CCA Tissues Research Articles

Upregulated FFAR4 correlates with the epithelial-mesenchymal transition and an unfavorable prognosis in human cholangiocarcinoma.

Free fatty acid receptor 4 (FFAR4) is associated with the epithelial mesenchymal transition (EMT) and is involved in the progression of several types of cancer. However, the role of FFAR4 in cholangiocarcinoma (CCA) remains unclear. The present study evaluated the diagnosis and prognosis of CCA using FFAR4 as a biomarker. Immunohistochemistry was employed to detect expression of FFAR4 in 98samples of CCA tissues and adjacent tissues. In addition, expression of E-cadherin, vimentin, Snail-1, CK7 and CK19 in the 98samples of CCA tissues was detected, and relationships with FFAR4 were analyzed. Correlation between FFAR4 and clinical pathological factors and prognosis was also analyzed. FFAR4 was highly expressed in 72.4% (71/98) of CCA tissues and 29.6% (29/98) of adjacent tissues, with a statistically significant difference between the two tissue types (P< 0.05). A negative correlation between high expression FFAR4 and E-cadherin expression in CCA tissues was also observed (r=-0.445, P< 0.001), and high expression of FFAR4 was positively correlated with vimentin (r= 0.354, P< 0.001), Snail-1(r= 0.496, P< 0.001), CK7(r= 0.494, P< 0.001) and CK19 (r= 0.532, P< 0.001). Moreover, the degree of FFAR4 expression was associated with aggressive clinicopathological characteristics, such as histological grade, perineural invasion (PNI), lymph node metastasis (LNM), advanced TNM stage and preoperative serum CA19-9 level (P< 0.05). In terms of prognosis, CCA patients with high FFAR4 expression showed shorter disease-free survival (DFS) (P< 0.05) and overall survival (OS) (P< 0.05) than did patients with low FFAR4 expression. FFAR4 overexpression may mediate the process of CCA EMT. In addition, FFAR4 is promising as a new diagnostic molecule and therapeutic target for CCA.

Comprehensive analysis of aberrantly expressed profiles of lncRNAs, miRNAs and mRNAs with associated ceRNA network in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly malignant and poorly differentiated bile duct cancer with an extremely poor prognosis, but the pathogenesis of CCA remains not well-known. Attention has been increasingly focused on long noncoding RNAs, which plays an important role in tumorigenesis. However, the roles of cancer specific lncRNA and its related competitive endogenous RNAs (ceRNA) network in CCA remain elusive. In this study, we comprehensively integrated expression profiles, including data on mRNAs, lncRNAs and miRNAs obtained from 36 CCA tissues and 9 normal tissues in The Cancer Genome Atlas. 1434 cancer specific lncRNAs, 68 miRNAs and 3538mRNAs (|logFC|> 1, p< 0.05) were identified. Based on bioinformatics generated from miRcode, starBase, miRTarBase, TargetScan and miRDB, we constructed an lncRNA-miRNA-mRNA network (ceRNA network) in CCA. We constructed the lncRNA-miRNA-mRNA ceRNA network consisting of 206 molecules and 454 interactions. In addition, we used Cytoscape software to visualize the ceRNA network in WGCNA, 22 mRNA network modules were identified, five of which were significantly related to tumor grade and survival time. Moreover, three lncRNAs COL18A1-AS1, SLC6A1-AS1 and HULC were found to be significantly associated with overall survival. The present study provides novel insight for better understanding of lncRNA-related ceRNA network in CCA and useful resource for identifcation of novel biomarkers of CCA.

Tetraspanin 1 promotes epithelial-to-mesenchymal transition and metastasis of cholangiocarcinoma via PI3K/AKT signaling

