Invasion Of Cholangiocarcinoma Cells Research Articles

Matricellular proteins in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is typically characterized by a prominent desmoplastic stroma that is often the most dominant feature of the tumor. This tumor reactive stroma is comprised of a dense fibro-collagenous-enriched extracellular matrix (ECM) surrounding the cancer cells, together with other ECM proteins/peptides, specifically secreted matricellular glycoproteins and proteolytic enzymes, growth factors, and cytokines. Moreover, as enjoined by cholangiocarcinoma cells, this enriched tumor microenvironment is populated by various stromal cell types, most prominently, cancer-associated myofibroblasts (CAFs), along with variable numbers of tumor-associated macrophages (TAMs), inflammatory and vascular cell types. While it is now well appreciated that the interplay between cholangiocarcinoma cells, CAFs, and TAMs in particular play a critical role in promoting cholangiocarcinoma progression, therapeutic resistance, and immune evasion, it is also becoming increasingly evident that over-expression and secretion into the tumor microenvironment of functionally overlapping matricellular glycoproteins, including periostin, osteopontin, tenascin-C, thrombospondin-1, mesothelin and others have an important role to play in regulating or modulating a variety of pro-oncogenic cellular functions, including cholangiocarcinoma cell proliferation, invasion, and metastasis, epithelial-mesenchymal transition, ECM remodeling, and immune evasion. Matricellular proteins have also shown promise as potential prognostic factors for iCCA and may provide unique therapeutic opportunities particularly in relation to targeting iCCA pre-metastatic and metastatic niches, tumor cell dormancy, and immune evasion. This review will highlight timely research and its translational implications for salient matricellular proteins in terms of their structure-function relationships, as modulators of intrahepatic cholangiocarcinoma microenvironment and progression, and potential clinical value for iCCA prognosis and therapy.

YY1 and eIF4A3 are mediators of the cell proliferation, migration and invasion in cholangiocarcinoma promoted by circ-ZNF609 by targeting miR-432-5p to regulate LRRC1.

Cholangiocarcinoma is a highly aggressive malignant tumor, and its incidence is increasing all over the world. More and more evidences show that the aberrant expression of circular RNAs play important roles in tumorigenesis and progression. Current studies on the expression and function of circRNAs in cholangiocarcinoma are scarce. In this study, circ-ZNF609 was discovered as a novel circRNA highly expressed in cholangiocarcinoma for the first time. The circ-ZNF609 expression is connected with the advanced TNM stage, lymphatic invasion and survival time in cholangiocarcinoma patients, and can be used as an independent prognostic factor for the patients. Circ-ZNF609 can promote the cholangiocarcinoma cells proliferation, migration and invasion in vitro, it can also catalyze the xenograft growth in vivo. The promoting effect of circ-ZNF609 on cholangiocarcinoma is achieved via oncogene LRRC1 up-regulation through targeting miR-432-5p by endogenous competitive RNA mechanism. In addition, transcription factor YY1 can bind to the promoter of ZNF609 to further facilitate the transcription of circ-ZNF609. RNA binding protein eIF4A3 can bind to the pre-mRNA of circ-ZNF609 which promotes the circ-ZNF609 circular formation. Overall, YY1/eIF4A3/circ-ZNF609/miR-432-5p/LRRC1 have a significant role in progression of cholangiocarcinoma, and circ-ZNF609 is expected to become a novel biomarker for targeted therapy and prognosis evaluation of cholangiocarcinoma.

Value of glycogen synthase 2 in intrahepatic cholangiocarcinoma prognosis assessment and its influence on the activity of cancer cells

