Thirty-two paired human CRC tissue samples and matched tumor-adjacent tissues were obtained from CRC patients and his topathologically diagnosed at the First Hospital of Jilin University (Changchun, China). The specimens were collected at surgery, immediately frozen in liquid nitrogen and stored at −80°C until total RNAs or proteins were extracted. The patient characteristics and clinicopathological features were collected and are described in Table I. The present study was approved by the Ethics Committee of Jilin University. Written informed consent for use of tissue samples was obtained from all patients before surgery.

Four human CRC cell lines, LoVo, SW1116, SW480 and HCT-116, and a normal colonic cell line (NCM460) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL, Gaithersburg, MD, USA), 100 U/ml penicillin or 100 mg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C.

Total RNA of the tissues and cultured cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and RNA molecules <200 nucleotides in size were purified by the mirVana miRNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. For detection of miR-874, first-strand cDNA was synthesized using miScript reverse transcription kit (Qiagen, Germany), and were then quantified using the miScript SYBR-Green PCR kit (Qiagen) under the 7900 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The miRNA sequence-specific reverse transcription-quantitative PCR (RT-qPCR) primers for miR-874 and endogenous control U6 were purchased from Qiagen. To quantify X-linked inhibitor of apoptosis protein (XIAP), complementary DNA (cDNA) was synthesized using M-MLV reverse transcriptase kits (Promega, Madison, WI, USA) according to the manufacturer's instructions. Real-time PCR was performed using SYBR Premix Ex Taq (Takara, Dalian, China) under the 7900 Real-Time PCR system. Primers for XIAP and GAPDH were used as previously described (18). The fold-change in target mRNA or miRNA expression was calculated using the 2^−ΔΔCt method following normalization to GAPDH or U6 expression, respectively.

The miR-874 mimic (miR-874) and corresponding negative control (miR-NC) were purchased from GenePharma (Shanghai, China). The plasmid encoding XIAP-siRNA (pSi-XIAP) and the plasmid encoding non-specific siRNA (pSi-NC) were gifted by Dr Yang Qu (Jilin University).

For transfection, the cells were plated in 6- or 12-well plates 24 h before transient transfection. Transfection was performed using Lipofectamine 2000 reagent (Invitrogen) in Opti-MEM media (Gibco) using 50 nM miR-874/miR-NC or 50 µg pSi-XIAP/pSi-NC according to the manufacturer's instructions. Analyses were performed 48–72 h after transfection.

Cell proliferation was determined by MTT assay. Briefly, the transfected cells (5×10³ cells/well) were seeded into 96-well microplate and cultured in DMEM including 10% FBS. At the indicated time (24, 48, 72 and 96 h), 20 µl MTT solution (5 mg/ml) was added to each well and cultured for 4 h, and then 200 µl of dimethyl sulfoxide (DMSO; Sigma) was added to each well followed by shaking for 15 min to dissolve the crystals. The absorption at 570 nm was measured under a microplate reader (Molecular Devices Corp., Sunnyvale, CA, USA). All experiments were performed in triplicate.

Analysis of cell apoptosis was performed using a phycoerythrin (PE)-Annexin V apoptosis detection kit (BD Pharmingen, San Jose, CA, USA). Briefly, 4×10⁵ transfected cells were seeded in 6-well plates and cultured for 48 h. Then, the cells in the suspension that were adherent, were harvested and labeled with Annexin V for 15 min in a dark place, and then 50 µg/ml propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA) was added to each sample. The cell apoptosis ratio was analyzed using a FACSCalibur flow cytometer (BD Biosciences, Mansfield, MA, USA).

SW480 cells (2×10⁴ each well) were seeded in each well of 12-well plates. The cells were then transfected with the miR-874 mimic or miR-NC for 48 h and incubated with different concentrations of 5-fluorouracil (5-FU) for an additional 48 h. Then cell proliferation was determined by the MTT assay as described above. The IC₅₀ values were calculated.

In addition, the effects of miR-874 on cell proliferation and apoptosis were investigated in SW480 cells exposed to an IC₅₀ value of 5-FU. Briefly, the SW480 cells were transfected with miR-874 mimic or miR-NC for 48 h, followed by exposure to an IC₅₀ value of 5-FU for an additional 48 h. Cell colony formation and apoptosis were determined in the above cells.