BackgroundNumerous studies have demonstrated that tetraspanin 1 (TSPAN1), a transmembrane protein, functions as an oncoprotein in many cancer types. However, its role and underlying molecular mechanism in cholangiocarcinoma (CCA) progression remain unclear.MethodsIn the present study, the expression of TSPAN1 in human CCA and adjacent nontumor tissues was examined using real-time PCR, western blot and immunohistochemistry. The effect of TSPAN1 on proliferation and metastasis was evaluated by functional assays both in vitro and in vivo. A luciferase reporter assay was performed to investigate the interaction between microRNA-194-5p (miR-194-5p) and TSPAN1 3′-untranslated region. Co-immunoprecipitation (co-IP) was used to confirm the interaction between TSPAN1 protein and integrin α6β1 and western blot was used to explore TSPAN1 mechanism.ResultsWe found that TSPAN1 was frequently upregulated in CCA and high levels of TSPAN1 correlated with TNM stage, especially metastasis in CCA. TSPAN1 overexpression promoted CCA growth, metastasis, and induced epithelial-to-mesenchymal transition (EMT), while its silencing had the opposite effect both in vitro and in vivo. To explore the differential expression of TSPAN1, we screened miR-194-5p as the upstream regulator of TSPAN1. A combination of high-level TSPAN1 and low-level miR-194-5p predicted poor prognosis in patients with CCA. Furthermore, in accordance with the functional characteristics of the TSPAN superfamily, we proved that TSPAN1 interacted with integrin α6β1 to amplify the phosphoinositide-3-kinase (PI3K)/AKT/glycogen synthase kinase (GSK)-3β/Snail family transcriptional repressor (Snail)/phosphatase and tensin homolog (PTEN) feedback loop.ConclusionThe results indicate that TSPAN1 could be a potential therapeutic target for CCA.

MiR-365 Suppresses Cholangiocarcinoma Cell Proliferation and Induces Apoptosis by Targeting E2F2.

Cholangiocarcinoma (CCA) is one of the most malignant adenocarcinomas arising from bile duct epithelial cells. However, the molecular mechanism regulating CCA development and progression still needs to be investigated. Here we found that miR-365 was downregulated in CCA tissues compared with adjacent normal tissues. By functional experiments, we found that overexpression of miR-365 significantly inhibited CCA cell proliferation and promoted cellular apoptosis in vitro. Furthermore, administration with miR-365 markedly suppressed the growth of tumor tissues in vivo. Mechanistically, we identified E2F2 as the target gene of miR-365 in CCA cells. We found that overexpression significantly inhibited the expression of E2F2 in CCA cells, and there was an inverse correlation between the expression levels of E2F2 and miR-365 in CCA tissues. We also found that E2F2 was highly expressed in CCA tissues and cell lines. Restoration of E2F2 in miR-365-overexpressing CCA cells promoted cell viability and reduced cellular apoptosis in CCA. Collectively, our study demonstrated the essential role of miR-365 and its functional mechanism in CCA cells, which provided a new insight on the design of therapeutic targets for CCA treatment.

Global identification and characterization of lncRNAs that control inflammation in malignant cholangiocytes

BackgroundLong noncoding RNAs (lncRNAs) are known to play important roles in different cell contexts, including cancers. However, little is known about lncRNAs in cholangiocarcinoma (CCA), a cholangiocyte malignancy with poor prognosis, and associated with chronic inflammation and damage to the biliary epithelium. This study determined whether lncRNAs were dysregulated and participated in disease diagnosis or pivotal inflammation pathways through a genome-wide lncRNA screening and functional analysis.ResultsWe firstly identified a large number of lncRNAs abnormally expressed between 9 pairs of cancerous and adjacent tissues of CCA, and between intra-hepatic CCA and extra-hepatic CCA through a genome-wide profiling. A set of aberrant differentially expressed lncRNAs were further validated in a training set (16 pairs) and a test set (11 pairs) of CCA patient samples. Following assessment of the diagnostic value of the 7 differentially expressed lncRNAs, we confirmed the optimal combination of H19, C3P1, AC005550.3, PVT1, and LPAL2 with area under the curve of 0.8828 [95% CI: 0.7441–1.021, P < 0.001], with 93.75% sensitivity and 81.25% specificity, at the cutoff point of − 0.2884 to distinguish the CCA tissue from the normal ones, suggesting that specific lncRNAs may have potential for detecting CCA. More importantly, the genome-wide locus and lncRNA/mRNA co-expression analyses revealed a set of lncRNAs that participated in inflammation and oxidative stress response pathways by regulating genes in cis or in trans. Finally, APOC1P1, PVT1, and LPAL2 were validated to regulate the migration and some pivotal inflammation genes under the CCA pathogenesis.ConclusionsOur findings are the first to show that lncRNAs may not only be potential biomarkers of CCA progression but also respond to inflammation in CCA.

