CCA Tissues Research Articles

Sirtuin7 has an oncogenic potential via promoting the growth of cholangiocarcinoma cells

Accumulating evidence indicates that sirtuin7 (SIRT7) plays an oncogenic role in the main types of liver cancer, hepatocellular carcinoma (HCC). Nevertheless, the clinical significance of SIRT7 and its role in cholangiocarcinoma (CCA) is largely undiscovered. Here, we found that SIRT7 displayed higher expression in CCA tissues compared to intrahepatic normal bile duct and surrounding liver tissues based on The Cancer Genome Atlas (TCGA) data. Our data further confirmed that SIRT7 was overexpressed in CCA patient tissues and cell lines. Clinical analysis revealed that high SIRT7 expression was correlated with large tumor size and advanced tumor-node-metastasis (TNM) stage. Furthermore, SIRT7 overexpression independently predicted poor prognosis of CCA patients. Functionally, we demonstrated that SIRT7 knockdown suppressed proliferation and cell cycle progression of HUCCT1 cells in vitro and in vivo. SIRT7 restoration promoted the growth of QBC-939 cells. Mechanistically, SIRT7 reduced p21 expression and increased the levels of Cyclin D1 and cyclin dependent kinase 2 (CDK2) in CCA cells. Furthermore, microRNA-125b-5p (miR-125b-5p) was recognized as a direct negative regulator of SIRT7 and reduced SIRT7 abundance in CCA cells. Notably, miR-125b-5p restoration showed similar effects to SIRT7 knockdown on the growth of CCA cells. Taken together, we demonstrate for the first time that miR-125b-5p regulation of SIRT7 functions as an oncogene and a potential prognostic biomarker in CCA.

Downregulated circular RNA hsa_circ_0001649 regulates proliferation, migration and invasion in cholangiocarcinoma cells

Cholangiocarcinoma (CCA) is one of the most aggressive malignancies with increasing worldwide incidence and is characterized by unfavorable prognosis due to its early invasive characteristics and poor response to chemotherapy or radiotherapy. Accumulating evidence has indicated that aberrantly expressed circular RNAs (circRNAs) are involved in cancer development and progression. However, their clinical values and biological roles in CCA remain unclear. Hsa_circ_0001649, a novel cancer-related circRNA, has been previously reported to be downregulated in hepatocellular carcinoma and gastric cancer. In the present study, qRT-PCR was carried out to measure the expression of hsa_circ_0001649 in CCA tissue samples and cell lines, and the correlation between hsa_circ_0001649 expression and clinicopathologic features was analyzed. The biological functions of hsa_circ_0001649 in CCA cells were evaluated both in vitro and in vivo. As a result, hsa_circ_0001649 was aberrantly downregulated in CCA tissues and cells, and this downregulation was associated with tumor size and differentiation grade in CCA. In addition, hsa_circ_0001649 overexpression caused tumor suppressive effects via inhibiting cell proliferation, migration and invasion; inducing cell apoptosis in KMBC and Huh-28 cells. On the contrary, silencing of hsa_circ_0001649 caused the opposite phenotypes. Furthermore, tumor xenograft study confirmed the in vitro results. Collectively, our findings suggest that hsa_circ_0001649 might be a rational CCA-related therapeutic target.

Long non-coding RNA CCAT2 promotes cholangiocarcinoma cells migration and invasion by induction of epithelial-to-mesenchymal transition

Cholangiocarcinoma (CCA) is one of the most aggressive malignancies in humans. Emerging evidence has indicated that abnormally expressed long non-coding RNAs (lncRNAs) could conduce to tumorigenesis and progression. Specifically, colon cancer-associated transcript 2 (CCAT2) has been reported to be overexpressed in several carcinomas. However, its clinical significance and functional roles in CCA is still unknown. qRT-PCR experiments were conducted to assess the CCAT2 expression in CCA tissue samples and cell lines. In addition, the link between CCAT2 expression and clinicopathological characteristics was analyzed. The potential effects of CCAT2 in CCA cells was evaluated in vitro including cell proliferation, colony-forming ability, apoptosis, migration, invasion and epithelial-to-mesenchymal transition (EMT). As a result, CCAT2 was aberrantly overexpressed in CCA tissue samples and cells, and this upregulation was correlated with tumor size, lymph node invasion, TNM stage and postoperative recurrence in CCA patients. Overexpression of CCAT2 could serve as an independent prognostic indicator for CCA. Additionally, overexpression of CCAT2 was a dismal prognostic indicator for patients with CCA. Furthermore, CCAT2 silencing caused tumor suppressive effects via reducing cell proliferation, migration and invasion, inducing cell apoptosis and reversing the EMT process in HuCCT1 and CCLP1 cells. Collectively, our data illustrated that lncRNA CCAT2 played an oncogenic role in CCA and may offer a potential therapeutic target for treating this fatal disease.

