The present study was approved by the Ethics Committee of The Military General Hospital of Beijing PLA (Beijing, China). Written informed consent was obtained from all patients, prior to enrollment in the present study. The patients with CRC had not received chemotherapy or radiotherapy prior to surgery. A total of 59 pairs of CRC tissues and their normal adjacent tissues (NATs) were obtained from patients with CRC, who underwent surgical resection at The Military General Hospital of Beijing PLA. CRC tissues and the NATs were rapidly frozen in liquid nitrogen and stored in at −80°C.

A total of four human CRC cell lines (HCT116, HT29, SW480 and SW620), the normal human colon epithelium cell line FHC and the HEK293T cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained in Dulbecco's modified Eagle's medium or RPMI-1640 supplemented with 10% fetal bovine serum (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 mg/ml penicillin and 100 mg/ml in a humidified atmosphere of 5% CO₂ at 37°C.

In order to investigate the function of miR-330 in CRC, CRC cells were transfected with miR-330 mimics or the negative scrambled control (NC), purchased from GenePharma, Inc. (Sunnyvale, CA, USA). Furthermore, TYMS small interfering (si)RNA and NC siRNA were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China) and transfected into CRC cells. The sequence of the miR-330 mimic was 5′-GCAAAGCACACGGCCUGCAGAGA-3′ and the sequence of the miR-NC mimic was 5′-UUCUCCGAACGUGUCACGUTT-3′. The sequence of the TYMS siRNA was 5′-TACGTCCAAGGTCGGGCAGGAAGA-3′ and the NC siRNA sequence was 5′-AACAGGCACACGTCCCAGCGT-3′. Cell transfection was performed using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc), according to the manufacturer's protocol.

Total RNA was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. In order to detect miR-330 expression level, reverse transcription was performed using the TaqMan^® MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), followed by RT-qPCR using the TaqMan miRNA assay (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The temperature protocol for reverse transcription was as follows: 16°C for 30 min; 42°C for 30 min; and 85°C for 5 min. The thermocycling conditions for qPCR were as follows: 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 1 min; followed by a final elongation step at 72°C for 10 min.

For analysis of TYMS mRNA expression level, reverse transcription was performed using the M-MLV Reverse Transcription system (Promega Corp., Madison, WI, USA), according to the manufacturer's protocol. The temperature protocol for reverse transcription was as follows: 95°C for 2 min; 20 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min; and 72°C for 5 min. Subsequently, SYBR^®-Green Master mix was used to determine the mRNA expression level. The thermocycling conditions of qPCR were as follows: 95°C for 10 min; and 40 cycles of 95°C for 15 sec and 60°C for 1 min. U6 small nuclear RNA was used for normalization of miRNA expression and GADPH was used as an internal control for mRNA expression. RT-qPCR was performed using a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative expression of miR-330a and TYMS was analyzed by use of the 2^−ΔΔCq method (24). This assay was performed in triplicate and repeated three times.

Cell proliferation rates were evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay. Cells were seeded into 96-well plates at a density of 3,000 cells/well. Subsequently, cells were transfected and incubated in a humidified atmosphere of 5% CO₂ at 37°C for 24–96 h. Every 24 h post-transfection, CCK-8 assays was performed. A total of 10 µl CCK8 assay solution was added to each well. Cells were incubated for 2 h at 37°C and the absorbance of each well was determined at 450 nm by an automatic multi-well spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were performed in triplicate.

Cells were seeded into 96-well plates at a density of 3,000 cells/well. Subsequently, cells were transfected and incubated for 48 h in a humidified atmosphere of 5% CO₂ at 37°C. Cells were then treated with 5-FU (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) at various concentrations (0–32 µM) at room temperature. Following incubation for a further 48 h, a chemosensitivity assay was performed using a CCK8 assay, as aforementioned. The dose-response curve was charted at various concentrations. All experiments were performed in triplicate.