Overexpression of CD44 Variant 9: A Novel Cancer Stem Cell Marker in Human Cholangiocarcinoma in Relation to Inflammation

Various CD44 isoforms are expressed in several cancer stem cells during tumor progression and metastasis. In particular, CD44 variant 9 (CD44v9) is highly expressed in chronic inflammation-induced cancer. We investigated the expression of CD44v9 and assessed whether CD44v9 is a selective biomarker of human cholangiocarcinoma (CCA). The expression profile of CD44v9 was evaluated in human liver fluke Opisthorchis viverrini-related CCA (OV-CCA) tissues, human CCA (independent of OV infection, non-OV-CCA) tissues, and normal liver tissues. CD44v9 overexpression was detected by immunohistochemistry (IHC) in CCA tissues. There was a higher level of CD44v9 expression and IHC score in OV-CCA tissues than in non-OV-CCA tissues, and there was no CD44v9 staining in the bile duct cells of normal liver tissues. In addition, we observed significantly higher expression of inflammation-related markers, such as S100P and COX-2, in OV-CCA tissues compared to that in non-OV and normal liver tissues. Thus, these findings suggest that CD44v9 may be a novel candidate CCA stem cell marker and may be related to inflammation-associated cancer development.

The Importance of CYP19A1 in Estrogen Receptor-Positive Cholangiocarcinoma.

CYP19A1, also called aromatase, is a key enzyme for converting androgens to estrogens of estrogen synthesis. Elevated serum estrogen and high expression levels of estrogen-related proteins are found in cholangiocarcinoma (CCA; bile duct cancer). However, the expression of CYP19A1 in relation to estrogen-related proteins, including estrogen receptors (ERα, ERβ, and GPR30) and an estrogen response protein (TFF1), has never been explored in CCA. In this study, we investigated the expressions of CYP19A1 and estrogen-related proteins in CCA tissues (n = 74; 51 males and 23 females) using immunohistochemistry. The results showed that CYP19A1 was overexpressed in CCA cells compared with that in normal bile duct cells in the adjacent tissues. High expression of CYP19A1 was correlated with the metastatic status of the patients. High CYP19A1 expression was also positively correlated with GPR30 expression. Correlation between high CYP19A1 expression in the tumor tissues and shorter survival time was more prominent in male than in female CCA patients. To elucidate further, the effect of CYP19A1 knockdown on a CCA cell line was examined using a specific siRNA. When CYP19A1 gene expression was suppressed, migration and proliferation activities of CCA cells were significantly reduced. Moreover, the cell proliferation of high CYP19A1-expressing KKU-213 cells was more profoundly suppressed by CYP19A1 inhibitors (exemestane and letrozole) than low CYP19A1-expressing KKU-100 cells. Thus, CYP19A1 promotes CCA progression with aggressive clinical outcomes via increased migration and proliferation activities of cancer cells. CYP19A1 can be a potential chemotherapeutic target for CCA, especially in male patients.

Lentivirus-mediated overexpression of HSDL2 suppresses cell proliferation and induces apoptosis in cholangiocarcinoma.