Tip60 Suppresses Cholangiocarcinoma Proliferation and Metastasis via PI3k-AKT

Background/Aims: Aberrant expression of Tip60 is associated with progression in many cancers. However, the role of Tip60 in cancer progression remains contradictory. The aim of this study was to investigate the clinical significance, biological functions and underlying mechanisms of Tip60 deregulation in cholangiocarcinoma (CCA) for the first time. Methods: Quantitative real-time PCR (QRT-PCR), western blotting and immunohistochemistry staining (IHC) were carried out to measure Tip60 expression in CCA tissues and cell lines. Kaplan–Meier analysis and the log-rank test were used for survival analysis. In vitro, cell proliferation was evaluated by flow cytometry and CCK-8, colony formation, and EDU assays. Migration/ invasion was evaluated by trans-well assays. Phosphokinase array was used to confirm the dominant signal regulated by Tip60. Tumor growth and metastasis were demonstrated in vivo using a mouse model. Results: Tip60 was notably downregulated in CCA tissues, which was associated with greater tumor size, venous invasion, and TNM stage. Down-regulation of Tip60 was associated with tumor progression and poorer survival in CCA patients. In vitro and in vivo studies demonstrated that Tip60 suppressed growth and metastasis throughout the progression of CCA. We further identified the PI3K/AKT pathway as a dominant signal of Tip60 and suggested that Tip60 regulated CCA cell proliferation and metastasis via PT3K-AKT pathway. Pearson analysis revealed that PTEN was positively correlated with the Tip60 level in CCA tissues. Conclusion: Tip60, as a tumor suppressor in CCA via the PI3K/AKT pathway, might be a promising therapeutic target or prognostic marker for CCA.

Progranulin modulates cholangiocarcinoma cell proliferation, apoptosis, and motility via the PI3K/pAkt pathway.

Progranulin (PGRN) is a growth factor normally expressed in rapidly cycling epithelial cells for growth, differentiation, and motility. Several studies have shown the association of PGRN overexpression with the progression of numerous malignancies, including cholangiocarcinoma (CCA). However, the underlying mechanisms on how PGRN modulates CCA cell proliferation and motility is not clear. In this study, we investigated the prognostic significance of PGRN expression in human CCA tissue and the mechanisms of PGRN modulation of CCA cell proliferation and motility. We found that CCA tissues with high PGRN expression were correlated with poor prognosis and likelihood of metastasis. PGRN knockdown KKU-100 and KKU-213 cells demonstrated a reduced rate of proliferation and colony formation and decreased levels of phosphatidyl inositol-3-kinase (PI3K) and phosphorylated Akt (pAkt) proteins. Accumulation of cells at the G1 phase was observed and was accompanied by a reduction of cyclin D1 and CDK4 protein levels. Knockdown cells also induced apoptosis by increasing the Bax-to-Bcl-2 ratio. Increased cell apoptosis was confirmed by annexin V-FITC/PI staining. Moreover, suppression of PGRN reduced CCA cell migration and invasion in vitro. Investigating the biomarkers in epithelial–mesenchymal transition (EMT) revealed a decrease in the expression of vimentin, snail, and metalloproteinase-9. In conclusion, our findings imply that PGRN modulates cell proliferation by dysregulating the G1 phase, inhibiting apoptosis, and that it plays a role in the EMT affecting CCA cell motility, possibly via the PI3K/pAkt pathway.

