SW480 cells were seeded in 6-well plates and transfected with miR-193b mimic for 24 h at 37°C. Subsequently, cells were treated with 500 µl pyrolysis liquid with protease inhibitors for 5 min to lyse the cells, prior to incubation with 1 mg sample and 80 µg Glutathione S-transferase-Rafl-RBD (part of the detection kit) for 1 h at 4°C. Protein centrifugation (6,000 × g for 30 sec at 4°C) was performed followed by protein concentration determination (20). Following electrophoresis and transfer to the polyvinylidene fluoride membrane, the blot was incubated at 4°C overnight with primary antibody (anti-Ras antibody; 1:200), which was included in the Ras Pull-Down and Detection kit.

Targetscan (http://www.targetscan.org/), Pictar (http://pictar.mdc-berlin.de/) and miRanda (http://www.microrna.org/microrna/home.do) were used to predict the downstream target gene of miR-193b, through bioinformatics analysis. The RAB22A 3′ untranslated region (UTR) was identified at miR-193b binding sites, and wild-type and mutant RAB22A 3′UTR luciferase reporter gene plasmids (Shanghai GenePharma Co., Ltd., Shanghai, China) were constructed for further study. Luc-RAB22A-3′UTR wild-type or mutant-type 3′pGL3 vector were constructed by Shanghai GeneChem Co., Ltd. (Shanghai, China). HEK293T cells were plated onto 96-well plates at density of 2×10⁴ cells/ml for each well 24 h prior to transfection. The Luc-RAB22A-3′ UTR wild-type or mutant-type 3′ pGL3 vector and miR-193b mimics were co-transfected into 293T cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At the time of transfection, 50 µl RPMI-1640 medium, 50 µl Dual Glo Luciferase Reagent (premixed; Promega Corporation, Madison, WI, USA), and 100 µl Glo Stop & Glo Reagent were added to each well of a LockWell MaxiSorp test plate. After 48 h of transfection, the Firefly and Renilla luciferase fluorescence values were detected using a Dual-Luciferase^® Reporter Assay System (Promega Corporation). Results were normalized to that of Renilla luciferase and the experiment was repeated in triplicate.

SW480 Cells were harvested by trypsinization, washed with PBS and fixed in cold 70% EtOH at room temperature for 1 h. Then SW480 cells were washed with PBS. Staining was performed with propidium iodide (cat. no. 81845; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at room temperature for 15 min and cell cycle profiles were analyzed using a FACS Aria II flow cytometer (BD FACSAria II; BD Biosciences, San Jose, CA, USA). Data were acquired and analyzed using the FACS DiVa software program (BD Biosciences).

GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) were used for systemic analyses and plotting data. One-way Analysis of variance was used to determine significant differences between the test conditions. ImageJ 1.51p 22 software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the grey ratios of western blotting bands. The association between miR-193b and clinicopathological parameters was compared using a χ² test. Data are presented as the mean ± or + standard error of the mean as indicated. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was used to measure the expression levels of miR-193 in 62 colon cancer tissues and adjacent normal tissue. Of the 62 patients, 40 (64.5%) exhibited a significantly decreased level of miR-193b in colon cancer tissue specimens compared with adjacent normal tissue specimens (P=0.003; Fig. 1A), The median expression value in colon cancer tissues (64.32, normalized by the internal U6 gene) was significantly decreased compared with the median value (163.7, normalized to internal U6 gene) in the corresponding normal tissues (Fig. 1A). miR-193b was expressed at the lowest level (0.43±0.08) in the SW480 cell line compared with the other four cancer cell lines (HCT116, HT-29, LOVO and CACO-2) as indicated in Fig. 1B. The SW480 cell line was therefore selected for further study. The relative expression of miR-193b in the miR-193b mimic group was significantly downregulated compared with the NC group (P<0.05), with no significant differences between the NC and BC groups (Fig. 1C).

miR-193b expression levels were significantly altered in accordance with TNM stage and lymph node metastasis status (Table I). Results indicated the miR-193b expression levels in patients with stage III+IV were significantly lower compared with those at stage I+II (P=0.030). Furthermore, patients with lymph node metastasis exhibited significantly decreased miR-193b expression levels compared with those with no lymph node metastasis (P=0.007). However, no significant differences were observed relating to age, histological type or tumor size (Table I).

A Transwell migration assay at 48 h was performed to evaluate the function of miR-193b in SW480 cancer cells (Fig. 2). Results revealed that, following transfection of miR-193b mimic in SW480 cells group, the wound closure rate was 38.3±4.4%, whereas the wound closure rate of the NC and BC groups (compared with miR-193b mimic; P<0.05) were significantly increased in comparison (64.1±6.7 and 66.9±5.2%, respectively; Fig. 2A and C).

Additionally, Transwell assay results indicated that miR-193b may have a negative correlation with invasion. The results suggested that, compared with the NC (25±4) and BC groups (22±3), the miR-193b mimic group cell migration number (11±2) was significantly decreased (P<0.05, Fig. 2B and D). No significant difference was detected between the NC and BC groups. These results suggest that exogenous miR-193b may significantly inhibit SW480 cell migration.

SW480 cycle cell cycle results indicated that transfection with miR-193b mimic compared with the other groups was significantly different (P<0.05); the proportion of cells in the G0/G1 phase of the miR-193b mimic group was significantly increased, whereas the proportion of cells in the S phase was significantly reduced compared with the NC and BC groups, respectively (P<0.05; Fig. 2E and F). There was no significant difference between the groups in the G2/M phase (data not shown).

miR-193b mimic and pMIR-REPORT luciferase vector containing the RAB22A 3′UTR binding site fragment were co-transfected for 24 h. Results indicated that the luciferase activity compared with the NC or BC group, the enzyme activity was significantly reduced by ~40% in the mimic co-transfection group (P<0.05; Fig. 3A). The luciferase assay directly demonstrated that RAB22A may be a target of miR-193b (Fig. 3A).

The primary downstream target gene of miR-193b was predicted to be RAB22A according to bioinformatics analysis. Following transfection with miR-193b mimic into SW480 cells, RAB22A expression was significantly decreased compared with that of the NC group (0.35±0.11 vs. 1.11±0.19; P<0.01; BC group densitometry value=1) via western blot analysis. No marked differences were detected between the BC and NC groups (Fig. 3B).

The association of miR-193b and RAB22A was further analyzed by exploring the role of the Ras signaling pathway. Results indicated that the miR-193b mimic markedly reduced the protein expression levels of Ras-GTP compared with the NC and BC groups (Fig. 3C). Furthermore, ERK protein is a key Ras signaling factor (21), and the phosphorylation of ERK1/2 was evaluated by western blot analysis. In SW480 cells transfected with miR-193b, the protein expression levels of p-ERK l/2 were markedly decreased compared with the NC and BC groups (Fig. 3D).