In the present study, the gene expression of 30 patients with colorectal cancer, including 17 patients (11 men and 6 women; range, 51–68 years; mean age, 59±7 years) with oxaliplatin-sensitive colorectal cancer and 13 patients (8 males and 5 females; range 56–69 years) with oxaliplatin-resistant colorectal cancer were examined. All the participants received oxaliplatin-based chemotherapy and were enrolled in Xingtai People's Hospital (Xingtai, China) between February 2013 and March 2015. Tumor response and progression were assessed according to the Response Evaluation Criteria in Solid Tumors version 1.1 (23). According to the outcome of therapy, 17 patients were classified as responders (complete or partial response) and 13 patients were classified as non-responders (no change and progressive disease). Written consents regarding participation in the present study and use of their tissues were obtained from all patients and the experiments were conducted with the approval of and under the supervision of the Ethics Committee of Xingtai People's Hospital.

The kidney derived 293T cell line and human colorectal cancer cell lines, HCT116 and HT-29, which were commonly used to study oxaliplatin sensitivity of colorectal cancer (24–25), were obtained from the American Type Culture Collection and used within 6 months. HT-29 cell line was authenticated by STR profiling. The HCT116 and HT-29 cell lines were maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in an incubator at 37°C with 5% CO₂.

For oxaliplatin treatment, indicated concentrations (2, 4, 6 and 8 µmol/l) of oxaliplatin (Selleck Chemicals) were added into the culture medium of HCT116 and HT-29 cells and sustained for 48 h at 37°C then subjected to the following procedures.

Cell viability was assessed with an MTT assay. Briefly, HCT116 and HT-29 cells were cultured at 37°C in 96-well plates (1×10³ cells/well) and treated with oxaliplatin for the indicated lengths of time (0, 25, 50, 75 and 100 h). Subsequently, HCT116 and HT-29 cells were stained with thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich; Merck KGaA) for 2 h at 37°C and absorbance at 570 nm was detected following purple precipitates being dissolved with MTT detergent reagent on a microplate reader (BioTek Instruments, Inc.). Cell viability was calculated as the ratio of the absorbance values of treated samples to those of controls.

RNA was extracted from HCT116 and HT-29 cells and tissues from patients using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA with a PrimeScript™ RT reagent kit (Takara Bio, Inc.), according to manufacturer's protocol. For mRNA expression analysis of FOXQ1, the RT-qPCR was performed on a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Inc.) with SYBR^® Premix Ex Taq™ II (Takara Bio, Inc.). GAPDH was used as an endogenous reference gene.

For miRNA expression analysis, RT was performed with a miScript II RT kit (Qiagen GmbH). Expression levels of miR-106a were detected using miScript primer assays with a miScript SYBR Green PCR kit (Qiagen GmbH), according to the manufacturer's protocol. U6 was used as an endogenous reference gene. The thermocycling conditions were as follows: 95°C for 15 min, followed by denaturation: 95°C for 10 sec, annealing for 30 sec and extension for 30 sec (40 cycles). The relative expression of miRNA and mRNA were calculated using 2^−ΔΔCq method (26). The primer sequences were listed as follows: FOXQ1-Forward: 5′-CACGCAGCAAGCCATATACG-3′; FOXQ1-Reverse: 5′-CGTTGAGCGAAAGGTTGTGG-3′; MMP-2-Forward: 5′-GGCCCTGTCACTCCTGAGAT-3′; MMP-2-Reverse: 5′-GGCATCCAGGTTATCGGGGA-3′; VEGF-A-Forward: 5′-AGGGCAGAATCATCACGAAGT-3′; VEGF-A-Reverse: 5′-AGGGTCTCGATTGGATGGCA-3′; GAPDH-Forward: 5′-CTCTGATTTGGTCGTATTGGG-3′; GAPDH-Reverse: 5′-TGGAAGATGGTGATGGGATT-3′; miR-106a-RT: 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCTACCTG-3′; miR-106a-Forward: 5′-ATCCAGTGCGTGTCGTG-3′; miR-106a-Reverse: 5′-TGCTAAAAGTGCTTACAGTG-3′; U6-Forward: 5′-CTCGCTTCGGCAGCACA-3′; U6-Reverse: 5′-AACGCTTCACGAATTTGCGT-3′.

The FOXQ1 antibody (cat. no. SC-166265; dilution, 1:2,000) and vascular endothelial growth factor-A (VEGF-A; cat. no. SC-365578; dilution, 1:2,000) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against matrix metallopeptidase-2 (MMP-2; cat. no. 40994; dilution, 1:1,000) was purchased from Cell Signaling Technology, Inc. The GAPDH antibody was obtained from Sigma-Aldrich (Merck KGaA). The secondary antibodies against mouse and rabbit (HRP-conjugate; dilution, 1:10,000) were purchased from ProteinTech Group, Inc. Proteins were extracted from HCT116 and HT-29 cells using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA). The concentration of protein lysates was determined by bicinchoninic acid assay (BCA assay) using a BCA protein assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Western blot analysis was performed as follows: Equal amounts (20 µg) of samples were loaded into each lane on an 8% SDS-PAGE gel. The proteins were separated and transferred on a polyvinylidene fluoride membrane. Following blocking in 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with secondary antibodies for 1 h at room temperature and then developed with Enhanced Chemiluminescent Western Blotting Substrate (Pierce; Thermo Fisher Scientific, Inc.). Images were captured with ImageQuant LAS 4000 (GE Healthcare Life Sciences). GAPDH was used as endogenous control.