MiR-191 Inhibition Induces Apoptosis Through Reactivating Secreted Frizzled-Related Protein-1 in Cholangiocarcinoma

Background/Aims: Cholangiocarcinoma (CCA) is one of the most common malignant tumors of the biliary tract originating from biliary epithelial cells. Although many therapeutic strategies have been developed to treat CCA, the survival rate for CCA patients is still quite low. Thus it is urgent to elucidate the pathogenesis of CCA and to explore novel therapeutic targets. miR-191 has been shown to be associated with many human solid cancers, but the function of miR-191 in CCA is still poorly understood. Methods: We first investigated the expression level of miR-191 in human CCA tissues and cell lines with quantitative real-time PCR (qRT-PCR). The effects of miR-191 on CCA cells were determined by Cell Counting Kit-8 assay, colony formation assay and acridine orange/ethidium bromide staining. Finally, we utilized qRT-PCR, western blot and luciferase reporter assays to verify the miR-191 target gene. Results: We showed that miR-191 was up-regulated in CCA cell lines and patients. Knockdown of miR-191 by transfection of its inhibitor sequence blocked RBE cells viability and induced apoptosis of RBE cells. Both qRT-PCR and western blot analysis showed that the secreted frizzled-related protein-1 (sFRP1) level was negatively correlated with that of miR-191. Luciferase assay validated that sFRP1 was a direct target of miR-191. Moreover, knockdown of miR-191 led to suppression of Wnt/β-catenin signaling activation. Co-transfection of sFRP1 small interfering RNA (siRNA) and miR-191 inhibitor re-activated the Wnt/β-catenin signaling pathway as detected by an increased level of β-catenin and phosphorylation of GSK-3β, and restored the expression of survivin and c-myc in RBE cells. Co-transfection of sFRP1 siRNA with miR-191 inhibitor restored the colony formation ability and viability of RBE cells. Conclusion: Taken together, our results demonstrate a novel insight into miR-191 biological function in CCA. Our findings suggest that miR-191 is a potential therapeutic target of CCA treatment.

Fyn knockdown inhibits migration and invasion in cholangiocarcinoma through the activated AMPK/mTOR signaling pathway.

Cholangiocarcinoma (CCA) is a rare and fatal tumor. In previous decades, there has been a steady increase in the incidence and mortality rates of this tumor worldwide. Metastasis is regarded as the major factor that contributes to poor prognosis in CCA patients. Studies therefore aim to develop novel therapeutic targets to control CCA metastasis. Fyn is known to enhance expression and promote metastasis in various cancers, including pancreatic cancer, prostate cancer and colorectal cancer. However, the exact function and mechanism of Fyn in CCA metastasis remains unclear. In the present study, mRNA and protein expression levels of Fyn, AMP-activated protein kinase (AMPK), phosphorylated (p-)AMPK, mammalian target of rapamycin (mTOR) and p-mTOR were measured, using the reverse transcription-quantitative polymerase chain reaction and western blot analysis, in CCA tissues and cell lines. In addition, Transwell assays were used to determine the migratory and invasive abilities of human CCA QBC939, following transfection. In the present study, it was found that Fyn was overexpressed in CCA cell lines. Fyn knockdown inhibited CCA cell migration and invasion. Furthermore, it was demonstrated that Fyn knockdown induces phosphorylation of AMPK, inhibits downstream phosphorylation of mTOR, and activate the AMPK/mTOR signaling pathway. Compound C, an AMPK inhibitor, inhibited the AMPK/mTOR signaling pathway, and reversed the effect of Fyn knockdown on migration and invasion of CCA cells. In conclusion, the present study suggests that Fyn knockdown inhibits cell migration and invasion by regulating the AMPK/mTOR signaling pathway in CCA cell lines and that Fyn knockdown is a potential target for anti-CCA therapy.

