Intrahepatic cholangiocarcinoma tissue and adjacent non-cancer tissue were surgically obtained between January 2010 and May 2012 from 30 patients at The First Affiliated Hospital of Nanjing Medical University. Written informed consent was obtained from all the patients before sample collection. The intrahepatic cholangiocarcinoma cells (HuCCT1), extrahepatic cholangiocarcinoma cells (QBC939) and the normal intrahepatic biliary epithelial cells (HIBEC) obtained from American Type Culture Collection (ATCC; Rockville, MD) were grown in Dulbecco’s medium essential medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) under cell culture conditions (5% CO₂, 95% relative humidity, and 37°C).

Total RNA was purified from cultured cells using the RNAiso™ Plus kit (Takara, Tokyo, Japan). cDNA was synthesized using the PrimeScript RT Master Mix (Takara) in a 10-μl reaction mixture according to the manufacturer’s protocol. cDNA (2 μl) was used as a template in a 20-μl reaction using the SYBR Premix Ex Taq real-time PCR kit (Takara). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. The following primers were used for the detection of TPM1 expression: 5′-ACGAACAACTTGAAGTCACTAA-3′ (sense) and 5′-TTTAGTTACTGACCTCTCCGCA-3′ (anti-sense). The primers for GAPDH were: 5′-ACGGATTTGGTC GTATTGGGC-3′ (sense) and 5′-TTGACGGTGCCATGG AATTTG-3′ (antisense). PCR was performed using the following cycles: 95°C for 30 sec, 40 cycles of 95°C for 5 sec, 60°C for 31 sec and the dissociation stage: 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec.

Cells were lysed, and equal amounts of protein were electrophoresed on a 12% SDS-polyacrylamide gel and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST) for 1 h at room temperature. The membranes were incubated with the following primary antibodies at 4°C overnight: TPM1 (1:400; ab55915, Abcam, Hong Kong, China), β-actin (1:800; Beijing Biosynthesis Biotechnology, Beijing, China), and α-tubulin (1:500; Beijing Biosynthesis Biotechnology). The membranes were then washed three times with PBST, incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000; Beijing Biosynthesis Biotechnology) for 2 h at room temperature. After three PBST washes, the membranes were developed using ECL Plus (Millipore, MA, USA) and exposed to X-ray film for visualization of protein bands. β-actin and α-tubulin were used as internal loading controls.

Immunohistochemistry was carried out on 30 cholangiocarcinoma tissues and 30 adjacent non-cancer tissues by overnight incubation at 4°C using a polyclonal antibody to TPM1 (ab55915, Abcam). The sections were subsequently treated with goat anti-mouse secondary antibody, followed by further incubation with streptavidin-HRP complex. Diaminobenzidine (Dako, Glostrup, Denmark) was used as a chromogen, and sections were lightly counterstained with hematoxylin. Immunostaining intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) as well as the percentage of cells stained (0, no cells; 1, <10% of cells; 2, 11–50% of cells; 3, 51–80% of cells; 4, >80% of cells) were evaluated, and the respective scores were multiplied, resulting in intensity values ranging from 0 to 12. The tissues showing immunostaining intensity values in the rage (0–6) were grouped as negative and those showing immunostaining intensity values in the rage (7–12) were grouped as positive.

Total RNA was extracted from cultured cells using the RNAiso Plus (Takara) and mature miR-21 was detected using the miRCURY LNA™ Universal RT microRNA PCR kit (Roche, Germany). The RT-PCR results were analyzed and expressed as relative Ct (threshold cycle) values, which were then converted to fold-changes. Each sample was measured in triplicate. The small nuclear RNA U6 (U6 snRNA) was used as an endogenous control.