Cells were seeded into 6-well plates at a confluence of between 60 and 70%. Cells were transfected and incubated for 24 h in a humidified atmosphere of 5% CO₂ at 37°C. Cells were subsequently treated with 5-FU at a concentration of 8 µM. Following a 48-h incubation, the rate of cell apoptosis was determined by Annexin V-fluorescein isothiocyanate (FITC; BD Biosciences, Franklin Lakes, NJ, USA; cat. no. 556419) and propidium iodide (PI; BD Biosciences; cat. no. 556463) staining, according to the manufacturer's protocol. In brief, cells were harvested and washed with ice-cold PBS (Gibco; Thermo Fisher Scientific, Inc.) three times. Cells were resuspended in 100 µl binding buffer, followed by treatment with 2 µl Annexin V-FITC and 5 µl of PI at room temperature. Following a 15 min incubation at room temperature in the dark, 400 µl of binding buffer was added and the mixture was analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) using FCS Express software (version 3.0; De Novo Software, Glendale, CA, USA).

Transfected cells were washed with ice-cold PBS three times and lysed with radioimmunoprecipitaion assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with protease inhibitors (Promega Corp.). Protein concentration was quantified using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (20 µg) were separated by 10% SDS-PAGE, followed by transference to polyvinylidene difluoride membranes. The membranes were blocked in TBS-Tween-20 containing 5% non-fat dry milk for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with the goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:3,000 dilution; cat. no. sc-ab6789; Abcam, Cambridge, MA, USA) for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence solution (Pierce; Thermo Fisher Scientific, Inc.). The primary antibodies used in the present study were mouse anti-human monoclonal TYMS (cat. no. sc-33679) and mouse anti-human GADPH (cat. no. sc-59540) (both 1:1,000 dilution; both Santa Cruz Biotechnology, Inc., Dallas, TX, USA). GADPH was used as an internal control. Band intensities were imaged using the FluorChem imaging system (alpha innotec GmbH, Kasendorf, Germany) and analyzed using Image Lab version 5.2.1 (Bio-Rad Laboratories, Inc.).

The Dual-Luciferase Reporter assay (Promega Corporation) was performed in HEK293T cells. PGL3-TYMS-3′UTR wild-type (Wt) and PGL3-TYMS-3′UTR mutant (Mut) were purchased from GenePharma, Inc. Cells were seeded into 24-well plates at a confluence of between 60 and 70%. Following overnight incubation, cells were co-transfected with miR-330 mimics or NC, and PGL3-TYMS-3′UTR Wt or PGL3-TYMS-3′UTR Mut, using Lipofectamine 2000. The firefly and Renilla luciferase activities were determined by Dual-Luciferase reporter assay 48 h following transfection. Luciferase activity was measured at a wavelength of 560 nm using an xMark™ Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories, Inc.). Renilla luciferase activities were used as an internal control. All experiments were performed in triplicate.

Data are presented as the mean ± standard deviation, and compared with the Student's t-test or a one-way analysis of the variance using SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA). Two-tailed P<0.05 was considered to indicate a statistically significant difference.

In order to investigate whether miR-330 was downregulated in CRC tissues, the miR-330 expression level was determined by RT-qPCR in CRC tissues and paired NATs. As presented in Fig. 1A, miR-330 was significantly downregulated in CRC tissues compared with paired NATs (P<0.05). Furthermore, the expression level of miR-330 was evaluated in CRC and FHC cell lines. Consistent with the result that the miR-330 expression level was decreased in CRC tissues, miR-330 was downregulated in the HCT116, HT29, SW480 and SW620 cell lines, compared with the FHC cell line (all P<0.05; Fig. 1B). These findings suggested that miR-330 may act as a tumor suppressor in CRC.

HCT116 and HT29 expression levels were lower compared with SW480 and SW620 expression levels. Therefore, the present study transfected miR-330 mimics or NC into HCT116 and HT29 cells in order to explore the functions of miR-330 in CRC cells. RT-qPCR was performed to determine the miR-330 expression level following transfection. As presented in Fig. 1C, miR-330 was significantly upregulated in miR-330 mimic transfected HCT116 and HT29 cells, compared with in NC cells (both P<0.05).

