Five CRC cell lines, consisting of the HT-29, HCT-116, SW480, SW620 and LoVo cell lines (American Type Culture Collection, Rockville, MD, USA), and the healthy human colonic epithelial HCoEpiC cell line (Sciencell Research Laboratories, Carlsbad, CA, USA) were used in the present study. The cells were maintained under the recommended conditions.

In total, 6 human CRC, 2 adjacent non-tumor and 2 healthy colorectal tissue samples were obtained in March 2014 from patients that were diagnosed with colon adenocarcinoma at the Department of General Surgery, The Third Xiangya Hospital of Central South University (Changsha, Hunan, China). The specimens were obtained subsequent to surgical resection and were immediately frozen at −80°C until use. Written informed consent was obtained from all patients. The methodology used in the present study conformed to the standards set by the Declaration of Helsinki (22) and was approved by the ethics committee of The Third Xiangya Hospital of Central South University.

To evaluate the expression of miR-133b, all cell lines were treated with 3 µM of the demethylation agent 5-Aza-CdR, 3 mM histone deacetylase (HDAC) inhibitor (PBA), or 3 µM 5-Aza-CdR (Sigma-Aldrich, St. Louis, MO, USA) and 3 mM PBA (Sigma-Aldrich) for 24, 48 and 72 h. The cells were seeded 24 h prior to treatment.

Total RNA was extracted from the CRC cell lines and HCoEpiC cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and then reverse-transcribed into complementary DNA. HmiRQP0167 primers were obtained from GeneCopoeia, Inc. (Rockville, MD, USA). The PCR protocol consisted of 35 cycles at 95°C for 3 min, 95°C for 12 sec and 58°C for 30 sec. The relative expression level of miRNA was normalized to the expression of the internal control U6 small nuclear RNA using the 2^−ΔΔCt method (23).

MSP was used to analyze the methylation status of healthy and CRC tissues, as well as healthy, HT-29 and SW620 cell lines, as described previously (24). Genomic DNA was extracted from tissues or cells using the DNA Extraction Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Extracted DNA samples underwent sodium bisulfite modification using a Universal CpGenome™ DNA Modification Kit (Merck Millipore, Darmstadt, Germany). MethPrimer software (available from www.methdb.de) was used to design the two sets of primers, as follows: methylated miR-133b forward, 3′-TTTATTTAAAATATAAAAATA GCGG-5′ and reverse, 3′-TCACCCAAACTAAAATACAAT AACG-5′; and unmethylated miR-133b forward, 3′-TTTTTA TTTAAAATATAAAAATAGTGG-5′ and reverse, 3′-ACC CAAACTAAAATACAATAACACT-5′. MSP was performed using the previously described negative controls and conditions (25).

The methylation pattern of miR-133b was analyzed using COBRA, as previously described (26). Briefly, the process involved sodium bisulfite treatment, which was followed by PCR, restriction digestion and quantitation (27). The following primers were designed using MethPrimer software to specifically amplify the methylated sequence of miR-133b: Forward, 3′-GTATTT AGTATAAAGAAAGTAA-5′ and reverse, 3′-ACCAATACC CATAAACAACG-5′.

The cells were seeded into 96-well plates at a density of 1×10³ cells/well. Following treatment, cell viability was evaluated at 0, 24, 48 and 72h using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer's instructions. Optical density was measured at a wavelength of 450 nm and all data is based on the results of three independent experiments with six replicates.

Transwell migration chambers (BioCoat Matrigel Invasion Chamber; BD Biosciences, Franklin Lakes, NJ, USA) were used to evaluate cell invasion. A total of 2.5×10⁵ cells/well were starved in serum-free medium (Invitrogen Life Technologies) for 24 h, then resuspended in serum-free medium. The cells were added to the upper chamber, while the lower chamber was filled with basic medium containing 10% fetal bovine serum. Following incubation for 24 h, the cells that had migrated across the membrane were fixed with 75% alcohol and stained with crystal violet (Amresco LLC, Solon, OH, USA) for 20 min. Migrated cells were then counted using an inverted microscope (TS100; Nikon Corporation, Tokyo, Japan). All experiments were repeated in triplicate.

Flow cytometry was used to analyze cell cycle distribution, as previously described (27). Briefly, 5-Aza-CdR or PBA-treated cells were fixed in ice-cold 70% ethanol and stained with propidium iodide. The cell cycle profiles were assayed using EPICS Elite ESP flow cytometry (Beckman Coulter, Inc., Brea, CA, USA) at a wavelength of 488 nm and the data were analyzed using the BD CellQuest software (BD Biosciences, San Jose, CA, USA).

Data in the present study are expressed as the mean ± standard deviation of a minimum of three independent experiments, unless otherwise stated. Analyses were conducted using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). An unpaired Student's t-test was used for comparisons between two groups. P<0.05 was considered to indicate a statistically significant difference.

The expression of miR-133b was previously reported to be decreased in CRC tissues and two colon adenocarcinoma cell lines (21). Therefore, the present study used RT-qPCR to determine miR-133b expression levels in healthy colon tissues, human CRC tissue and adjacent non-tumor tissue, as well as five CRC cell lines, consisting of the HT-29, HCT-116, SW480, SW620 and LOVO cell lines, and the healthy HCoEpiC colon cell line. miR-133b expression was notably downregulated or silenced in all the investigated CRC tissues and cell lines (P=0.013), but miR-133b was broadly expressed in all healthy tissues and HCoEpiC cell lines (Fig. 1A and B).

The present study investigated whether miR-133b silencing in CRC was caused by CpG methylation in the promoter region. MSP analysis revealed that miR-133b was frequently methylated in the investigated CRC tissues and in the SW260 and HT-29 colon cancer cell lines, and was strongly associated with expression levels. However, no miR-133b methylation was identified in the healthy colon tissue samples or healthy colon cell line (Fig. 2A and B). This finding was supported by results obtained from the RT-qPCR and COBRA analyses (Fig. 2C and D), which indicated that the expression of miR133b was increased in SW620 and HT-29 cells treated with Aza, PBA and Aza+PBA. CCK-8 was also used to assess the effects of demethylation agents on SW620 and HT-29 cell proliferation. It was observed that inhibition of proliferation was induced by Aza or PBA treatment, whilst a significantly higher level of inhibition was induced by Aza+PBA treatment (Fig. 2E).

The present study investigated the effects of miR-133b expression on the cell cycle and invasive ability of the HT-29 and SW620 CRC cell lines. Representative results of cell cycle distribution in control and 5-Aza-CdR/PBA-treated cells are shown in Fig. 3A–D. Flow cytometry analysis revealed a statistically significant increase in the number of 5-Aza-CdR/PBA-treated cells in the G0/G1 phase (13% in HT-29 cells and 12% in SW620 cells) compared with the number of control cells in the G0/G1 phase (P<0.05). This was accompanied by a significant decrease in the number of 5-Aza-CdR/PBA-treated cells in the S and G2-M phase compared with the control cells (P=0.027; Fig. 3E).

The effect of miR-133b methylation on tumor metastasis was evaluated by performing a cell invasion assay. Analysis of the Transwell invasion chamber assay data indicated that the invasive ability of cells in the miR-133b demethylation group treated with 5-Aza-CdR/PBA was significantly inhibited compared with the control group (P<0.05; Fig. 4).

The aforementioned results indicate that miR-133b methylation reduced CRC cell cycle progression and the invasiveness of CRC tumor cells.