In total, 42 patients with CRC (23 male, 19 female; median age, 63 years) and 42 healthy individuals (24 male, 18 female; median age, 60 years) were recruited from The First Hospital of Hebei Medical University (Shijiazhuang, China) between October 2013 and March 2014. No previous local or systemic treatment had been administered to the 42 patients with CRC prior to surgery. Written informed consent was obtained from all of the patients, in accordance with the protocols of the Ethics Review Board at Hebei Medical University (Shijiazhuang, China), which approved the present study.

A total of 42 pairs of CRC and non-tumor adjacent tissues were obtained from patients that underwent radical resection between October 2013 and March 2014 at The First Hospital of Hebei Medical University. The corresponding normal tissues were obtained from a section of the resected specimen ≥5 cm from the edge of the tumor. The samples were stored in liquid nitrogen or an −80°C freezer until use.

Plasma samples were collected from 42 CRC patients prior to surgical resection and 7 days following surgery respectively. Furthermore, 42 plasma samples were obtained from 42 cases of age-matched healthy individuals as the control. Cell-free plasma was extracted from all blood samples within 2 h of collection using a two-step protocol at 2,140 × g for 10 min and 12,840 × g for 2 min. Plasma was transferred to fresh tubes and stored in a −80°C freezer until use.

The human CRC cell line HCT116 was obtained from Professor Xiaofeng Sun (Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden). Cells were cultured in McCoy's 5A medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomcyin (Invitrogen; Thermo Fisher Scientific, Inc.) in a 37°C humidified incubator containing 5% CO₂.

Total RNA was extracted from the frozen tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, and resuspended in 60 µl pre-heated (95°C) nuclease-free water.

Total RNA was extracted from 400 µl plasma samples using the miRNeasy Serum/Plasma kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol, and eluted with 105 µl pre-heated (95°C) double distilled water.

The concentration and purity of all RNA samples were detected using a NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). All RNA was stored at −80°C until use following determination of the concentration RNA using a spectrophotometer.

Reverse transcription was carried out using the One Step PrimeScript^® miRNA cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol.

Quantitative polymerase chain reaction (qPCR) was performed using an All-in-One^™ miRNA qPCR kit (QP101; GeneCopoeia, Inc., Guangzhou, China) with an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. All-in-OneTM miRNA qPCR Primers were used, including a hsa-miR-106a-5p primer pair (HmiRQP0026) and a HsnRNA U6 primer pair (HmiRQP9001; GeneCopoeia, Inc.). The PCR reaction mixture included 10 µl 2X All-in-One^™ qPCR mix, 2 µl All-in-One^™ miRNA qPCR Primer, 2 µl Universal Adaptor PCR Primer, 2 µl template cDNA and 4 µl double distilled water, for a total volume of 20 µl. The reaction mixture was incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. All RT-qPCR reactions were performed in triplicate.

U6 small nuclear RNA was used as a housekeeping gene to normalize miRNA expression, and the relative expression of miR-106a in CRC cells was determined using the comparative threshold cycle (Cq) method (2^−ΔΔCq) (17).

HCT116 cells were seeded into 96-well plates and then transfected with miR-106a mimic (miR10000103-1-5), miR-106a mimic control (miR01101-1-5), miR-106a inhibitor (miR20000103-1-5) or miR-106a inhibitor control (miR02101-1-5; all RiboBio, Guangzhou, China) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 24 h after culture, the cells were harvested and absorbance values at 450 were determined using CCK-8 reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. Tests were performed in triplicate.

Fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) staining were performed by means of the Annexin V-FITC Apoptosis Detection kit (Neobioscience, Shenzhen, China). Following transfection with miR-106a mimic or inhibitor, or negative control, cells were cultured for 48 h, resuspended in 1X binding buffer, washed twice with PBS, and treated with Annexin V-FITC and PI. Following 10 min of incubation at room temperature in the dark, cells were submitted to flow cytometry with a BD FACSCalibur flow cytometer calibrated with CaliBRITE beads, and the results were analyzed using CellQuest software (all BD Biosciences, San Jose, CA, USA). Tests were performed in quadruplicate.

Early apoptotic cells were located in the lower-right quadrant, late apoptotic or necrotic cells were located in the upper-right quadrant, normal cells were located in the lower-left quadrant and mechanically damaged cells were located in the upper-left quadrant of the flow cytometric dot plots (18).