ABSTRACT Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver tumor with increasing incidence worldwide. Metabolic reprogramming caused by metabolic related gene disorders is a prominent hallmark of tumors, among which Glycogen Synthase 2 (GYS2) is the key gene responsible for regulating cellular energy metabolism, and its expression disorders are closely related to various tumors and glycometabolic diseases. However, we still know nothing about its role in ICC. This study is intended to reveal the functional role of GYS2 in the ICC progress and explore the underlying mechanism. Based on the integrated pan-cancer analysis of GYS2 in the GEPIA database, the expression of GYS2 in paired ICC and adjacent non tumor tissues was detected by qPCR. It was found that the expression of GYS2 was significantly down-regulated in ICC. Further analysis showed that its low expression was not only associated with the degree of pathological differentiation, tumor size, microvascular invasion and lymph node metastasis, but also an independent risk factor for unfavorable prognosis. Functional studies have shown that GYS2 overexpression can significantly impair the proliferation, replication, cloning, migration and invasion of cholangiocarcinoma cells, while the silencing GYS2 dramatically promotes the development of the aforementioned phenotypes, the underlying mechanism may be that GYS2 activates the P53 pathway. In conclusions,low GYS2 expression in ICC predicted unfavorable patient outcomes; GYS2 overexpression could significantly impair the proliferation, migration and invasion of cholangiocarcinoma cells via activating the P53 pathway and GYS2 was expected to become a potential therapeutic target for such patients.

MicroRNA-206 Suppresses Cholangiocarcinoma Cell Growth and Invasion by Targeting Jumonji AT-Rich Interactive Domain 2.

The current study set out to elucidate the specific role of microRNA (miR)-206 in cholangiocarcinoma (CCA) cell biological activities by negatively modulating jumonji AT-rich interactive domain 2 (JARID2). Firstly, human intrahepatic biliary epithelial cells and CCA cell lines were selected via the analysis of miR-206 and JARID2 expression patterns in CCA by qRT-PCR. Next, the target relation between miR-206 and JARID2 was predicted by Targetscan and validated using dual-luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay. Subsequently, CCK-8 method, colony formation assay, scratch test, Transwell assay, and western blot analysis were performed to evaluate cancer cell development after the overexpression of miR-206 and/or JARID2, with levels of invasion-related proteins assessed. In addition, xenograft transplantation was also employed to confirm the role of miR-206 in vivo. Lastly, Ki-67 expression pattern was also quantified with immunohistochemistry. It was found that miR-206 was poorly expressed and JARID2 was highly expressed in CCA cell lines. Also, miR-206 overexpression brought about a suppressive effect on cancer cell proliferation, migration, and invasion. Furthermore, miR-206 was observed to target JARID2. Meanwhile, JARID2 overexpression promoted cell growth, while simultaneous overexpression of miR-206 and JARID2 impeded malignant cancer progression, indicating that miR-206 overexpression inhibited cell progression via targeting JARID2. Finally, in vivo experimentation illustrated that miR-206 overexpression suppressed tumor growth and weight, and inhibited the expressions of JARID2 N-cadherin, vimentin, and Ki-67. Altogether, our findings clarified that miR-206 inhibited CCA malignancy by negatively regulating JARID2.

Anti-tumor activity of rice bran hydrolysates on migration, invasion and angiogenesis

Objective: To investigate anti-tumor effect of rice bran hydrolysates (RBH) on proliferation, migration, invasion, and angiogenesis of cholangiocarcinoma (CCA) cells, and elucidate the underlying mechanisms. Methods: RBH was prepared from Tubtim Chumprae rice (Oryza sativa L.) by hydrothermolysis followed by protease digestion. Phenolic content in RBH was analyzed by high-performance liquid chromatography. Human CCA cells, KKU-156, KKU-452, and KKU-100, were used to study the effects of RBH on proliferation, migration, invasion, and adhesion by wound healing, Transwell chamber, and fibronectin cell adhesion assays. Angiogenesis was evaluated using human umbilical vein endothelial cells. Proteins associated with cancer progression were analyzed by immunobloting assays. Results: RBH contained carbohydrates, proteins, lipids, and various phenolic compounds and flavonoids. RBH did not inhibit CCA proliferation, but strongly suppressed migration, invasion, adhesion of CCA cells, and the formation of tube-like capillary structures of human umbilical vein endothelial cells. Moreover, RBH down-regulated phosphorylation of FAK, PI3K, and Akt, suppressed NF-κB nuclear translocation, decreased the expression of ICAM-1, vimentin and vascular endothelium growth factor (VEGF), and increased the expression of E-cadherin. Conclusions: RBH suppresses CCA cell migration and invasion and decreases expression of proteins involved in cancer metastasis. RBH is a potential food supplement for cancer prevention.