Decrease of miR-622 expression promoted the proliferation, migration and invasion of cholangiocarcinoma cells by targeting regulation of c-Myc

ObjectiveTo explore the mechanism of miR-622 in regulating the proliferation, migration and invasion of cholangiocarcinoma (CCA) cells. Materials and methodsQuantitative real-time PCR was conducted to measure the expression of miR-622 and c-Myc in CCA tissues and cell lines. Protein level of c-Myc was measured by Western blot. The effect of miR-622 on cell proliferation, migration and invasion was analyzed by MTT assay and Transwell chamber migration assay. Luciferase reporter assay was performed to measure the effect of miR-622 on c-Myc. ResultsmiR-622 expression was downregulated in both CCA tissues and cell lines, while c-Myc expression was uregulated. Overexpression of miR-622 in CCA cells was statistically correlated with a decrease of cell proliferation, migration and invasion, while inhibition of miR-622 made an inverse result. We also proved c-Myc was identified as a target gene of miR-622 in CCA. Moreover, we found overexpression of c-Myc can strengthen the effects of miR-622 on the proliferation, migration and invasion of CCA cells. ConclusionDecrease of miR-622 promotes the proliferation, migration and invasion of CCA cells by directly targeting c-Myc.

Prolonged oxidative stress down-regulates Early B cell factor 1 with inhibition of its tumor suppressive function against cholangiocarcinoma genesis

Early B cell factor 1 (EBF1) is a transcription factor involved in the differentiation of several stem cell lineages and it is a negative regulator of estrogen receptors. EBF1 is down-regulated in many tumors, and is believed to play suppressive roles in cancer promotion and progression. However, the functional roles of EBF1 in carcinogenesis are unclear. Liver fluke-infection-associated cholangiocarcinoma (CCA) is an oxidative stress-driven cancer of bile duct epithelium. In this study, we investigated EBF1 expression in tissues from CCA patients, CCA cell lines (KKU-213, KKU-214 and KKU-156), cholangiocyte (MMNK1) and its oxidative stress-resistant (ox-MMNK1-L) cell lines. The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) was used as an oxidative stress marker. Our results revealed that EBF1 expression was suppressed in cancer cells compared with the individual normal bile duct cells at tumor adjacent areas of CCA tissues. CCA patients with low EBF1 expression and high formation of 8-oxodG were shown to correlate with poor survival. Moreover, EBF1 was suppressed in the oxidative stress-resistant cell line and all of CCA cell lines compared to the cholangiocyte cell line. This suggests that prolonged oxidative stress suppressed EBF1 expression and the reduced EBF1 level may facilitate CCA genesis. To elucidate the significance of EBF1 suppression in CCA genesis, EBF1 expression of the MMNK1 cell line was down-regulated by siRNA technique, and its effects on stem cell properties (CD133 and Oct3/4 expressions), tumorigenic properties (cell proliferation, wound healing and cell migration), estrogen responsive gene (TFF1), estrogen-stimulated wound healing, and cell migration were examined. The results showed that CD133, Oct3/4 and TFF1 expression levels, wound healing, and cell migration of EBF1 knockdown-MMNK1 cells were significantly increased. Also, cell migration of EBF1-knockdown cells was significantly enhanced after 17β-estradiol treatment. Our findings suggest that EBF1 down-regulation via oxidative stress induces stem cell properties, tumorigenic properties and estrogen responses of cholangiocytes leading to CCA genesis with aggressive clinical outcomes.

Long non-coding RNA UCA1 indicates an unfavorable prognosis and promotes tumorigenesis via regulating AKT/GSK-3β signaling pathway in cholangiocarcinoma.

Long non-coding RNAs (lncRNAs) have been documented to play key roles in a wide range of pathophysiological processes, including cancer initiation and progression. Recently, the aberrant expression of urothelial carcinoma associated 1 (UCA1) was observed in many types of cancers. However, its clinical relevance and exact effects in cholangiocarcinoma (CCA) remains unknown. In the present study, we aimed to investigate the clinical significance of UCA1 and evaluate its prognostic value in patients with CCA. Besides, the functional roles of UCA1 were detected both in vitro and in vivo. Moreover, potential signaling pathways were explored to clarify the molecular mechanisms underlying CCA cell proliferation. The results indicated that UCA1 transcription is enhanced in both CCA tissue samples and cell lines, and this overexpression is associated with tumor stage (P = 0.007), lymph node invasion (P = 0.027), TNM stage (P = 0.004) and postoperative recurrence (P = 0.033) of CCA patients. Besides, UCA1 could function as an independent prognostic predictor for overall survival in patients with CCA (P = 0.014). For the part of functional assays, knockdown of UCA1 could attenuate CCA cell growth both in vitro and in vivo. Besides, UCA1 facilitates apoptosis via Bcl-2/caspase-3 pathway. In addition, UCA1 regulates migration and invasion potential of CCA cells by affecting EMT. Furthermore, AKT/GSK-3β axis was activated to upregulate CCND1 expression due to overexpression of UCA1 in CCA. To summary, UCA1 might be a potentially useful prognostic biomarker and therapeutic target for CCA.

