IHCC tissues and paired adjacent non-tumorous tissues (n=106) were collected from the patients with IHCC who underwent curative surgical resection between January 2008 and October 2015 at The Fourth Hospital of Hebei Medical University (Shijiazhuang, China). Tumor and adjacent non-tumorous tissues were isolated. Non-tumorous tissues were obtained from ≥1 cm away from the tumor border and were confirmed to contain no existing tumor cells. Following resection, the collected paired tissue samples were frozen with liquid nitrogen and stored at −80°C until further use. No patients received anticancer therapies prior to surgery. Patients diagnosed with more than two malignances were excluded from the study. Written informed consent was obtained from each patient. This study was approved by The Ethics Committee of The Fourth Hospital of Hebei Medical University (Shijiazhuang, China).

The IHCC cell lines HUH28, HuCCT1, RBE and HCCC9810 and the immortalized human cholangiocyte-derived cell line H69 were all purchased from The Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in RPMI-1640 medium (HuCCT1, HCCC9810 and RBE) or Dulbecco's modified Eagle's medium (DMEM) (HUH28), supplemented with 10% fetal bovine serum (all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 1% penicillin/streptomycin in an atmosphere of 5% CO₂ at 37°C.

lncRNA CCAT2 overexpression vector (pcDNA3.1-CCAT2), control vector (pcDNA3.1-vector) and non-targeting small interfering (si)RNA negative control (siNC) were all purchased from Genewiz, Inc. (South Plainfield, NJ, USA). CCAT2 siRNA (iCCAT2.1 and siCCAT2.2) were designed using the Custom RNAi Design Tool on the Integrated DNA Technologies (IDT) website (http://sg.idtdna.com/site) and synthesized by Genewiz, Inc. Transfection was performed with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's instructions. Subsequent experiments were performed 24 h after transfection.

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from clinical specimens or cultured cells, according to the manufacturer's instructions. The concentration and purity of the RNA were determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was reverse transcribed using 2 µg extracted RNA with the PrimeScript One-Step RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed using a SYBR PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd.) with the Mx3005P qPCR System (Agilent Technologies, Inc., Santa Clara, CA, USA), according to the manufacturer's instructions. The PCR cycling conditions were as follows: initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 30 sec, annealing at 50°C for 30 sec and extension at 72°C for 30 sec. The expression of lncRNA CCAT2 from tissue samples or cultured cells was normalized to GAPDH and compared using the 2^−ΔΔCq method (17). The primer sequences were: CCAT2 forward, 5′-CCCTGGTCAAATTGCTTAACCT-3′ and reverse, 5′-TTATTCGTCCCTTTTTATGGAT-3′; GADPH forward, 5′-ACCCACTCCTCCACCTTTGAC-3′ and reverse, 5′-TGTTGCTGTAGCCAAATTCGTT-3′.

Cells (2,000/well) were seeded into 96-well plates for incubation at 37°C, and MTT (10 µl, 10 mg/ml) was added to the medium at the indicated time (0, 24, 48, 72 and 96 h following the initial incubation). The plates were incubated for another 4 h at 37°C, and dimethylsulfoxide (100 µl) was added to dissolve the purple formazan crystals. The absorbance was subsequently measured by a microplate spectrophotometer (Dynex Technologies, Inc., Sullyfield, VA, USA) at a wavelength of 490 nm. Each assay was conducted in triplicate and repeated at least three times.

Cells (1,500 cells/dish) were seeded in 6 cm dishes and incubated in an atmosphere of 5% CO₂ at 37°C for 7 days until the colonies were large enough to be clearly discerned. Colonies were defined as groups of >50 cells and were counted by light microscopy (×10). Each assay was conducted in triplicate and repeated at least three times.

Cell migratory ability was evaluated using a Transwell system (pore size, 8.0 µm; Sigma-Aldrich; Merck KGaA) Cells (3×10⁴) were suspended in serum-free medium (RPMI-1640 medium for HuCCT1, HCCC9810 and RBE; DMEM medium for HUH28) and plated in the upper chamber of the Transwell system, and the lower chamber was filled with medium containing 10% FBS. Following incubation for 24 h at 37°C, the cells in the upper chamber were removed and the cells in the lower chamber were fixed by pure cold methanol (−4°C) for 30 min and subjected to Giemsa staining (1:10 dilution) for another 30 min at room temperature. Cells were subsequently counted with a light microscope (×10). The Matrigel assay procedure to assess cell invasive ability was identical to the Transwell assay, except the Transwell membrane was precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). All experiments were performed in triplicate.

