Tissue samples (n=8) were collected between January 2008 and December 2010 at the Second Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China) and confirmed to be colorectal cancer pathologically. All participants provided written consent of their information to be stored in the hospital database and for samples to be used for research. This research was approved by the Institutional Review Boards of the Second Affiliated Hospital, Zhejiang University School of Medicine.

The Y human colorectal cancer cell line was established in our laboratory, as described previously (2). The HT29 and SW620 human colorectal adenocarcinoma cell line was obtained from the Cell Bank of the China Academy of Medical Sciences (Beijing, China). Y and HT29 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). SW620 cells were cultured in Leibovitz L15 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with FBS (Gibco; Thermo Fisher Scientific, Inc.). All cells were maintained at 37°C in a humidified 5% CO₂ atmosphere.

CRC cells (<2×107) were labeled with an anti-human monoclonal CD133 antibody conjugated to R-phycoerythrin (R-PE; cat. no. 130-098-826; 10 µl per 107 cells; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in PBS containing 2% FBS for 10 min in the dark at 4°C. A blank control and mouse IgG1-R-PE isotype control (cat. no. 130-098-845; 10 µl per 107 cells; Miltenyi Biotec GmbH) served as controls. CD133+ cells were identified and sorted using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA).

Total RNA was isolated from CD133+ and CD133- CRC cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The quantity and the quality of RNA were evaluated using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Inc.). The miRNA expression profile of CD133+ and CD133- CRC cells was assessed using an Affymetrix miRNA array (Affymetrix GeneChip miRNA 2.0 array; Affymetrix, Inc., Santa Clara, CA, USA).

Total RNA was isolated from cell lines and tissues using TRIzol reagent according to the manufacturer's protocol. The quantity and the quality of RNA were evaluated using a Nanodrop spectrophotometer. The RNA was reverse-transcribed to cDNA using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega Corporation, Madison, WI, USA). The expression of miR-141, sex determining region Y-box 2 (Sox2), octamer-binding transcription factor 4 (Oct4), nanog homeobox (Nanog) and B cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) was measured using the SYBR^®-Green PCR Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). All samples were run in triplicate. The miR-141 expression levels were normalized to those of U6, which served as an internal control. The Sox2, Oct4, Nanog and Bmi1 expression levels were normalized to those of GAPDH, which served as an internal control. The relative expression of target genes were calculated using the 2^−ΔΔCq method (18). The primer sequences used were as follows: SOX2, F 5′-AACCCCAAGATGCACAACTC-3′, R 5′-CGGGGCCGGTATTTATAATC-3′; Oct4, F 5′-TTCTCAGGGGGACCAGTGTC-3′; R 5′-CCCATTCCTAGAAGGGCAGG-3′; Nanog, F 5′-CCAGTGACTTGGAGGCTGC-3′, R 5′-AAGGATTCAGCCAGTGTCC-3′; Bmi1, F 5′-TGACAAATGCTGGAGAACTG-3′, R 5′-AAGATTGGTGGTTACCGCT-3′; GADPH, F 5′-ACAGTCAGCCGCATCTTCTT-3′; R 5′-TGGAAGATGGTGATGGGATT-3′. Results were expressed as relative quantitation.

CRC cells were transfected with the negative control mimic (5′-UUCUCCGAACGUGUCACGUTT-3′), an miR-141 mimic (5′-UAACACUGUCUGGUAAAGAUGG-3′) or an anti-miR-141 inhibitor (5′-CCAUCUUUACCAGACAGUGUUA-3′), all obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated for 12 h. Cells were subsequently seeded at a density of 3×103 cells per well in 0.2 ml RPMI 1640 medium containing 10% FBS in a 96-well plate. MTS reagent (20 µl; Promega Corporation) was added to each well and the cells were incubated at 37°C for 4 h. The absorbance values were measured at a wavelength of 490 nm on a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and assessed every day for 3 consecutive days.

CRC cells were transfected as described above. After a further incubation of 24 h, cells were reseeded in 96-well plates and treated with various concentrations of 5-fluorouracil (5-FU; Nantong Jinghua Pharmaceutical Co., Ltd., Nantong, China) or oxaliplatin (Sanofi S.A., Gentilly, France) for 48 h. A proliferation assay was subsequently performed as described above, to determine the individual inhibitory concentration 50 values (50% cell growth inhibitory concentrations).

Cells were trypsinized and fixed in 70% ethanol. The DNA content was assessed by analyzing incorporated propidium iodide (PI) (10 µl PI in 1.5 ml 1×106 cells; Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China) using a FACScanto II. The cell populations in G0/G1, S and G2/M phases were determined using ModFit LT software v2.0 (Verity Software House, Inc., Topsham, ME, USA).