To construct a lentiviral vector (LV) containing the miR-21 knockdown (LV-anti-miR21), as well as the negative control (LV-GFP), a complementary sequence that targeted miR-21 was designed and two suitable PCR primers that targeted the miR-21 sequence were annealed in order to obtain the fragment insert. The PCR primers were as follows: forward 5′-CCGGTCAACATCAGTCTGATAAGCTATTTTTG-3′ and reverse: 5′-AATTCAAAAATAGCTTATCAGACTGATGTTGA-3′. The fragment was cloned into the GV159 vector, after which the GV159 vector (20 μg), the pHelper 1.0 vector (15 μg), and the pHelper 2.0 vector (and 10 μg; Genechem Co., Shanghai, China) were cotransfected into 293T cells (purchased from ATCC) using 100 μl Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). The supernatants were collected 48 h later, were filtered (pore size, 0.45-μm) and stored at −80°C. HuCCT1 cells were infected with either LV-anti-miR21 or LV-green fluorescent protein (GFP) in the presence of 5 μg/ml polybrene (Sigma, St. Louis, MO, USA). The medium was refreshed after 6 h, and the efficiency of infection was measured 72 h later under a fluorescent microscope based on GFP expression.

Cells were treated with manumycin A (Cayman Chemicals, MI, USA; 1, 5, 10, 25, 50 and 75 μmol/) for 24 and 48 h, LY294002 (Sigma; 5, 10, 20 and 50 μmol) for 48 and 96 h, U0126 (Sigma; 10, 20, 50 and 75 μmol) for 48 and 96 h, 5-aza-2-deoxycytidine (DAC) (Sigma; 0.1, 0.5, 1, 5, 25 and 100 μmol) for 24 and 48 h (DAC was replaced with fresh drug every 24 h), and trichostatin A (TSA) (Sigma; 0.1, 0.25, 0.5, 1.0, 1.5 and 2.0 μmol) for 24 and 48 h.

Cells (4×10³) were seeded in 96-well plates in 100 μl medium containing 10% FBS. After 24 h, when the cells were grown to 50–60% confluency, manumycin A, U0126, LY294002, DAC or TSA were added. Cell proliferation was assessed at 24 and 48 h or 48 and 96 h by using the Cell Counting kit-8 (CCK-8; Beyotime, Jiangsu, China) according to the manufacturer’s protocol.

Cells (4×10⁵) were seeded in 6-well plates. After 24 h, cells were treated with manumycin A (10 μmol), U0126 (75 μmol) or TSA (0.25 μmol) for 24 h and LY294002 (75 μmol) and DAC (25 μmol) for 48 h. Apoptosis was detected using the Annexin V-FITC Apoptosis Detection kit (Keygen, Nanjing, China). Results were represented as the percentage of apoptotic cells in all the counted cells.

Cells (6×10⁵) were seeded in 6-well plates. After 24 h, when the cells were grown to 90–100% confluency, wounds were generated in cells by scraping with a plastic tip across the cell monolayer. The medium was replaced with serum-free medium, and the cells were treated with manumycin A (5 μmol), U0126 (10 μmol), LY294002 (5 μmol), DAC (25 μmol), TSA (0.1 μmol) for 24 h. Phase-contrast images were recorded at the time of wounding (0 h) and at 24 h thereafter. Untreated cells served as controls.

Data were analyzed using the SPSS 13.0 software and are presented as mean ± standard deviation (SD). Statistical comparisons between groups were performed using one-way analysis of variance (ANOVA) followed by a Student’s t-test. P<0.05 was considered statistically significant.

TPM1 expression was detected in HuCCT1 and QBC939 cell lines using western blot analysis and quantitative RT-PCR; HIBEC cells were used as a control. At the mRNA level, TPM1 was downregulated in HuCCT1 cells and upregulated in QBC939 cells compared with HIBEC cells (control). At the protein level, TPM1 was expressed at a much lower level in HuCCT1 cells compared with HIBEC cells, and was expressed at a higher level in QBC939 cells compared with HIBEC cells (Fig. 1A), indicating that the different expression of TPM1 in cholangiocarcinoma cells, is at the translational and transcriptional level. The immunohistochemistry results showed no staining (5/30) (Fig. 1B) or a weak staining (25/30) (Fig. 1C) for TPM1 in the intrahepatic cholangiocarcinoma tissues; however, all the adjacent non-cancer tissues (30/30) showed a weak staining for TPM1 (Fig. 1D). For statistical analysis, no staining and weak staining were grouped as negative and there was no significant difference between the staining results of intrahepatic cholangiocarcinoma tissues (0/30) and adjacent non-cancer tissues (0/30) (P>0.05).