BackgroundCholangiocarcinoma (CCA) is a malignant tumor of the bile duct epithelium, including intrahepatic, perihilar, and distal CCA based on anatomical location. Hydroxysteroid dehydrogenase-like 2 (HSDL2) belongs to the SDR subfamily of oxidoreductases, and it is involved in glioma oncogenesis, as it can promote cell proliferation and inhibit cell apoptosis. The purpose of this study was to explore the underlying molecular mechanisms of HSDL2 in the process of CCA.MethodsHSDL2 expression levels were observed in CCA and adjacent (normal control) tissues by analyzing data from The Cancer Genome Atlas and Gene Expression Omnibus databases. A receiver operating characteristic curve analysis was carried out. In vitro, we overexpressed HSDL2 in RBE cells (a human CCA cell line) using a stable lentivirus-mediated transduction strategy. We then used quantitative real-time-PCR and Western blotting methods to detect the efficiency of HSDL2 overexpression. Cell proliferation was assessed using a Celigo Image Cytometer, MTT assays, and the expression of PCNA. Cell apoptosis was assessed by flow cytometry analysis, caspase3/7 activity, and the expression of the apoptotic markers BCL-2 and BAX.ResultsWe observed a downregulation of HSDL2 in CCA tissues based on The Cancer Genome Atlas and Gene Expression Omnibus data analysis. The receiver operating characteristic curve analysis showed that HSDL2 could be an excellent efficacy biomarker for CCA. In vitro, HSDL2 overexpression largely suppressed the proliferation of RBE cells. In addition, apoptosis was induced by HSDL2 overexpression.ConclusionThe results of the data analysis indicated that, compared with adjacent tissues, HSDL2 was downregulated in CCA tissues, and overexpressing HSDL2 in CCA cells suppressed growth and proliferation, which involved activating apoptosis. This helps to understand the underlying HSDL2-related molecular mechanisms in the process of CCA.

A Novel lncRNA Panel Improves the Prediction of Survival in High-Risk Cholangiocarcinoma

Background: There is increasing evidence that long non-coding RNA are involved in the occurrence and progression of various tumors as carcinogenic or tumor suppressor regulators. Aims: Our study purposed to identify a novel lncRNA signature panel and evaluate its predictive value on cholangiocarcinoma (CCA) prognosis. Methods: Differentially expressed lncRNA were identified in CCA patients from The Cancer Genome Atlas database. The edgeR package and Cox regression analysis was used to establish a lncRNA prognostic model, which allowed patients to be divided into high-risk and low-risk groups. Through Receiver operating curves analysis, 90 CCA patients from the First Affiliated Hospital of Wenzhou Medical University (WMU) was performed to investigate the specificity and sensitivity of the model. By using Gene Ontology function and Kyoto Encyclopedia Gene and Genome pathway enrichment analysis, we explored the potential function of prognosis lncRNA. Results: A total of 1192 differential expression of lncRNA were identified in CCA and normal tissues. Five lncRNAs (HULC; AL359715.5; AC006504.8; AC090114.2; AP00943.4) were screened to establish a predictive model, whose high predictive power was validated in 90 CCA patients from WMU. Further analysis showed the independent prognostic capability of 5-lncRNA in the model, which had high sensitivity and specificity. Moreover, functional enrichment analysis suggested prognostic lncRNAs were primarily associated with CCA-related various biological processes. Conclusions: Our data established a novel lncRNA prognostic model for CCA, provided prognostic more useful information that went beyond traditional clinical pathology to predict survival in CCA patients. In addition, it revealed new molecular mechanisms of CCA. Funding: This work was supported by the grants from the Natural Science Foundation of Zhejiang Province (LY17H160047), the Public Projects of Zhejiang Province (2016C37127, 2018C37114), the National Natural Science Foundation of China (81470868, 81772628, 81703310). Declaration of Interest: The authors declare no conflict of interests. Ethical Approval: This study was approved by WMU’s Ethics Committee.

Dysregulation of KCNQ1OT1 promotes cholangiocarcinoma progression via miR-140-5p/SOX4 axis

It is commonly recognized that aberrant expression of long non-coding RNAs (lncRNAs) is an important cause of cancer progression. The oncogenic property of KCNQ1OT1 has been identified in several malignant tumors. Here, we decided to explore the biological function and molecular mechanism of KCNQ1OT1 in cholangiocarcinoma (CCA). The expression conditions of KCNQ1OT1 in different tissues and cell lines were examined with qRT-PCR analysis. As expected, KCNQ1OT1 was highly expressed in CCA tissues and cell lines. Results of functional assays revealed the oncogenic function of KCNQ1OT in cholangiocarcinoma progression. The positive effect of KCNQ1OT1 on cell proliferation, invasion and epithelial-mesenchymal transition was identified by performing MTT assay, colony formation assay, transwell invasion assay and western blotting. Whereas, the negative effect of KCNQ1OT1 on the cell apoptosis was tested with flow cytometry analysis. Mechanism investigation revealed that KCNQ1OT1 can act as a ceRNA to improve CCA progression by regulating miR-140-5p/SOX4 axis. Recue assays were conducted to demonstrate the actual effects of KCNQ1OT1-miR-140-5p-SOX4 pathway on CCA progression.

