Primary CCA tissues and matched adjacent paracancerous tissues (distance, 1 cm) were surgically obtained from 27 patients with CCA 20 males and 7 females with an age range of 56–78 years (mean age, 66.4 years), including 9 cases of highly differentiated CCA and 18 cases of less differentiated CCA) at The Second Hospital of Hebei Medical University (Shijiazhuang, China) from January 2016 to December 2017. Ethics approval (approval no. 2014018) was obtained from The Second Hospital of Hebei Medical University and all samples were collected with written informed consent from the patients.

RBE cells (Qiao Xinzhou Biotechnology Co., Ltd.) were cultured at 37°C with 5% CO₂ in DMEM (Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Biological Industries; Sartorius AG) and 1% penicillin and streptomycin (Sigma-Aldrich; Merck KGaA). Cells in the logarithmic growth phase were used for experiments.

Total RNA was extracted from CCA and paracancerous tissue and RBE cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and first-strand cDNA was synthesized using PrimeScript™ RT Reagent kit (Takara Biotechnology Co., Ltd.). qPCR was performed in triplicate with SYBRII qPCR Master Mix (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol using GAPDH as a control. RT was performed at 37°C for 15 min and 85°C for 5 min. Thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 31 sec. The relative mRNA levels of the target genes were calculated with the 2^−ΔΔCq method (22). The primer sequences used in qPCR are listed in Table I.

Western blotting was performed as previously described (18). Briefly, total protein was extracted from RBE (5×10⁶) cells, CCA tissues and paracancerous tissues with cold RIPA buffer containing protease and phosphatase inhibitors (all Beijing Solarbio Science and Technology Co., Ltd.), followed by quantification of protein concentration using the BCA assay. Protein samples (30 µg/lane) were heated to 100°C for 10 min in SDS-PAGE loading buffer (Thermo Fisher Scientific, Inc.) and separated on 10% SDS-PAGE. The separated proteins were transferred at 200 mA to a PVDF membrane (MilliporeSigma) for 2 h at 4°C. After blocking the membrane with TBS containing 5% skimmed milk for 1.5 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies used were as follows: β-actin (rabbit monoclonal; 1:2,000; Abcam), Bax (rabbit monoclonal; 1:500; Abcam), Bcl2 (rabbit monoclonal antibody, dilution: 1:500; Abcam), cleaved caspase-3 (rabbit monoclonal antibody dilution: 1:500; Abcam), cleaved caspase-8 (rabbit polyclonal antibody, dilution: 1:500; Abcam), cleaved caspase-9 (rabbit polyclonal; 1:500; Abcam), MMP2 (rabbit polyclonal antibody, dilution: 1:500; Abcam), MMP9 (rabbit polyclonal antibody, dilution: 1:500; Abcam), ICAM-1 (rabbit polyclonal antibody, dilution: 1:500; Abcam); VEGFA (rabbit polyclonal; 1:500; Abcam); E-cadherin (rabbit polyclonal; 1:500; Abcam); N-cadherin (rabbit polyclonal antibody, dilution: 1:500; Abcam); TWIST (mouse monoclonal antibody, dilution: 1:500, Santa Cruz Biotechnology, Inc.); Vimentin (mouse monoclonal antibody, dilution: 1:500; Santa Cruz Biotechnology, Inc.); CSRC (rabbit monoclonal; 1:500; Abcam); p-CSRC (rabbit monoclonal antibody, dilution: 1:500; Abcam); PI3K, p-PI3K, AKT, p-AKT (rabbit monoclonal antibody, dilution: 1:500; Cell Signaling Technology, Inc.). Appropriate horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, dilution: 1:5,000 and goat anti-mouse IgG, dilution: 1:3,000) at 25°C for 1 h. The bands were detected using SuperECL Plus detection reagents (LI-COR Biosciences) and quantified with a Bio-Rad ChemiDoc imaging system (Bio-Rad Laboratories, Inc.) using β-actin as an internal control.

RBE cells (30–40% confluence) were transfected with Lipofectamine^® RNAiMAX Reagent (Thermo Fisher Scientific, Inc.) and 20 nM small interfering RNA (siRNA) (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions for 4 h at 37°C. The sequences of all specific and control siRNAs are as follows: siRNA-1 CCUCUACAAAGAGGUGGACAGAGAATT; siRNA-2 CCAUGGUGAGAAAUUGACGACUUCATT; siRNA-3 CACCACUGCCAAGCUCACUAUUGAATT and control, CCUCUUACCUCAGUUACAAUUUAUATT. Three separate CEACAM6-specific siRNA sequences were used, and an unrelated control siRNA sequence was transfected using the same protocol, which had no effect on CEACAM6 expression. Non-transfected cells were also used as a negative control; these were treated with serum-free DMEM for 4 h at 37°C. Knockdown of CEACAM6 expression was confirmed at 48 h by western blotting.

The viability of RBE cells was determined using a Cell Counting Kit (CCK)-8 assay (Abcam) according to the manufacturer's instructions. Briefly, transfected cells (2×10³) in 96-well plates were incubated for 24 h at 37°C and 5% CO₂. CCK-8 reagent (10 µl) was added for 1 h at 37°C and optical density was measured at 460 nm.

RBE cells (80–90% confluence) transfected with siRNAs for 48 h were washed twice with PBS (both 30 sec at room temperature) and then resuspended in staining buffer containing 0.025 mg/ml Annexin V-FITC and 1 mg/ml propidium iodide (Shanghai Yisheng Biotechnology Co., Ltd.). Double staining was performed for 10 min at room temperature in the dark, and the number of apoptotic cells was then determined by flow cytometry using a BD FACSCanto™ ІІ flow cytometer (BD Biosciences). The software used for data analysis is FlowJo (7.6.1; FlowJo, LLC.).