Previous studies have shown that the Ras signaling pathway is involved in TPM1 downregulation in urinary bladder carcinoma and breast cancer (8,12). It is known that the K-ras gene is highly mutated in cholangiocarcinoma (13,14). Furthermore, studies have also shown that the HuCCT1 cell line contains K-ras mutations that are located at codons 12 and 61, but not at codon 13 and that the mutated Ras family genes may contribute to the growth of cholangiocarcinoma (15,16). The Ras family genes exert their functions by farnesylating themselves and manumycin offers promise as a specific inhibitor of the farnesylation process (17). It can exert an antiproliferative effect on human tumor cells that harbor a mutated K-ras gene (18). Based on the previous use of manumycin in abolishing the function of Ras in hepatocellular carcinoma (18) and pancreatic cancer (19,20), we treated HuCCT1 cells with manumycin A to determine whether the activated Ras can participate in the regulation of TPM1. Western blot analysis and RT-PCR of HuCCT1 cells, which were incubated with manumycin A (5 and 10 μmol/l) for 24 h, showed a significant restoration of TPM1 expression (Fig. 2A). This finding supports the role of activated Ras in the downregulation of TPM1.

It has been shown that the RAS/MEK/ERK pathway participates in the downregulation of TPM1 (8,12). In the present study, U0126, a specific MEK inhibitor, was used to investigate the role of this pathway in the downregulation of TPM1. HuCCT1 cells were incubated for 24 h with U0126 (5 and 10 μmol/l). At the mRNA level, the TPM1 expression increased, as revealed by RT-PCR results. Similarly, western blot analysis suggested that U0126 can restore the TPM1 expression in HuCCT1 cells at the protein level when compared with that in untreated controls (Fig. 2B).

Previous studies have reported that the PI3K/AKT pathway does not affect the expression of TPM1 (8,21). However, contradictory to this study, we found that the expression of TPM1 was decreased both at the mRNA level and at the protein level after a 24-h treatment with the PI3K/AKT-specific inhibitor LY294002 (20 and 50 μmol/l), as shown by western blot analysis and RT-PCR (Fig. 2C).

The present study showed that genetic mutations in the Ras signaling pathway play an important role in the regulation of TPM1 expression. The underlying epigenetic mechanisms have been found in breast cancer (6,22) and melanoma (23). To elucidate the epigenetic mechanisms responsible for TPM1 regulation in cholangiocarcinoma, we treated HuCCT1 cells with the demethylating agent, DAC or with the histone deacetylase inhibitor, TSA and subsequently analyzed TPM1 expression using western blots and RT-PCR. Evaluation of TPM1 expression before and after treatment of HuCCT1 cells with DAC and TSA showed that TPM1 mRNA and protein expression (Fig. 2D) was significantly upregulated after a 24-h treatment with DAC (1 and 5 μmol/l) compared to untreated cells. The TPM1 mRNA expression was slightly upregulated in HuCCT1 cells after a 24-h treatment with 0.1 μmol/l TSA, but not after a 24-h treatment with 0.25 μmol/l TSA. However, contradictory to the mRNA levels, the TPM1 protein level in TSA-treated cells was significantly upregulated compared to untreated cells (Fig. 2E).

TaqMan quantitative RT-PCR analysis demonstrated an overexpression of miR-21 in cholangiocarcinoma cell lines compared with non-malignant biliary epithelial cells (Fig. 3A). This result was in agreement with a previous study showing that miR-21 functions as an oncogene in cholangiocarcinoma (24). We also observed an inverse correlation between the levels of miR-21 and TPM1 in HuCCT1 cells, which suggests that miR-21 may also be involved in the loss of function of TPM1 in HuCCT1 cells.