14-3-3ζ/atypical protein kinase C-ιsynergistically facilitate the invasion and metastasis of cholangiocarcinoma via epithelial-mesenchymaltransition

Objective To illuminate the mechanism of 14-3-3ζ/atypical protein kinase C-iota (aPKC-ι) to promote the invasion and metastasis of cholangiocarcinoma (CCA). Methods The expression of 14-3-3ζ and aPKC-ιwereexamined in the samples of 64 CCA, peritumoural and normal tissues respectively by immunohistochemistry, Western blotting and quantitative real-time polymerase chain reaction. And the independent prognostic factors were analyzed by Kaplan-Meier survival analysis and Multivariate Cox regression analysis. Furthermore, theepithelial-mesenchymaltransition (EMT) model of CCA mediated by Transforming Growth Factor-β1 (TGF-β1) was established to analyze the regulation mechanism of 14-3-3ζand aPKC-ι. Finally, the capacity of invasion, migration, proliferation and metastasis of CCA cells with14-3-3ζdepleted were assessed by transwell cell invasion, wound healing, colony formation assays in vitro. Results Overexpression of 14-3-3ζ and aPKC-ιwas detected in CCA tissue (56.25%, 36/64, and 51.56%, 33/64), there was a significant positive correlation between 14-3-3ζ and aPKC-ι(r=0.406, P=0.001). Low expression of14-3-3ζ or aPKC-ιhad a better overall survival compared with those of overexpression (14-3-3ζ: χ2=10.140, P=0.001; aPKC-ι: χ2=12.575, P=0.000). Moreover, both 14-3-3ζ and aPKC-ιwere the independent prognostic factors of CCA. Interfering with 14-3-3ζ or aPKC-ιsignificantly suppressed the changes in EMT-related markers in CCA cells. In vitro, the inhibition of 14-3-3ζ impaired CCA cell invasion, metastasis and proliferation. Conclusion Our study demonstrated that 14-3-3ζ may combine with aPKC-ιandsynergistically facilitate EMT of CCA to promote CCA invasion and metastasis. Key words: 14-3-3ζ; Atypical protein kinase C-iota; Cholangiocarcinoma; Epithelial-mesenchymal transition; Invasion and metastasis

Forced overexpression of FBP1 inhibits proliferation and metastasis in cholangiocarcinoma cells via Wnt/β-catenin pathway

AimTo investigate the effect of fructose-1,6-bisphosphatase 1 (FBP1) on the malignant phenotypes of cholangiocarcinoma (CCA) cells, and to explore the underlying mechanism. Main methodsThe expression of FBP1 in clinical CCA tissues was detected by real-time PCR, Western blot and immunohistochemistry staining. FBP1 was overexpressed by transfection of a forced expression plasmid. MTT, plate colony formation assay, Hoechst staining, flow cytometry, Western blot, wound healing, transwell assays and xenograft were performed to detect the growth, proliferation, cell cycle, apoptosis, migration, invasion and tumorigenesis in RBE and HCCC-9801 cells. In addition, the Wnt/β-catenin signaling was detected. Key findingsFBP1 was downregulated in clinical CCA specimens and cell lines, compared to paired para-carcinoma tissues or normal cholangetic epithelial cells. Gain-of-function experiments demonstrated that the forced expression of FBP1 inhibited the proliferation, colony formation, and blocked cell cycle of RBE and HCCC-9801 cells. Apoptosis of CCA cells was significantly enhanced by FBP1 overexpression, evidenced by upregulation of cleaved caspase-3, cleaved PARP and Bax levels, while downregulation of Bcl-2 level. Moreover, overexpression of FBP1 decreased the migratory and invasive ability in RBE and HCCC-9801 cells. However, FBP1-induced phenotypic changes were eliminated by overexpression of β-catenin. Finally, the forced overexpression of FBP1 inhibited tumorigenesis in vivo. SignificanceOur findings demonstrate that FBP1 is downregulated in CCA tissues and cell lines, and the overexpression of FBP1 inhibits the proliferation, migration, invasion and tumorigenesis of CCA cells partly via inactivation of Wnt/β-catenin pathway. FBP1 may be a novel early diagnosis marker and therapeutic target for CCA.