The effect of miR-330 expression on CRC cell proliferation was evaluated by performing a CCK-8 assay. As presented in Fig. 2, the upregulation of miR-330 resulted in significant growth inhibition of HCT116 and HT29 cells (both P<0.05) at 96 h compared with the NC. The results suggested that miR-330 may be a negative regulator of cell proliferation in CRC cells.

In order to evaluate the effect of miR-330 on CRC cell chemosensitivity to 5-FU, a chemosensitivity assay was performed. As presented in Fig. 3, enforced miR-330 expression decreased the survival rate of HCT116 and HT29 cells, compared with NC-transfected HCT116 and HT29 cells (P<0.05). These results indicated that miR-330 increased cell chemosensitivity of CRC cells to 5-FU.

Cell-cycle arrest at the G₁ phase and/or apoptosis of cancer cells is enhanced by 5-FU (25,26). Therefore, the rate of cell apoptosis induced by 5-FU was evaluated. As presented in Fig. 4, the overexpression of miR-330 significantly increased the rate of apoptosis of HCT116 and HT29 cells induced by 5-FU (P<0.05) compared with the NC. This supports the observation that miR-330 increases HCT116 and HT29 cell chemosensitivity to 5-FU. These results suggested that miR-330 increased cell chemosensitivity of HCT116 and HT29 cells to 5-FU via the cell apoptosis pathway.

In order to investigate the molecular mechanisms underlying the functions of miR-330 in CRC cells, bioinformatic predication algorithms (TargetScan; www.targetscan.org) were used to determine the direct target gene of miR-330. Amongst the hypothetical target mRNAs identified for miR-330, TYMS was selected for further investigation (Fig. 5A). In addition, a Dual-Luciferase reporter assay was performed to determine whether TYMS was a direct target mRNA of miR-330 in vitro. As presented in Fig. 5B, miR-330 significantly inhibited PGL3-TYMS-3′UTR Wt luciferase activity in HEK293T cells (P<0.05) compared with the NC, whereas PGL3-TYMS-3′UTR Mut demonstrated no significant response to miR-330 (P>0.05).

In order to further investigate the potential effects of miR-330 in the regulation of TYMS, RT-qPCR and western blot analysis were performed to evaluate TYMS mRNA and protein levels following transfection with miR-330 mimics or NC. As presented in Fig. 5C, TYMS mRNA expression levels were not significantly altered following transfection with miR-330 mimics (P>0.05). However, TYMS protein expression was significantly downregulated in miR-330 mimic-transfected HCT116 and HT29 cells, compared with NC groups (both P<0.05; Fig. 5D). This indicated that miR-330 regulated TYMS expression at the post-transcriptional level. Therefore, TYMS was a direct target gene of miR-330 in CRC cells.

In order to investigate the function of TYMS in CRC, HCT116 and HT29 cells were transfected with TYMS siRNA or NC siRNA. As presented in Fig. 6A, TYMS siRNA decreased the expression level of TYMS in HCT116 and HT29 cells (P<0.05). Following transfection with TYMS siRNA, CCK-8, chemosensitivity and cell apoptosis assays were performed. As presented in Fig. 6B, relative cell growth was significantly suppressed in TYMS siRNA transfected HCT116 and HT29 cells, compared with NC siRNA cells (both P<0.05). In addition, chemosensitivity assays demonstrated that TYMS siRNA significantly improved chemosensitivity of HCT116 and HT29 cells to 5-FU (both P<0.05; Fig. 6C). Furthermore, TYMS siRNA significantly increased the rate of HCT116 and HT29 cell apoptosis induced by 5-FU (both P<0.05; Fig. 6D). These results revealed that the effects of TYMS siRNA were similar to those induced by miR-330 in CRC cells, suggesting that TYMS was a functional target of miR-330 in CRC cells.