Upregulation of TCTP is associated with cholangiocarcinoma progression and metastasis.

In order to investigate the role of translationally-controlled tumor protein (TCTP) in cholangiocarcinoma (CCA) progression and metastasis, TCTP protein staining in paraffin-embedded sections of human CCA tissue samples was examined using immunohistochemistry, and its expression was subsequently compared with clinicopathological parameters. Small interfering RNA (siRNA) targeting TCTP (siTCTP) were transfected into CCA cell lines to evaluate its effects on cellular functions. The proliferation, tumorigenicity and migration abilities of the transfected cells were measured using sulforhodamine B, clonogenic and would healing assays, respectively. The protein levels of TCTP and its associated molecules were evaluated by western blot analysis. Of the 119 individual cases of CCA tissues analyzed, high TCTP scores were significantly correlated with overall metastasis (P=0.044) and a shorter survival time (P<0.001). Multivariate proportional hazards analysis revealed that TCTP is an independent indicator of poor prognosis in CCA (hazard ratio =2.864; P<0.001). siTCTP transfection suppressed CCA cell growth and migration abilities, compared with the control cells (P<0.01). The siTCTP reduced the protein levels of focal adhesion kinase (FAK), phospho-FAK, nuclear factor kappa-light-chain-enhancer of activated B cells and matrix metalloproteinase 9, suggesting potential roles of TCTP in regulating CCA progression and metastasis. In conclusion, the upregulation of TCTP is clinically significant in patients with CCA, serving roles in CCA progression, particularly in cell survival and metastasis. Suppression of TCTP may serve as a potential target in CCA prevention and treatment.

Epigenetic Silencing of miRNA-34a in Human Cholangiocarcinoma via EZH2 and DNA Methylation: Impact on Regulation of Notch Pathway

Aberrant expression and regulation of miRNAs have been implicated in multiple stages of tumorigenic processes. The current study was designed to explore the biological function and epigenetic regulation of miR-34a in human cholangiocarcinoma (CCA). Our data show that the expression of miR-34a is decreased significantly in CCA cells compared with non-neoplastic biliary epithelial cells. Forced overexpression of miR-34a in CCA cells inhibited their proliferation and clonogenic capacity invitro, and suppressed tumor xenograft growth in severe combined immunodeficiency mice. We identified three key components of the Notch pathway, Notch1, Notch2, and Jagged 1, as direct targets of miR-34a. Our further studies show that down-regulation of miR-34a is caused by Enhancer of zeste homolog 2 (EZH2)-mediated H3 lysine 27 trimethylation as well as DNA methylation. Accordingly, treatment with the EZH2 inhibitor, selective S-adenosyl-methionine-competitive small-molecule (GSK126), or the DNA methylation inhibitor, 5-Aza-2'-deoxycytidine, partially restored miR-34a levels in human CCA cells. Immunohistochemical staining and Western blot analyses showed increased EZH2 expression in human CCA tissues and cell lines. We observed that GSK126 significantly reduced CCA cell growth invitro and intrahepatic metastasis invivo. Our findings provide novel evidence that miR-34a expression is silenced epigenetically by EZH2 and DNA methylation, which promotes CCA cell growth through activation of the Notch pathway. Consequently, these signaling cascades may represent potential therapeutic targets for effective treatment of human CCA.