MicroRNA-494-dependent WDHDI inhibition suppresses epithelial-mesenchymal transition, tumor growth and metastasis in cholangiocarcinoma

BackgroundCholangiocarcinoma (CCA) represents a devastating malignancy characterized by high mortality, and notoriously problematic to diagnose. Recently, microRNAs (miRs) have been intensively investigated due to their potential usefulness from a tumor treatment perspective. AimsThe current study was aimed to investigate whether miR-494 influences epithelial-mesenchymal transition (EMT), tumor growth and metastasis of CCA. MethodsThe regulatory miRNAs of WDHD1 in CCA expression chip were predicted, followed by determination of the miR-494 and WDHD1 expression in normal cholangiocyte tissues and CCA tissues. The related protein levels were determined. CCA cell migration, invasion, viability, and cell cycle distribution and the dosage-dependent effect of miR-494 on CCA cell growth were subsequently detected. Finally, tumorigenicity and lymph node metastasis (LNM) were measured. ResultsInitially, miR-194 affected the CCA development via negatively regulating WDHD1 and miR-494 which were downregulated while WDHD1 was upregulated in CCA. In addition, miR-494 overexpression elevated E-cadherin expression while decreased expressions of WDHD1, N-cadherin, Vimentin, Snail, Twist and MMP-9. Finally, overexpressed miR-494 was observed to suppress EMT, cell viability, migration, invasion, arrest cell cycle progression, tumor formation, and LNM while accelerating cell apoptosis in vivo. ConclusionThis study indicated that miR-494 overexpression suppresses EMT, tumor formation and LNM while promoting CCA cell apoptosis through inhibiting WDHD1 in CCA.

Long non-coding RNA HOTAIR promotes tumorigenesis and forecasts a poor prognosis in cholangiocarcinoma

Cholangiocarcinoma (CCA) arising from the neoplastic transformation of cholangiocytes with increasing incidence in the worldwide. Unfortunately, a large amount of CCA patients lost their chance for surgery because it is hard to diagnose in the early stages. Long non-coding RNAs (lncRNAs) is closely associated with development and progression of various malignant tumors. Hox transcript antisense intergenic (HOTAIR), a negative prognostic factor for patients with gastric, liver and pancreatic carcinoma. Its transcription levels and functional roles in CCA is still unknown. Therefore, we aimed to explore the effect of HOTAIR in CCA including cell proliferation, apoptosis, migration, invasion and epithelial-to-mesenchymal transition (EMT). The results showed that HOTAIR was highly expressed both in CCA tissue samples and cell lines compared with corresponding normal bile duct tissues and Human intrahepatic biliary epithelial cells (HIBEC). Its overexpression was closely correlated with Tumor size, TNM stage and postoperative recurrence in CCA patients. Moreover, up-regulation of HOTAIR has correlation with prognosis in CCA patients. Knockdown of HOTAIR by siRNAs significantly decreased the migration and invasion but increased apoptosis of CCA cells in vitro. Overall, our study revealed that HOTAIR may play as a new potential therapeutic target and forecast poor prognosis for this fatal disease.

Long Noncoding RNA NEAT1 Promotes Growth and Metastasis of Cholangiocarcinoma Cells.

Long noncoding RNAs (lncRNAs) are known to play important roles in cancers. However, little is known about lncRNAs in cholangiocarcinoma (CCA), a cholangiocyte malignancy with poor prognosis. We investigated the role of nuclear paraspeckle assembly transcript 1 (NEAT1) lncRNA in promoting CCA. qRT-PCR analysis of patient samples showed that NEAT1 expression was higher in CCA tumors than in matched adjacent nontumor tissue. NEAT1 levels were also higher in CCA cell lines than in a normal biliary epithelium cell line (HIBEpic). NEAT1 knockdown in CCA cell lines using shNEAT1 reduced cell proliferation and colony formation in CCK-8 and colony formation assays, respectively. CCA cells transfected with shNEAT1 also exhibited reduced metastasis and invasiveness in Transwell assays. NEAT1 knockdown cells produced smaller tumors, demonstrating that NEAT1 promotes tumor growth in vivo. Silencing of NEAT1 increased E-cadherin expression in vitro, and E-cadherin expression was inversely correlated with NEAT1 expression in CCA tissue samples. RIP and ChIP assays suggest that NEAT1 is recruited to the E-cadherin promoter by EZH2 (enhancer of zeste homolog 2), where it represses E-cadherin expression. These findings indicate that NEAT1 exerts oncogenic effects in CCA. We postulate that NEAT1 is a potentially useful diagnostic and therapeutic target for CCA.

