The present study was approved by the Ethical Committee of The Third Xiangya Hospital of Central South University (Changsha, China). A total of 15 ICC tissue samples and matched adjacent normal tissue samples were obtained from the Department of Hepatobiliary and Pancreatic Surgery, the Third Xiangya Hospital of Central South University. The patients providing the samples consisted of 11 males and 4 females with an average age 53.5 years old (41–64 years old). ICC was diagnosed by doctors from the Department of Pathology and tissues were obtained by surgical resection. The patients were recruited between June 2011 and January 2012. All patients provided their written consent for the present study. All tissues were immediately snap-frozen in liquid nitrogen following surgical removal, and stored at −70°C until further use.

The ICC-9810 human ICC cell line and normal human intrahepatic biliary epithelial cells (HIBEC) were obtained from the Institute of Cell Biology at the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies) at 37°C in a humidified incubator containing 5% CO₂.

For mRNA detection, total RNA was extracted from cells and tissues using TRIzol^® reagent (Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. The tissues were homogenized with liquid nitrogen in a mortar (Ziyi Company, Shanghai, China). RevertAid First-Strand cDNA Synthesis kit (Fermentas, Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to reverse transcribe RNA into cDNA (1 µg used). Subsequently, iQ™ SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to perform qPCR with the ABI 7500 thermocycler (Applied Biosystems Life Technologies, Foster City, CA, USA). The cycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec and 60°C for 30 sec. The sequences of the specific primers from Shanghai Shenggong Co., Ltd. (Shanghai, China) were as follows: VEGF-C, forward 5′-GGCTGGCAACATAACAGAGAA-3′, reverse 5′-CCCCACATCTATACACACCTCC-3′; and GAPDH, forward 5′-ACAACTTTGGTATCGTGGAAGG-3′, and reverse 5′-GCCATCACGCCACAGTTTC-3′. GAPDH was used as an internal reference. Relative expression was analyzed using the 2^−ΔΔCt method.

For miR detection, total RNA was extracted using TRIzol^® reagent, according to the manufacturer's instructions. miRNA Reverse Transcription kit (Life Technologies) was used to convert RNA into cDNA (1 µg used). Subsequently, qPCR was conducted using the miRNA QRT-PCR Detection kit (GeneCopoeia, Rockville, MD, USA) and the ABI 7500 ther-mocycler. The cycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec and 60°C for 30 sec. U6 was used as an internal reference. Relative expression was analyzed using the 2^−ΔΔCt method.

Tissues and cells were solubilized in cold radioimmunoprecipitation lysis buffer (Invitrogen Life Technologies). The tissues were homogenized with liquid nitrogen in a mortar (Ziyi Company, Shanghai, China). Proteins were separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology, Shanghai, China), and transferred onto a polyvinylidene difluoride (PVDF; Invitrogen Life Technologies) membrane. The PVDF membrane was then incubated with phosphate-buffered saline containing 5% milk at 4°C overnight. Subsequently, the PVDF membrane was incubated with monoclonal mouse anti-human VEGF-C (ab63221; 1:100) and monoclonal mouse anti-human GAPDH (ab8245; 1:100) primary antibodies (Abcam, Cambridge, UK) at room temperature for 3 h. The membrane was then incubated with rabbit anti-mouse secondary IgG antibodies (Abcam) at room temperature for 1 h. An Enhanced Chemiluminescence kit (Pierce Biotechnology, Inc., Rockford, IL, USA) was then used to perform chemiluminescent detection. The relative protein expression was analyzed using Image-Pro plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and was presented as the density ratio versus GAPDH.

Lipofectamine^® 2000 (Life Technologies) was used to perform cell transfection, according to the manufacturer's instructions. For miR-101 and VEGF-C functional analysis, ICC-9810 cells (5×10⁶ cells) were transfected for 48 h at 37°C with 100 mM each of scrambled miR as a negative control (NC), miR-101 mimics, miR-101 inhibitor (Life Technologies), VEGF-C-small interfering (si)RNA, or VEGF-C plasmid (Nlunbio, Changsha, China), respectively. Non-transfected ICC-9810 cells were used as control.

Bioinformatics analysis was conducted to predict the putative targets of miR-101 using Targetscan online software (www.targetscan.org).

A mutant (MUT) 3′-UTR of VEGF-C was generated using the Directed Mutagenesis kit (Stratagene, Agilent Technologies, Inc., Santa Clara, CA, USA), according to the manufacturer's instructions. The wild type (WT) or MUT 3′-UTR of VEGF-C (Nlunbio) was inserted into the psiCHECK™2 vector (Promega Corporation, Madison, WI, USA). Subsequently, ICC-9810 cells (5×10⁶ cells) were transfected with psiCHECK™2-WT VEGF-C 3′-UTR or psiCHECK™2-MUT VEGF-C 3′-UTR vector, with or without miR-101 mimics (100 nM), and then incubated at 37°C in an atmosphere containing 5% CO₂ for 48 h. The Dual-Luciferase Reporter Assay system (Promega Corporation) was then used to determine the luciferase activities on an LD400 luminometer (Beckman Coulter, Inc., Brea, CA, USA). Renilla luciferase activity was normalized to firefly luciferase activity.