The human XIAP 3′UTR oligonucleotides containing the wild-type (Wt) or mutant (Mut) miR-874 binding site were cloned by PCR and inserted into the pGL3 vector (Ambion) at the NheI and XhoI sites. For the luciferase assay, the SW480 cells were seeded in 24-well plates for 24 h, and then co-transfected with 100 ng of luciferase reporter vectors (Wt/Mut) and 50 nM of miR-874 or miR-NC. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter assay (Promega, Madison, WI, USA) at 48 h after transfection.

Total protein was extracted from the cells or tissue samples using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). The concentrations of protein were determined with a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Equivalent amounts of protein (30 µg each lane) were electrophoresed on SDS-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoide (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline for 2 h and then incubated with antibodies against XIAP or GAPDH (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. The membranes were washed thrice with TBS buffer and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. The protein bland was analyzed on X-ray film (Denville Scientific) using chemiluminescent reagents. GAPDH was used as the internal control.

Twenty 6-week-old male BALB/c nude mice (18–20 g) were purchased from the Experimental Animal Center of Changchun Institute for Biological Sciences (Changchun, China), and maintained under specific pathogen-free (SPF) conditions. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Jilin University.

Equal numbers of SW480 cells (2×10⁶) with stable expression of the miR-874 mimic or miR-NC were suspended in 100 µl serum-free DMEM and subcutaneously injected into the right rear flank of each mouse (n=10), respectively. Tumor volume (V) was measured every 5 days, and was calculated according to the formula: V = 0.5 × L (length) × W² (width). The mice were sacrificed 30 days after injection. The tumor tissues were dissected and weighed. Part of each tumor tissue was harvested and stored for analysis of the expression of XIAP and miR-874.

All data are expressed as means ± standard deviation (SD) from at least three independent experiments. Statistical analysis between two samples was performed using the Student's t-test, and analysis of more than two groups was performed using one-way ANOVA. Statistical analysis was performed using Statistical Package for Social Science (SPSS for Windows version 16.0; SPSS, Inc., Chicago, IL, USA). A P-value <0.05 was considered to indicate a statistically significant result.

The expression levels of miR-874 were detected by RT-qPCR in 32 pairs of CRC tissues and their matched adjacent tissues. miR-874 expression levels were significantly decreased in the CRC tissues when compared with these levels in the adjacent normal tissues (P<0.01; Fig. 1A). To further investigate the clinical significance of miR-874 in CRC, 32 patients were divided into two groups: a high expression group (>0.46, n=17) and a low expression group (<0.46, n=15), according to the median value (0.46) of the miR-874 expression level in the CRC tissues. The results showed that the level of miR-874 expression was significantly negatively correlated with lymph node metastasis (P<0.01) and TNM stage (P<0.01) (Table I), which are indicators of poor prognosis. However, there were no significant correlations between miR-874 expression and other factors including patient age and gender, and tumor size. In addition, the expression levels of miR-874 were quantified in four human CRC cell lines and a normal colonic cell line (NCM460) by RT-qPCR. The expression level of miR-874 was obviously downregulated in the CRC cell lines compared to the expression level in the normal colonic cell line (NCM460) (Fig. 1B). Additionally, the expression level of miR-874 in the SW480 cell line was the lowest among the four cell lines; thus, the SW480 cell line was selected for subsequent study. These results suggest that miR-874 plays crucial roles in CRC progression.

To assess the biological effects of miR-874 on CRC cells, the miR-874 mimic was transfected into the SW480 cells. The transfection of miR-874 was successful, and a significant increase in the miR-874 expression level was achieved in the SW480 cells (Fig. 2A). Then, cell proliferation, colony formation and apoptosis were investigated in the SW480 cells after transfection with miR-874 or miR-NC. The results showed that restoration of miR-874 expression significantly inhibited cell proliferation (Fig. 2B) and colony formation (Fig. 2C), as well as induced cell apoptosis (Fig. 2D) in the SW480 cells.