Enhanced expression of lncRNA TP73-AS1 predicts adverse phenotypes for cholangiocarcinoma and exerts oncogenic properties in vitro and in vivo

Cholangiocarcinoma (CCA) is one of the most aggressive malignancies with increasing incidence worldwide. Various evidence documents that abnormally expressed long non-coding RNAs (lncRNAs) play important roles in tumorigenesis and progression. TP73-AS1 is a novel cancer-related lncRNA that contributes to the development of several malignancies. However, its clinical value and potential effects on CCA remains unknown. RT-qPCR was used to measure the expression levels of TP73-AS1 in CCA tissues and paired non-tumor tissues and the association between TP73-AS1 expression and clinicopathological characteristics was analyzed. In addition, the functional roles of TP73-AS1 in CCA were detected both in vitro and in vivo. The results illustrated that TP73-AS1 transcription is enhanced in both CCA tissue samples and cell lines, and this upregulation is closely associated with larger tumor size (p=0.008) and advanced TNM stage (p=0.026) in patients with CCA. For the part of functional assays, silencing of TP73-AS1 could attenuate CCA cell growth both in vitro and in vivo. Additionally, silencing of TP73-AS1 facilitates apoptosis via activating caspase-3 and caspase-9. Importantly, TP73-AS1 expression did not affect HIBEC cell growth and apoptosis. Moreover, TP73-AS1 could also facilitate migration and invasion potential of CCA cells. Collectively, these findings may help to develop a potential therapeutic target for the patients with CCA.

SP1-induced upregulation of lncRNA SPRY4-IT1 exerts oncogenic properties by scaffolding EZH2/LSD1/DNMT1 and sponging miR-101-3p in cholangiocarcinoma

BackgroundAccumulating evidence has indicated that long non-coding RNAs (lncRNAs) behave as a novel class of transcription products during multiple cancer processes. However, the mechanisms responsible for their alteration in cholangiocarcinoma (CCA) are not fully understood.MethodsThe expression of SPRY4-IT1 in CCA tissues and cell lines was determined by RT-qPCR, and the association between SPRY4-IT1 transcription and clinicopathologic features was analyzed. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were performed to explore whether SP1 could bind to the promoter region of SPRY4-IT1 and activate its transcription. The biological function of SPRY4-IT1 in CCA cells was evaluated both in vitro and in vivo. ChIP, RNA binding protein immunoprecipitation (RIP) and luciferase reporter assays were performed to determine the molecular mechanism of SPRY4-IT1 in cell proliferation, apoptosis and invasion.ResultsSPRY4-IT1 was abnormally upregulated in CCA tissues and cells, and this upregulation was correlated with tumor stage and tumor node metastasis (TNM) stage in CCA patients. SPRY4-IT1 overexpression was also an unfavorable prognostic factor for patients with CCA. Additionally, SP1 could bind directly to the SPRY4-IT1 promoter region and activate its transcription. Furthermore, SPRY4-IT1 silencing caused tumor suppressive effects via reducing cell proliferation, migration and invasion; inducing cell apoptosis and reversing the epithelial-to-mesenchymal transition (EMT) process in CCA cells. Mechanistically, enhancer of zeste homolog 2 (EZH2) along with the lysine specific demethylase 1 (LSD1) or DNA methyltransferase 1 (DNMT1) were recruited by SPRY4-IT1, which functioned as a scaffold. Importantly, SPRY4-IT1 positively regulated the expression of EZH2 through sponging miR-101-3p.ConclusionsOur data illustrate how SPRY4-IT1 plays an oncogenic role in CCA and may offer a potential therapeutic target for treating CCA.