miR-NC mimics and miR-106a mimics were purchased from Shanghai GenePharma Co., Ltd. The sequences were: miR-NC mimics, 5′-UUCUCCGAACGUGUCACGUTT-3′; miR-106a mimics, 5′-AAAAGUGCUUACAGUGCAGGUAG-3′. miR-NC mimics or miR-106a mimics were transfected at a final concentration of 40 nM with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. In brief, miR-NC mimics or miR-106a mimics and Lipofectamine^® 2000 were mixed (to a concentration of 200 nM) in Opti-MEM medium (Thermo Fisher Scientific, Inc.) and incubated for 20 min at 37°C. Subsequently, the mixture was added to HCT116 and HT-29 cells in DMEM. The medium was replaced with fresh DMEM after 6 h and sustained for 18 h before subjected to following experiments.

Sequencing alignment was performed using miRDB database (www.mirdb.org). FOXQ1 3′UTR was amplified from cDNA of 293T cells and ligated into a pGL3 plasmid (Promega Corporation). A total of two-point mutations were introduced into FOXQ1 3′UTR with a Site-directed mutagenesis kit (Agilent Technologies, Inc.), in order to construct pGL3-FOXQ1-3′UTR-Mut.

For the dual luciferase reporter assay, 293T and HCT116 cells were plated on 24-well plates. On the following day, cells were co-transfected with pGL3-FOXQ1-WT or pGL3-FOXQ1-3′UTR-Mut, miR-NC mimics or miR-106a mimics and pRL-TK plasmid (Invitrogen; Thermo Fisher Scientific, Inc.) using Lipofectamine^® 2000. The assay was performed 48 h after with a Dual-luciferase Assay system (Promega Corporation). The firefly luciferase activity was normalized to the Renilla luciferase activity value.

The data were calculated with GraphPad Prism v6 (GraphPad Software, Inc.) and presented as the mean ± standard deviation. Comparisons between two different groups were determined with unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

In order to evaluate the role of miR-106a in colorectal cancer cells, the HCT116 and HT-29 cell lines were selected. miR-106a was significantly overexpressed in these cell lines via transfection of miR-106a mimics (P<0.001; Fig. 1A). A cell viability assay was conducted to detect cell proliferation ability upon miR-106a overexpression. As depicted in Fig. 1B and C, miR-106a mimics significantly increased MTT absorbance compared with cells transfected with miR-NC mimics (P<0.01), indicating miR-106a overexpression promoted cell proliferation of HCT116 and HT-29 cells. However, in cells treated with increasing concentrations of oxaliplatin, overexpression of miR-106a significantly reduced cell viability, compared with control groups, in the cell lines tested (P<0.001; Fig. 1D and E). These results indicated that miR-106a overexpression could enhance oxaliplatin sensitivity in colorectal cancer cells.

FOXQ1 is frequently overexpressed in colorectal cancer and its expression is associated with oxaliplatin resistance (27). Transfection of miR-106a mimics significantly decreased FOXQ1 mRNA level in HCT116 and HT-29 cells (P<0.001; Fig. 2A). Western blot analysis demonstrated that the protein expression level of FOXQ1 was also reduced following miR-106 overexpression (Fig. 2B). These results indicated that miR-106a may regulate oxaliplatin sensitivity via repression of FOXQ1 level.

FOXQ1 is a transcriptional factor and can activate VEGF-A and MMP-2 transcription via regulation of the Wnt signaling pathway in colorectal cancer (27). In addition, overexpression of miR-106a significantly reduced VEGF-A (P<0.05) and MMP-2 (P<0.001) mRNA in HCT116 and HT-29 cells (Fig. 3A and B), and the protein levels of VEGF-A and MMP-2 were also reduced in the two cell lines (Fig. 3C). These results indicated that miR-106a could inhibit FOXQ1, and repress transcription of its target genes.

The aim of the present study was to investigate whether miR-106a directly regulates FOXQ1 expression. Sequence alignment demonstrated that there were binding sites for miR-106a in the 3′UTR of FOXQ1 mRNA (Fig. 4A). In the dual luciferase assay, it was determined that transfection of miR-106a mimics significantly decreased relative luciferase activity of 293T cells transfected with FOXQ1 3′UTR-WT, but not FOXQ1 3′UTR-Mut (P<0.001; Fig. 4B and C). Similar results were observed in HCT116 cells (P<0.001; Fig. 4D and E). These data confirmed that miR-106a could bind to FOXQ1 mRNA and directly repress its expression.

To further evaluate the role of miR-106a and FOXQ1 in oxaliplatin sensitivity, the expression of miR-106a and FOXQ1 in tumor tissues from patients with colorectal cancer receiving oxaliplatin-based chemotherapy was determined. RT-qPCR demonstrated that miR-106a expression levels (P<0.05) were significantly decreased in tumor tissues from oxaliplatin non-responders, while FOXQ1 (P<0.001) mRNA levels were significantly elevated (Fig. 5A and B). Consistent with the present in vitro data, statistical analysis indicated that the expression of miR-106a was associated with FOXQ1 expression (r=−0.5535; P=0.0015; Fig. 5C). These results demonstrated that miR-106a regulates the expression of FOXQ1 in tumor tissues of patients with colorectal cancer.