Cell migration and invasion assays were performed using Transwell chambers (BD Biosciences, Franklin Lakes, NJ, USA). For the invasion assay, the Transwell chambers were pre-coated with Matrigel (BD Biosciences), whereas the chambers used for the migration assay were not. A cell suspension containing 5×10⁵ cells/ml was prepared in serum-free media, and 300 µl cell suspension was added into the upper chamber. A total of 500 µl DMEM supplemented with 10% FBS was added to the lower chamber. The cells were incubated for 24 h at 37°C. A cotton-tipped swab was then used to carefully wipe away the cells that did not migrate or invade through the pores. The filters were fixed in 90% alcohol and stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA). Cell number was determined in five fields randomly selected under an inverted microscope (IX73-A21PH; Olympus Corporation, Tokyo, Japan).

SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used to perform statistical analysis. All data are expressed as the mean ± standard deviation. Differences were analyzed using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

To determine the role of miR-101 in ICC, RT-qPCR was conducted to examine the expression levels of miR-101 in ICC tissue samples. miR-101 was markedly downregulated in the ICC tissue, as compared with in the matched adjacent normal tissue (Fig. 1A). Furthermore, the miR-101 expression levels were determined in ICC cells; miR-101 was also downregulated in the ICC-9810 human ICC cell line, as compared with in the normal HIBECs (Fig. 1B). These findings suggest that deregulation of miR-101 may be associated with the development of ICC.

Bioinformatics analysis suggested that VEGF-C is a potential target gene of miR-101. Therefore, the present study investigated whether miR-101 could mediate the expression of VEGF-C in ICC-9810 cells. As presented in Fig. 2A, the miR-101 levels in ICC-9810 cells transfected with miR-101 mimics were significantly upregulated compared with the control group. By contrast, the miR-101 levels in ICC-9810 cells transfected with the miR-101 inhibitor were markedly reduced compared with the control group. In addition, transfection with NC miR did not affect the miR-101 in ICC-9810 cells. These above data indicate that the transfection efficiency was satisfactory. Subsequently, the protein expression levels of VEGF-C were determined using western blotting. As shown in Fig. 2B, upregulation of miR-101 markedly decreased the protein expression levels of VEGF-C, whereas downregulation of miR-101 significantly increased the protein expression levels of VEGF-C in the ICC-9810 cells. To further verify whether miR-101 may directly bind to seed sequences in the VEGF-C 3′-UTR in ICC-9810 cells, WT and MUT VEGF-C 3′-UTRs were generated (Fig. 2C) and a luciferase reporter assay was performed. Luciferase activity was significantly reduced in the cells co-transfected with the WT VEGF-C 3′UTR and miR-101 mimics; however, there was no difference in luciferase activity in the cells co-transfected with the MUT VEGF-C 3′UTR and miR-101 mimics, as compared with the control group (Fig. 2D). These results indicate that VEGF-C is a direct target of miR-101, and the protein expression levels of VEGF-C are negatively regulated by miR-101 in ICC-9810 cells.

Since VEGF-C has been suggested to be involved in ICC metastasis, the present study further examined the roles of miR-101 and VEGF-C in the regulation of ICC cell migration. ICC-9810 cells were transfected with miR-101 mimics, VEGF-C siRNA, or co-transfected with miR-101 mimics and VEGF-C plasmid. As shown in Fig. 3A, transfection efficiency was satisfactory. Upregulation of miR-101 or knockdown of VEGF-C markedly suppressed the migration of ICC-9810 cells; however, the suppressive effects of miR-101 upregulation on ICC-9810 cell migration were significantly attenuated following overexpression of VEGF-C (Fig. 3B). These results suggest that miR-101 may inhibit ICC-9810 cell migration by directly targeting VEGF-C.

The present study also examined the effects of miR-101 and VEGF-C on ICC cell invasion. Concordant with the cell migration results, upregulation of miR-101 or knockdown of VEGF-C significantly inhibited ICC-9810 cell invasion; however, the suppressive effects of miR-101 overexpression on ICC-9810 cell invasion were reversed following upregulation of VEGF-C (Fig. 4). These results indicate that miR-101 may inhibit ICC-9810 cell invasion via direct inhibition of VEGF-C.

The present study further detected the mRNA and protein expression levels of VEGF-C in ICC tissue samples and cell lines. As shown in Fig. 5A and B, the mRNA and protein expression levels of VEGF-C were frequently upregulated in ICC tissue, as compared with in the matched adjacent normal tissue. Furthermore, the mRNA and protein expression levels of VEGF-C were upregulated in the ICC-9810 cells, as compared with in the normal HIBECs (Fig. 5C and D). In addition, an inverse correlation was detected between miR-101 and VEGF-C expression in the ICC tissue (Fig. 5E).