Inhibition of KLHL21 prevents cholangiocarcinoma progression through regulating cell proliferation and motility, arresting cell cycle and reducing Erk activation

Kelch-like family member 21 (KLHL21) is involved in cell mitosis and motility. Nevertheless, the clinical significance and biological function of KLHL21 in cholangiocarcinoma (CCA) are elusive. This is the first study to describe a pivotal role for KLHL21 in the progression of CCA. The expression of KLHL21 was elevated in CCA tissues compared with paired normal bile duct tissues. In addition, immunohistochemical and statistical analyses demonstrated that the expression of KLHL21 correlated inversely with tumor histological grade (p < 0.05) and the overall survival of patients (p < 0.01). In CCA cells, we found that the inhibition of KLHL21 significantly reduced proliferation, migration and invasion. Further results indicated that inhibition of KLHL21 triggered G0/G1 cell cycle arrest, leading to the increased expression of P21 and P27 and decreased expression of Cyclin E1, which eventually resulted in proliferation suppression in CCA cells. Furthermore, KLHL21 knockdown alleviated the activation of the Erk signaling pathway via decreasing the expression of phospho-Erk1/2. Our data demonstrated that KLHL21 plays an essential role in the tumorigenesis and progression of CCA, implying that it might serve as a potential therapeutic target for CCA treatment.

Methylation-Mediated Silencing of GATA5 Gene Suppresses Cholangiocarcinoma Cell Proliferation and Metastasis

Cholangiocarcinoma (CCA) is one of the most common hepatic and biliary malignancies, accounting for about 3% of all gastrointestinal tumors. GATA5 is a transcription factor capable of suppressing the development of various human cancer types. Transcriptional inactivation and CpG island (CGI) methylation of GATA3 and GATA5, two members of the GATA family of transcription factors, have been observed in some human cancers. But whether high-density CGI methylation of GATA5 is associated with the clinical course of CCA patients has not been clarified. Herein, we observed reduced expression of GATA5 in CCA tissues compared with noncancerous tissues. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored GATA5 expression in CCA cell lines. Furthermore, GATA5 expression was downregulated after treatment with IL-6 in human intrahepatic biliary epithelial cells. Upregulated GATA5 inhibited CCA cell growth and metastasis. Mechanistically, GATA5 suppressed CCA cell growth and metastasis via Wnt/β-catenin pathway. Specific β-catenin inhibitor or siRNA abolished the discrepancy of the proliferation and metastasis capacity between GATA5-overexpression CCA cells and their control cells, which further confirmed that Wnt/β-catenin was required in GATA5-inhibited CCA cell growth and metastasis.

Mir-637 inhibits the proliferation of cholangiocarcinoma cell QBC939 through interfering CTSB expression.

To investigate the role of mir-637 on the proliferation, migration and apoptosis in human cholangiocarcinoma (CCA) cell line QBC939, and the impact of mir-637 on Cathepsin B (CTSB) expression. Expression of mir-637 and CTSB in CCA tissues and adjacent non-tumor tissues were measured by Real-time PCR. CTSB expression in human CCA cell lines was detected with or without mir-637 exogenous overexpression through fluorescent quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method. The efficiency of lentivirus-mir-637 vector infection, and the effect of mir-637 on the proliferation, migration ability, apoptosis and cell cycle of CCA cell were detected, respectively. The possibility of mir-637 targeting CTSB was also tested by bioinformatics method, correlation analysis and molecular biology method. Decreased mir-637 and increased CTSB expression were observed in CCA tissue compared with adjacent non-tumor tissues. CTSB expression in mir-637 exogenously overexpressed QBC939 cells decreased compared with negative control. Mir-637 overexpression caused significant decrease of proliferation and migration ability of QBC939 cell. In addition, mir-637 overexpression induced that the cell cycle was blocked in G0/G1 phase, and cell apoptosis rate increased significantly. mir-637 and CTSB play an important role in the proliferation and migration of CCA cells. Mir-637 could inhibit CTSB expression significantly, which in-turn down-regulates the proliferation, migration and invasion ability of QBC939 cell, and promotes apoptosis in QBC939 cell line.