Each experiment was conducted at least three times. All values are presented as the median and 25th and 75th percentiles or the mean ± standard deviation. Differences were tested for significance using a Student's t-test, unpaired t-test, Mann-Whitney U test or a two-way analysis of variance followed by Tukey's post hoc test in SPSS software (version 19.0; IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Our previous study (19) identified that miR-106a expression was increased in the plasma of patients with CRC compared with healthy controls. Therefore, it was hypothesized that miR-106a expression may be associated with the progress of CRC. In the present study, the effects of miR-106a on cell viability and apoptosis in CRC cells were analyzed. First, to measure the effect of miR-106a on cell viability, a CCK-8 assay was carried out. HCT116 cells were transfected with miR-106a mimic or mimic control, miR-106a inhibitor or inhibitor control. Cell viability curves and, at 24 h after transfection, miR-106a expression were examined (Fig. 1).

The expression of miR-106a and cell viability were significantly upregulated by the miR-106a mimic comparison with mimic control in HCT116 cells (P<0.01; Fig. 1A and B, respectively). Conversely, following transfection with miR-106a inhibitor, the viability of HCT116 cells was also significantly decreased compared with inhibitor control-transfected cells (P=0.0036; Fig. 1C) and the expression of miR-106a was significantly decreased (P=0.0004; Fig. 1D). These results indicated that the miR-106a expression level was positively associated with CRC cell viability.

The function of miR-106a on cell apoptosis was determined using flow cytometry. HCT116 cells were transfected with miR-106a mimic or mimic control, miR-106a inhibitor or inhibitor control. At 48 h after transfection, the apoptosis rates of HCT116 cells were determined (Fig. 2).

Following transfection with miR-106a mimic or mimic control, cell status was determined using flow cytometry, with early apoptotic cells located in the lower right quadrant of the plot (Fig. 2A and B). HCT116 cells transfected with miR-106a mimic exhibited a lower rate of early apoptosis compared with the control group. The mean ± standard deviation early apoptosis rates were significantly decreased for the miR-106a mimic compared with the mimic control (8.68±0.75 vs. 12.45±1.16%, respectively; P=0.0016; n=4; Fig. 2C).

Flow cytometry was also employed to analyze cells subsequent to transfection with an miR-106a inhibitor or an inhibitor control (Fig. 2D and E). HCT116 cells transfected with miR-106a inhibitor exhibited a higher rate of early apoptosis compared with the control group. The mean ± standard deviation early apoptosis rates were significantly increased for the miR-106a inhibitor compared with the inhibitor control (8.803±0.27 vs. 6.733±0.60%, respectively; P=0.0162; n=4; Fig. 2F).

These results indicated that the miR-106a mimic decreased early apoptosis in HCT116 cells, and that the miR-106a inhibitor increased early apoptosis in HCT116 cells, suggesting that miR-106a promoted cell viability by inhibiting early apoptosis in CRC cells.

In previous studies (19), miR-106a expression was upregulated in the serum of patients with CRC. In the present study, the expression of miR-106a in human CRC tissues and plasma was determined. It was identified that the expression level of miR-106a was significantly upregulated in tumor tissues compared with adjacent tissues [median (25th percentile, 75th percentile, 26.2882 (14.5358, 49.3492) vs. 8.7187 (5.4891, 13.6726); P<0.0001; Fig. 3A]. The association between miR-106a expression in cancer tissues and clinicopathological characteristics of patients with CRC was also analyzed (Table I).

miR-106a expression in plasma of patients with CRC or healthy controls, and pre- or post-surgery in patients with CRC, was also investigated. The expression level of miR-106a in plasma was significantly higher in patients with CRC compared with in the healthy control group, consistent with our previous study (19) (P=0.0041; Fig. 3B). The healthy control group contained 42 age-matched healthy individuals with patients with CRC (data not shown). miR-106a expression in plasma post-surgery was decreased compared with in plasma pre-surgery, but no statistically significant difference was identified (0.16±0.18 vs. 8.80±40.50; P=0.1600; Fig. 3C). No significant association was identified between miR-106a expression in plasma and the clinicopathological parameters of the patients with CRC (data not shown). The sensitivity and specificity of miR-106a were analyzed using a receiver operating characteristic curve in CRC diagnosis (Fig. 3D).

The expression of miR-106a in CRC tissues was identified to be associated with the differentiation and pathological type of tumor, by analyzing the clinicopathological characteristics of CRC patients. The expression level of miR-106a in CRC tissues was significantly increased in patients with adenocarcinoma compared with in patients with mucinous carcinoma (100.96±262.20 vs. 18.14±18.22; P=0.0348; Fig. 4A). miR-106a expression in CRC tissues was also increased in patients exhibiting moderate-well differentiation compared with that in patients exhibiting poor differentiation (108.87±272.79 vs. 18.12±19.20; P=0.0343; Fig. 4B).

The underlying molecular mechanism of increased miR-106a expression in adenocarcinoma and moderate-well differentiation CRC requires further investigation. No significant association was identified between miR-106a expression and other clinicopathological characteristics, such as sex, age, distant metastasis, Dukes staging, tumor location and lymph node metastasis.