Cell invasion assays were performed using Transwell inserts (Corning, Inc.) coated with Matrigel (37°C for 1 h) were performed as previously described (23). Briefly, RBE cells (1×10⁵) in 0.2 ml serum-free DMEM were placed in the upper chamber, and the lower chamber was loaded with 0.5 ml medium containing 15% FBS. Cells that migrated to the lower surface of the filters were stained with 0.005% crystal violet solution for 40 min at room temperature, and cells in five fields of view were counted after 24 h of incubation at 37°C and 5% CO₂ using light microscope. Three wells were examined for each cell type and condition, and the experiments were conducted in triplicate.

RBE cells (6×10⁵) were seeded in 6-well plates. When cells reached 90% confluency, a 100-µl pipette tip was used to scratch the serum-starved cell monolayer (time 0 h). Images of migrating cells during the closure of the wounded region were captured at 0 and 24 h using light microscope (magnification, ×10).

All statistical analyses were conducted using SPSS version 19.0 (IBM, Corp.). Data are presented as the mean ± standard error of the mean of three independent repeats. Comparisons of the means of different groups were performed by using two-way mixed ANOVA or one-way ANOVA. Pairwise comparisons with homogeneity of variance were performed using the post hoc Tukey's honestly significant difference and Student-Newman-Keuls tests. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, CEACAM6 was overexpressed in highly differentiated and less differentiated CCA tissues compared with its expression levels in highly differentiated and less differentiated paracancerous tissues (P<0.05). Furthermore, lesser differentiation degrees of CCA tissues resulted in higher CEACAM6 expression levels (P<0.05).

After determining that CEACAM6 was highly expressed in CCA tissues, the role of CEACAM6 in the invasion and migration of RBE cells was next verified. siRNA transfection was used to silence the expression of CEACAM6. The results of Fig. 2A-D show that CEACAM6-siRNA-2 exhibited the best knockdown efficiency after 48 h. Thus, CEACAM6-siRNA-2 was utilized in subsequent experiments. As shown in Fig. 2E, CEACAM6-siRNA-2 significantly decreased RBE cell viability by ~50 compared with that of the control group (P<0.05). In addition, Fig. 2F shows that the percentage of apoptotic cells increased after transfection of CEACAM6-siRNA-2 (34.4%) compared with that of the scramble (scr)-siRNA group (13.8%; P<0.05).

As shown in Fig. 3A and C, the invasion ability of RBE cells was decreased after transfection with CEACAM6-siRNA-2. Similar results were obtained regarding the migration ability of RBE cells, which was also declined upon CEACA-siRNA-2 transfection, as shown in Fig. 3B and D (P<0.05).

Since the percentage of apoptotic cells increased after transfection with CEACAM6-siRNA-2, the present study next investigated whether the mRNA and protein expression levels of apoptosis-related proteins were consistent with the aforementioned findings. The results showed that the mRNA and protein levels of anti-apoptotic Bcl-2 decreased, while those of pro-apoptotic Bax increased after transfection with CEACAM6-siRNA-2.

The caspase family is composed of a class of proteolytic enzymes that mediate apoptosis and are activated during cell apoptosis. Caspases-3, −8 and −9 are major members of the caspase family. The results showed that the protein of cleaved caspases-3, −8 and −9 and mRNA levels of caspases-3, −8 and −9 were significantly upregulated after transfection with CEACAM6-siRNA-2 (Fig. 4A-G).

There are four types of EMT markers (epithelial and interstitial cell, transcription factor and cytoskeleton); of them, the epithelial cell marker E-cadherin and the interstitial cell marker N-cadherin are the most studied. In the present study, E-cadherin expression was significantly increased, while N-cadherin expression was significantly decreased after CEACAM6-siRNA-2 transfection.

MMPs are a group of zinc ion (Zn²⁺)-dependent endopeptidases that degrade extracellular matrix (ECM) and then induce pathological processes, such as tumor cell invasion and migration. MMP-2 and MMP-9 are important MMPs, and the protein and mRNA expression levels of these endopeptidases were reduced after siRNA transfection.

The TWIST transcription factor enhances EMT by inhibiting E-cadherin expression and enhancing tumor cell migration and invasion. The protein and mRNA expression of TWIST decreased after transfection with CEACAM6-siRNA-2.

Vimentin is an intermediate filament protein in mesenchymal cells that regulates protein-protein interactions, such as those involving cytoskeletal proteins and cell adhesion molecules, which may participate in cell invasion, migration and signal transduction. After transfection with CEACAM6-siRNA-2, the expression level of this tumor-promoting molecule decreased.

Furthermore, tumor angiogenesis is associated with VEGFA overexpression in human iCCA and pCCA (24). In the present study, the protein and mRNA levels of VEGFA and tumorigenesis-promoting ICAM-1 decreased when CEACAM6-siRNA-2 was transfected into RBE cells. The aforementioned results are shown in Fig. 4H-R.

The present study demonstrated that there was no significant difference in the relative mRNA levels of molecules involved in the SRC/PI3K/AKT signal transduction pathway when CEACAM6 was knocked down (Fig. 5A). However, when CEACAM6-siRNA-2 was added to the cells, the levels of phosphorylated molecules involved in the SRC/PI3K/AKT signal transduction pathway was reduced (Fig. 5B-E).