Previous studies reported that miR-21 promoted tumor growth through the downregulation of TPM1 in breast cancer (25,26), tongue squamous cell carcinoma (27), prostate cancer (28) and glioma (29). In the present study, to investigate the relationship between miR-21 and TPM1 levels in HuCCT1 cells, a lentiviral vector containing the miR-21 knockdown (LV-anti-miR21) was constructed. In LV-anti-miR21-infected HuCCT1 cells, the miR-21 level was downregulated compared with that in LV-GFP-infected HuCCT1 cells (negative control) (Fig. 3B). RT-PCR and western blot analyses performed to investigate the effect of miR-21 on TPM1 expression showed that there was no difference in TPM1 expression in LV-anti-miR21-infected or LV-GFP (negative control)-infected HuCCT1 cells at the RNA level. However, at the protein level, there was a significant increase in TPM1 expression in LV-anti-miR21-infected HuCCT1 cells compared with that in LV-GFP (negative control)-infected HuCCT1 cells (Fig. 3C and D).

In the present study, HuCCT1 cells were exposed to the inhibitors manumycin A, LY294002, U0126, DAC and TSA to investigate their effect on cell growth inhibition in cholangiocarcinoma. There are no previous reports on the use of manumycin in the treatment of cholangiocarcinoma. Results of the CCK-8 assay showed that manumycin A inhibited HuCCT1 proliferation in a dose- and time-dependent manner (Fig. 4A). Because PI3K/AKT and MEK/ERK are two important signaling pathways in cholangiocarcinoma (30,31), the PI3K inhibitor LY294002 and the MEK inhibitor U0126 have been previously used to investigate their antiproliferative effect on cholangiocarcinoma (16,32–35). To our knowledge, our study is the first to analyze the effects of LY294002 and U0126 on the proliferation, migration, and apoptosis of HuCCT1 cells. We demonstrated that LY294002 and U0126 inhibited HuCCT1 proliferation in a dose- and in a time-dependent manner, in agreement with previous studies (16,32–35) (Fig. 4B and C). Although extrahepatic cholangiocarcninoma cells and gall-bladder carcinoma cells have been treated with the inhibitors DAC or TSA in previous studies (36,37), their effect on intrahepatic cholangiocarcninoma cells is unknown. In the present study, we made a novel observation that DAC inhibited the growth of HuCCT1 cells in a dose- and time-dependent manner (Fig. 4D). Our results were in agreement with those of a previous study showing that DAC inhibited the growth of QBC939 cells in a dose- and time-dependent manner (36). We also found that TSA inhibited the growth of HuCCT1 cells in a dose- and time-dependent manner (Fig. 4E).

Our results showed that manumycin A, LY294002, U0126, DAC and TSA were able to inhibit the growth of HuCCT1 cells. We further demonstrated that inhibitor-treated HuCCT1 cells underwent increased apoptosis compared with the untreated cells. Manumycin A(10 μmol/l), LY294002 (75 μmol/l) and U0126 (75 μmol/l), DAC (25 μmol/l) and TSA (0.25 μmol/l) significantly increased apoptosis of HuCCT1 cells (Fig. 4A, B, C, D and E respectively) compared with untreated cells (P<0.05).

Migration is one of the most important characteristics of malignant tumors, especially of cholangiocarcinomas. TPM1 plays an important role in cell motility and migration (10,11). We used specific inhibitors that blocked genetic and epigenetic mechanisms to investigate whether the migration of HuCCT1 cells is affected. HuCCT1 cells were wounded and treated with 5 μmol/l manumycin A, 5 μmol/l LY294002, 20 μmol/l U0126, 25 μmol/l DAC and 0.1 μmol/l TSA. Images were recorded at the time of wounding (0 h) and after 24 h. After 24 h, all the five inhibitors inhibited the migration of HuCCT1 cells in culture (Fig. 5).