Statistical analysis was performed using the SPSS 17.0 software package (SPSS, Inc., Chicago, IL, USA). All data is expressed as the mean ± standard deviation. For statistical analysis, categorical variables were compared using the χ² test and the Student's t-test was performed to compare the differences between continuous data. One-way analysis of variance and Bonferroni correction test were used to compare multiple groups. The overall survival (OS) and progression free survival (PFS) rate were analyzed with the Kaplan-Meier method and differences between groups were assessed with the log-rank test. The significance of the survival data was evaluated using univariate and multivariate Cox regression methods. The receiver-operating characteristic (ROC) curve was adopted to define the cut-off score for discriminating the specimens with high or low CCAT2 expression. The point on the curve with the shortest distance to the coordinate (0, 1) was selected as the threshold value to classify cases as high expression or low expression. P<0.05 was considered to indicate a statistically significant difference.

The analysis of lncRNA CCAT2 expression in IHCC cells lines by RT-qPCR assay revealed that CCAT2 expression was upregulated in IHCC cell lines compared with normal biliary epithelial cells (Fig. 1A). CCAT2 expression was higher in 70.8% (75/106) of IHCC samples compared with the paired adjacent non-tumorous tissues (Fig. 1B). Further statistical analysis confirmed that IHCC tissues had significantly higher relative CCAT2 expression compared with paired adjacent non-tumorous tissues (1 vs. 2.52±3.17; P<0.001; Fig. 1B), which indicated that lncRNA CCAT2 may be oncogenic in IHCC.

To better define the value of CCAT2 expression level in predicting the prognosis of patients with IHCC, the ROC curve was adopted to identify the optimal cut-off score for high/low CCAT2 expression (Fig. 1C and D). The area under the curves were 0.702 and 0.715 for OS and PFS, respectively, which suggested that CCAT2 may be a useful biomarker for the prognostic prediction of IHCC (P<0.001). The cutoff score was 4.4 for both OS and PFS.

To investigate the clinical significance of CCAT2 in IHCC, the association between CCAT2 expression and clinicopathological characteristics was statistically analyzed. High CCAT2 expression was revealed to be associated with microvascular invasion (P<0.001), differentiation grade (P=0.040), and tumor (T; P=0.012), lymph node (N; P<0.001), metastasis (M; P=0.045) and combined TNM (P<0.001) stages of IHCC (Table I). However, other clinicopathological parameters including age, sex, hepatitis B viral protein, hepatitis B surface antigen, hepatitis C virus, cancer antigen 19-9, tumor number, cirrhosis and encapsulation were not indicated to be statistically significant (P>0.05; Table I). These results suggested that CCAT2 overexpression may be associated with the development of IHCC.

The significance of CCAT2 in the prediction of prognosis was evaluated by Kaplan-Meier analysis. Patients with high CCAT2 expression were demonstrated to have a lower OS rate (P<0.001) and a shorter PFS period (P<0.001; Fig. 1E and F)

Furthermore, univariate analysis demonstrated that high CCAT2 expression was a risk factor for OS [hazard ratio (HR) = 3.184; 95% confidence interval (CI) = 1.882–5.385; P<0.001] and for PFS (HR = 2.926; 95% CI = 1.771–4.834; P<0.001) of patients with IHCC (Table II). Multivariate analysis also identified high CCAT2 expression as an independent risk factor of OS (HR=1.894; 95% CI=1.060–3.384; P=0.031) and PFS (HR=2.134; 95% CI=1.232–3.699; P=0.007) of patients with IHCC (Table III). Thus, lncRNA CCAT2 may be useful as a prognostic marker for OS and PFS of patients with IHCC.

To extend our understanding on the role of CCAT2 in IHCC, in vitro assays were performed in IHCC cells with either CCAT2 silencing and overexpression (Fig. 2A and B). As CCAT2 expression was relatively high in HuCCT1 cells and low in RBE cells (Fig. 1A) these cell lines were used to confirm the effects of siRNA-induced CCAT2 silencing and CCAT2 overexpression, respectively (Fig. 2A and B, respectively). The MTT assay revealed that the proliferative ability of HuCCT1 cells was significantly decreased following transfection with siCCAT2.1 and siCCAT2.2 compared with cells transfected with the siNC (Fig. 2C; P<0.05). The colony formation assay also revealed that CCAT2 silencing in HuCCT1 cells significantly decreased colony numbers compared with the siNC-transfected cells (Fig. 2D; P<0.05). In addition, CCAT2 overexpression significantly increased the proliferative ability (Fig. 2E; P<0.05) and the number of colonies (Fig. 2F; P<0.05) of RBE cells compared with cells transfected with the empty vector.

The migratory and invasive abilities of HuCCT1 cells were significantly inhibited in cells transfected with either CCAT2 siRNA compared with siNC-transfected cells (Fig. 3A; P<0.05) By contrast, the migratory and invasive abilities were significantly increased in RBE cells overexpressing CCAT2 compared with vector control cells (Fig. 3B; P<0.05). These data suggested that CCAT2 may promote proliferation and metastasis of IHCC cells.