Differential effects of FXR or TGR5 activation in cholangiocarcinoma progression

Background and aimsCholangiocarcinoma (CCA) is an aggressive tumor type affecting cholangiocytes. CCAs frequently arise under certain cholestatic liver conditions. Intrahepatic accumulation of bile acids may facilitate cocarcinogenic effects by triggering an inflammatory response and cholangiocyte proliferation. Here, the role of bile acid receptors FXR and TGR5 in CCA progression was evaluated. MethodsFXR and TGR5 expression was determined in human CCA tissues and cell lines. An orthotopic model of CCA was established in immunodeficient mice and tumor volume was monitored by magnetic resonance imaging under chronic administration of the specific FXR or TGR5 agonists, obeticholic acid (OCA) or INT-777 (0,03% in chow; Intercept Pharmaceuticals), respectively. Functional effects of FXR or TGR5 activation were evaluated on CCA cells in vitro. ResultsFXR was downregulated whereas TGR5 was upregulated in human CCA tissues compared to surrounding normal liver tissue. FXR expression correlated with tumor differentiation and TGR5 correlated with perineural invasion. TGR5 expression was higher in perihilar than in intrahepatic CCAs. In vitro, FXR was downregulated and TGR5 was upregulated in human CCA cells compared to normal human cholangiocytes. OCA halted CCA growth in vivo, whereas INT-777 showed no effect. In vitro, OCA inhibited CCA cell proliferation and migration which was associated with decreased mitochondrial energy metabolism. INT-777, by contrast, stimulated CCA cell proliferation and migration, linked to increased mitochondrial energy metabolism. ConclusionActivation of FXR inhibits, whereas TGR5 activation may promote, CCA progression by regulating proliferation, migration and mitochondrial energy metabolism. Modulation of FXR or TGR5 activities may represent potential therapeutic strategies for CCA.

The prognostic potential and carcinogenesis of long non-coding RNA TUG1 in human cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a fatal disease with increasing worldwide incidence and is characterized by poor prognosis due to its poor response to conventional chemotherapy or radiotherapy. Long non-coding RNAs (lncRNAs) play key roles in multiple human cancers, including CCA. Cancer progression related lncRNA taurine-up-regulated gene 1 (TUG1) was reported to be involved in human carcinomas. However, the impact of TUG1 in CCA is unclear. The aim of this study was to explore the expression pattern of TUG1 and evaluate its clinical significance as well as prognostic potential in CCA. In addition, the functional roles of TUG1 including cell proliferation, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT), were evaluated after TUG1 silencing. Our data demonstrated up-regulation of TUG1 in both CCA tissues and cell lines. Moreover, overexpression of TUG1 is linked to tumor size (p=0.005), TNM stage (p=0.013), postoperative recurrence (p=0.036) and overall survival (p=0.010) of CCA patients. Furthermore, down-regulation of TUG1 following RNA silencing reduced cell growth and increased apoptosis in CCA cells. Additionally, TUG1 suppression inhibited metastasis potential in vitro by reversing EMT. Overall, our results suggest that TUG1 may be a rational CCA-related prognostic factor and therapeutic target.

CD155 expression and its correlation with clinicopathologic characteristics, angiogenesis, and prognosis in human cholangiocarcinoma.

BackgroundCD155, an immunoglobulin-like adhesion molecule, plays an important role in carcinoma such as cells migration, proliferation, metastasis, and tumor immune. The upregulation of CD155 has been found in several human malignancies, but its expression in cholangiocarcinoma (CCA) still remains unclear. The aim of this study is to investigate CD155 expression and its correlations with clinicopathologic data, angiogenesis, and prognosis in the patients with CCA.Materials and methodsCD155 expression was investigated in 20 paired CCA tissues and corresponding paracancerous tissues by Western blotting and quantitative real-time polymerase chain reaction assays at protein and mRNA levels. Besides, this study evaluated the correlation between the tumor CD155 expression and the level of both vascular endothelial growth factor and intratumoral microvessel density by immunohistochemistry in 90 cases of CCA. Moreover, the clinical and prognostic significance of CD155 in CCA was assessed by immunohistochemistry.ResultsThe protein and mRNA levels of CD155 were higher in CCA tumor tissues compared with corresponding paracancerous tissues (P<0.05). Immunohistochemical staining showed that CD155 was located in the cytoplasm of carcinoma cells and overexpressed in 61.2% (55/90) CCA tissues. Obviously, CD155 expression level was significantly correlated with tumor histological grade (P=0.002), lymph node metastasis (P<0.001), and tumor-node-metastasis (P=0.03). Additionally, Spearman rank correlation test demonstrated that CD155 expression was positively associated with vascular endothelial growth factor (r=0.481, P<0.001) and microvessel density (r=0.442, P<0.001) in CCA tissues. More importantly, CCA patients with high CD155 expression had a markedly shorter overall survival (P<0.001) and disease-free survival (P<0.001) after surgical resection, and multivariate analysis showed that high CD155 expression was an independent poor prognostic predictor of overall survival and disease-free survival (P<0.001).ConclusionOur results revealed that upregulated CD155 correlated with aggressive clinicopathologic characteristics, angiogenesis, and poor prognosis in CCA and may be a promising prognostic biomarker for the CCA patients.