Total protein was extracted from cells lysed with the M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Inc.) supplemented with a protease inhibitor cocktail (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). Protein extracts (50 µl extract from 1×10⁶ cells) were separated by SDS-PAGE and transferred to a PVDF membrane. Following blocking with 5% non-fat milk in TBS containing Tween-20 (TBST) for 60 min, the membrane was incubated with anti-human-cyclin D2 (cat. no. 3741S; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-human GAPDH (cat no. KC-5G5; 1:2,000; Shanghai Kang-Chen Biotech, Shanghai, China). Primary antibodies were diluted in 5% bovine serum albumin (Amresco, LLC, Solon, OH, USA) in TBST and incubated overnight at 4°C. Membranes were incubated in secondary antibody [anti-rabbit horseradish peroxidase conjugated; cat. no. 70-GAR007; 1:5,000; Multi Sciences (Lianke) Biotech Co., Ltd.) for 1 h at room temperature. ECL chromogenic substrate [cat no. 70-P1421; Multi Sciences (Lianke) Biotech Co., Ltd.] was used to visualize the proteins.

Data are presented as the mean ± standard error using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). The RT-qPCR results from paired clinical samples were analyzed using a two-tailed paired Student's t-test and the remaining data were analyzed using a two-tailed unpaired Student's t-test or analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

CSCs are involved in tumor initiation, recurrence, metastasis and chemotherapy resistance (7,19). The present study assessed miRNA expression profiles of CD133+ and CD133- cells to identify differentially expressed miRNAs potentially involved in tumor progression. Microarray analysis identified four miRNAs that were upregulated (miR-373, miR-1246-star, miR-494-star and miR-455-3p-star) and 5 that were downregulated (miR-29b, miR-638, miR-19a, miR-101 and miR-141) in CD133+ cells compared with CD133- cells (Fig. 1A). Of these, miR-141 underwent the greatest decrease. In addition, the importance of the miR-200 family in cancer development has been demonstrated, therefore miR-141 was selected for further investigation. RT-qPCR confirmed the microarray results, demonstrating a 2.33-fold decrease in miR-141 expression levels in CD133+ cells compared with CD133- cells sorted from the Y cell line (Fig. 1B).

miR-141 expression levels were analyzed in CRC tissue samples. In the limited number of fresh tissue samples available (n=8), miR-141 expression was downregulated in the majority of CRC samples compared with adjacent healthy tissues (Fig. 1C). Thus, downregulation of miR-141 may be important during CRC development.

CRC cells were transfected with a negative control mimic, an miR-141 mimic or an anti-miR-141 inhibitor to investigate the biological functions of miR-141. RT-qPCR confirmed the transfection efficiency. Overexpression of miR-141 suppressed cell proliferation, whereas inhibiting miR-141 via an anti-miR-141 promoted cell proliferation (Fig. 2A).

The effect of miR-141 on drug resistance was examined in vitro. 5-FU or oxaliplatin as typical antitumor drugs were added into the culture medium of SW620 and Y cells. Transfection with anti-miR-141 enhanced drug resistance (Fig. 2B). Following treatment of SW620 and Y cells with various concentrations (2, 5 or 10 ug/ml) of 5-FU (Fig. 2C) or oxaliplatin (Fig. 2D), cell viability was markedly reduced in the miR-141 mimic-transfected, compared with the negative control, group. Transfection with anti-miR-141 demonstrated the opposite effect. These results indicated that miR-141 suppresses CRC proliferation and enhances drug sensitivity.

Sox2, Oct4, Nanog and Bmi1 are stem cell-associated genes, which are involved in self-renewal and the maintenance of stemness in stem cells (20). Following transfection of SW620 cells with anti-miR-141, Sox2, Oct4, Nanog and Bmi1 mRNA expression levels were elevated (Fig. 3A) indicating that miR-141 may promote stem cell differentiation.

Stem cell differentiation is associated with cell cycle progression. Flow cytometry revealed that, in SW620 and Y cells transfected with an miR-141 mimic, the proportion of cells in the G0/1 phase decreased, whereas the percentage in S phase increased (Fig. 3B).

Potential gene targets of miR-141 were analyzed using the TargetScan database (www.targetscan.org). Hundreds of potential miR-141 targets were further analyzed using the Kyoto Encyclopedia of Genes and Genomes pathway database (www.genome.jp/kegg/). Cyclin D2 was identified as a functional downstream target of miR-141. The cyclin D2 3′ UTR contains highly conserved miR-141 binding sites (Fig. 4A). Western blotting revealed that cyclin D2 protein expression levels were decreased in cells following transfection with an miR-141 mimic, and increased following transfection with an anti-miR-141 inhibitor (Fig. 4B).