It has been shown that various miRNAs can improve or decrease the resistance of cancer cells to chemotherapeutic agents. Thus, we tested whether miR-874 affects the resistance of CRC cells to 5-FU. SW480 cells were transfected with the miR-874 mimic or miR-NC for 48 h, followed by exposure to different concentrations of 5-FU for an additional 48 h. Cell proliferation was then determined using MTT assay. As shown in Fig. 3A, restoration of miR-874 reduced the resistance of SW480 cells to 5-FU compared to that in the miR-NC group. Compared with the IC₅₀ value of 5-FU in the miR-NC group (5.68±0.38 µg/ml), restoration of miR-874 significantly decreased the IC₅₀ value of 5-FU in the SW480 cells (2.69±0.21 µg/ml) (Fig. 3B). In addition, we also investigated the effect on colony formation and apoptosis in the miR-874-overexpressing cells exposed to an IC₅₀ (2.69 µg/ml) concentration of 5-FU. We found that the miR-874 mimic in combination with 5-FU treatment in the SW480 cells significantly decreased colony formation (Fig. 3C), and induced cell apoptosis (Fig. 3D) compared to single 5-FU treatment. These findings suggest that restoration of miR-874 decreased CRC cell resistance to 5-FU.

As the putative binding site of miR-874 in the mRNA 3′UTR region of XIAP was predicted using bioinformatic databases (TargetScan, PicTar) (Fig. 4A), a dual-luciferase assay was performed to determine the direct link between XIAP and miR-874. We found that luciferase activity was significantly reduced by the miR-874 mimic in the Wt XIAP group, while the miR-874 mimic had no effect on luciferase activity in the SW480 cells transfected with the mutated type (Mut) XIAP 3′UTR (Fig. 4B). Additionally, the expression of XIAP at the mRNA and protein levels was determined in the SW480 cells after transfection with miR-874 by RT-qPCR and western blotting, respectively. The expression of XIAP at the mRNA (Fig. 4C) and protein level (Fig. 4D) was inhibited in the SW480 after transfection with the miR-874 mimic. These results suggest that XIAP is a direct target of miR-874.

As XIAP was identified as the target of miR-874, we detected the expression of XIAP in the CRC and adjacent non-tumor tissues. The expression of XIAP at the mRNA (Fig. 5A) and protein level (Fig. 5B) was greatly increased in the CRC tissues when compared to these levels in the adjacent non-tumor tissues. Meanwhile, the correlation of miR-874 and XIAP expression was also investigated in the CRC tissues. Spearman's correlation analysis showed that XIAP expression at the mRNA level was inversely related to the expression of miR-874 (Fig. 5C; r=−0.540; P<0.01). In addition, XIAP protein expression was also detected in four human CRC cell lines and a normal colonic cell line (NCM460) by western blotting. The results showed that XIAP protein expression was obviously upregulated in the CRC cell lines compared to that in the normal colonic NCM460 cell line (Fig. 5D).

To investigate the biological functions of XIAP in CRC cells, endogenous expression of XIAP was downregulated in the SW480 cells with specific siRNA against XIAP (pSi-XIAP). RT-qPCR and western blot assay confirmed that XIAP expression at the mRNA (Fig. 6A) and protein level (Fig. 6B) was significantly inhibited in the SK480 cells by pSi-XIAP. Functional assays showed that downregulation of XIAP significantly inhibited cell proliferation (Fig. 6C) and colony formation (Fig. 6D), and induced cell apoptosis (Fig. 6E), as well as decreased 5-FU resistance (Fig. 6F) These findings suggest that inhibition of XIAP mimicked the inhibitory effects of miR-874 overexpression in the SW480 cells.

To investigate the biological role of miR-874 in vivo, an SW480 xenograft tumor model was established in BALB/c nude mice by injection of the SW480 cells with stable expression of miR-874 or miR-NC (SW480/miR-874 or SW480/miR-NC). Tumor volumes were determined every five days after injection. As shown in Fig. 7A, xenograft tumor growth in the SW480/miR-874 group was much lower than that noted in the SW480/miR-NC group. At five weeks post-injection, the mice were sacrificed, and tumor tissues were stripped and weighed. We found that the xenograft tumor weight in the SW480/miR-874 group was significantly smaller than that determined in the SW480/miR-NC group (Fig. 7B). In addition, miR-874 and XIAP expression in the xenograft tumors was detected by RT-qPCR. The results demonstrated that the miR-874 expression level was upregulated in the SW480/miR-874 group (Fig. 7C), while XIAP protein expression was downregulated in the SW480/miR-874 group (Fig. 7D). These results suggest that miR-874 suppresses tumor growth in nude mice at least in part by suppressing XIAP.