High expression of CCDC25 in cholangiocarcinoma tissue samples

Cholangiocarcinoma (CCA) is a malignant transformation of biliary epithelial cells. It is a slow growing tumor, but is also highly metastatic with a poor prognosis. Bile acids are known to transactivate the epidermal growth factor receptor (EGFR) in cholangiocytes and induce cyclooxygenase-2 expression. The protein expression profiles of bile acid-treated CCA cells were studied using a proteomic approach. To elucidate the possible mechanisms involved in the bile acid-mediated enhancement of CCA cell migration, the effects of six bile acids, including cholic, deoxycholic, taurocholic, taurodeoxycholic, glycocholic and glycodeoxycholic acid, on the migration of CCA cells were examined in vitro using wound healing assays. Subsequently, the possible proteins involved in enhanced CCA cell migration were investigated using a proteomic approach. Changes to the protein expression profiles of CCA cells following bile acid treatment was examined using two-dimensional electrophoresis and mass spectrometry. The results demonstrated that cholic and deoxycholic acid significantly enhanced the migration of CCA cells, compared with the treated MMNK-1 control cells. CCA cells had 77 overexpressed protein spots following cholic acid treatment, and 50 protein spots following deoxycholic acid treatment, compared with the treated MMNK-1 control cells. Liquid chromatography tandem-mass spectrometry analysis revealed that coiled-coil domain containing 25 (CCDC25) was significantly overexpressed in cholic acid-treated CCA cells compared with in cholic acid-treated control cells. When the expression levels of CCDC25 were investigated using western blot analysis, CCDC25 was demonstrated to be highly expressed in CCA tissues, but not in the adjacent non-cancerous tissue samples. The identified proteins were further analyzed for protein-chemical interactions using STITCH version 3.1 software. CCDC25 protein was identified to be associated with Son of sevenless homolog 1 and growth factor receptor-bound protein 2, which are involved in EGFR signaling. The results of the present study demonstrated that following cholic acid treatment, CCDC25 is overexpressed in CCA cells, which is associated with significantly enhanced cell migration. This suggests that CCDC25 is a potential therapeutic target for the treatment of patients with CCA.

Histone deacetylase 3 overexpression in human cholangiocarcinoma and promotion of cell growth via apoptosis inhibition

Histone deacetylase 3 (HDAC3) has an oncogenic role in apoptosis and contributes to the proliferation of cancer cells. MI192 is a novel HDAC3-specific inhibitor that displays antitumor activity in many cancer cell lines. However, the role of HDAC3 and the antitumor activity of its inhibitor MI192 are not known in cholangiocarcinoma (CCA). The present study aims to identify the target of MI192 in CCA as well as evaluate its therapeutic efficacy. CCK8 and colony formation assays showed that HDAC3 overexpression promotes proliferation in CCA cell lines. HDAC3 knockdown or treatment with MI192 decreased CCA cell growth and increased caspase-dependent apoptosis, while apoptosis was partially rescued by HDAC3 overexpression. We demonstrated that MI192 can inhibit the deacetylation activity of HDAC3 and its downstream targets in vitro, and MI192 inhibited xenograft tumor growth in vivo. Immunochemistry showed that HDAC3 was upregulated in CCA tissues compared with adjacent normal tissues, and this was correlated with reduced patient survival. Taken together, these results demonstrate for the first time that MI192 targets HDAC3 and induces apoptosis in human CCA cells. MI192 therefore shows the potential as a new drug candidate for CCA therapy.

Upregulated long noncoding RNA PANDAR predicts an unfavorable prognosis and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most malignant human cancers with increasing incidence worldwide. LncRNAs have emerged as gene regulators and prognostic biomarkers in a variety of neoplasms. PANDAR, a novel cancer-related lncRNA, has been reported to be upregulated in diverse human carcinomas. In this study, we aimed to investigate the clinical significance of lncRNA PANDAR in CCA and explore its functional roles in CCA cells including cell proliferation, apoptosis, migration, invasion and epithelial-to-mesenchymal transition (EMT). The results showed that PANDAR was significantly upregulated in CCA tissue specimens and cell lines, and its high expression was closely associated with lymph node invasion (P=0.004), TNM stage (P=0.034) and postoperative relapse (P=0.006) in patients with CCA. Thus, overexpression of PANDAR could serve as an independent prognostic biomarker of CCA. Furthermore, silencing of PANDAR followed by siRNA significantly inhibited cell proliferation and increased apoptosis in CCA cells. In addition, suppression of PANDAR impaired migration and invasion capacity in vitro partly by affecting EMT. Overall, our findings showed that lncRNA PANDAR serves as a novel prognostic biomarker and therapeutic target for CCA.

Clustering of patients with intrahepatic cholangiocarcinoma based on serum periostin may be predictive of prognosis.

An effective serum biomarker may improve cholangiocarcinoma (CCA) management. Periostin (PN) has been demonstrated to be associated with aggressive CCA. The current study evaluated PN in blood serum for its diagnostic and prognostic potential in patients with CCA. Sera of 68 patients with CCA were collected prior to treatment, and PN levels were measured using an ELISA. Sera from 50 normal controls, 6 patients with benign liver diseases, 2 with hepatocellular carcinoma and 21 with breast cancer were analyzed. Immunohistochemistry of PN in CCA tissues was also investigated. The data were analyzed using the Mann-Whitney U test, Kaplan-Meier log rank tests, Cox proportional hazard regression models and Fisher's exact tests. The median serum PN level in patients with CCA was significantly increased compared with that in healthy controls, patients with benign liver diseases and patients with breast cancer (all P<0.05). Using an optimal threshold value of 94 ng/ml PN, the diagnostic values for CCA compared with other conditions demonstrated a sensitivity level of 0.38 [95% confidence interval (CI), 0.27–0.51], specificity of 0.90 (95% CI, 0.81–0.96), accuracy of 0.66 (95% CI, 0.58–0.74), positive predictive value of 0.76 (95% CI, 0.59–0.89) and negative predictive value of 0.63 (95% CI, 0.53–0.72) (P<0.001). Furthermore, PN stain in stromal fibroblasts in CCA tissues was associated with serum PN levels (P=0.001), and patients with CCA were classified as low (≤94 ng/ml) or high PN (>94 ng/ml) accordingly. High serum and tissue PN levels were significantly associated with reduced survival rate (P<0.001 and P=0.033, respectively). Serum PN was an independent prognostic factor with a hazard ratio of 3.197 (P=0.001). In conclusion, serum PN may be used to divide patients with intrahepatic CCA into high and low PN groups. Elevated serum PN may be utilized as a marker of poor prognosis in patients with CCA.

Overexpression of long noncoding RNA H19 indicates a poor prognosis for cholangiocarcinoma and promotes cell migration and invasion by affecting epithelial-mesenchymal transition

Cholangiocarcinoma (CCA) is a deadly disease that poorly responds to chemotherapy and radiotherapy and whose incidence has increased worldwide. Furthermore, long noncoding RNAs (lncRNAs) play important roles in multiple biological processes, including tumorigenesis. Specifically, H19, the first discovered lncRNA, has been reported to be overexpressed in diverse human carcinomas, but the overall biological role and clinical significance of H19 in CCA remains unknown. In the present study, expression levels of H19 were investigated in CCA tissues and cell lines and were correlated with clinicopathological features. Moreover, we explored the functional roles of H19 depletion in QBC939 and RBE cells, including cell proliferation, apoptosis, migration, invasion and epithelial-to-mesenchymal transition (EMT). The results indicated that H19 was upregulated in CCA tissue samples and cell lines, and this upregulation was associated with tumor size, TNM stage, postoperative recurrence and overall survival in 56 patients with CCA. Moreover, knockdown of H19 followed by RNA silencing restrained cell proliferation and promoted apoptosis. In addition, H19 suppression impaired migration and invasion potential by reversing EMT. Overall, our findings may help to develop diagnostic biomarkers and therapeutics that target H19 for the treatment of CCA.