ITGB6-Knockout Suppresses Cholangiocarcinoma Cell Migration and Invasion with Declining PODXL2 Expression.

Intrahepatic cholangiocarcinoma (iCCA) is a heterogeneous bile duct cancer with a poor prognosis. Integrin αvβ6 (β6) has been shown to be upregulated in iCCA and is associated with its subclassification and clinicopathological features. In the present study, two ITGB6-knockout HuCCT1 CCA cell lines (ITGB6-ko cells) were established using the clustered regulatory interspaced short palindromic repeats (CRISPR), an associated nuclease 9 (Cas9) system, and single-cell cloning. RNA sequencing analysis, real-time polymerase chain reaction (PCR), and immunofluorescent methods were applied to explore possible downstream factors. ITGB6-ko cells showed significantly decreased expression of integrin β6 on flow cytometric analysis. Both cell lines exhibited significant inhibition of cell migration and invasion, decreased wound-healing capability, decreased colony formation ability, and cell cycle dysregulation. RNA sequencing and real-time PCR analysis revealed a remarkable decrease in podocalyxin-like protein 2 (PODXL2) expression in ITGB6-ko cells. Colocalization of PODXL2 and integrin β6 was also observed. S100 calcium-binding protein P and mucin 1, which are associated with CCA subclassification, were downregulated in ITGB6-ko cells. These results describe the successful generation of ITGB6-ko CCA cell clones with decreased migration and invasion and downregulation of PODXL2, suggesting the utility of integrin β6 as a possible therapeutic target or diagnostic marker candidate.

Long noncoding RNA LINC00184 facilitates the proliferation, metastasis, and adenine metabolism of cholangiocarcinoma via modulating hsa-miR-23b-3p/ANXA2 axis.

The purpose of this article was to probe the mechanism underlying long noncoding RNA (lncRNA)-LINC00184 in cholangiocarcinoma development and to investigate the effects of LINC00184 on cholangiocarcinoma. We used bioinformatics to analyze the expression of LINC00184, microRNA (miR)-23b-3p and ANXA2 in cholangiocarcinoma tissues. The levels of LINC00184, miR-23b-3p, and ANXA2 were detected by qRT-PCR. Cell proliferation was tested by CCK8. Transwell assay was used to detect cell invasion and migration. The target connection between LINC00184, miR-23b-3p, or ANXA2 was probed by luciferase reporter assay. RNA pull-down method was employed to test the relationship among LINC00184/miR-23b-3p/ANXA2 in cholangiocarcinoma cells. The Pearson correlation coefficient analyzed was applied to analyze the correlation among LINC00184, miR-23b-3p, and ANXA2. LC-MS/M analysis was used to explore whether the changes of adenine metabolism was affected by LINC00184 in cholangiocarcinoma cells. We discovered that LINC00184 expression was heightened in cholangiocarcinoma patients and cells. Knockdown of LINC00184 repressed cell proliferation, invasion, migration and adenine metabolism in cholangiocarcinoma cells. miR-23b-3p was regarded as a target of LINC00184 and its depletion perversed the inhibitive influence of LINC00184 silencing on cholangiocarcinoma cells. ANXA2 was a target of miR-23b-3p and was negatively modulated by miR-23b-3p. Moreover, ANXA2 was positively modulated by LINC00184 via sponging miR-23b-3p. In short, silencing of LINC00184 suppressed cell proliferation, invasion and migration through over-expression of miR-23b-3p and reducing of ANXA2 in cholangiocarcinoma cells. These findings contribute to understanding the influences of LINC00184, miR-23b-3p, and ANXA2 on cholangiocarcinoma and provide basis for cholangiocarcinoma treatment.

ESR1 as a recurrence-related gene in intrahepatic cholangiocarcinoma: a weighted gene coexpression network analysis

BackgroundIntrahepatic cholangiocarcinoma (iCCA) is the second most common malignant hepatic tumor and has a high postoperative recurrence rate and a poor prognosis. The key roles of most tumor recurrence-associated molecules in iCCA remain unclear. This study aimed to explore hub genes related to the postsurgical recurrence of iCCA.MethodDifferentially expressed genes (DEGs) between iCCA samples and normal liver samples were screened from The Cancer Genome Atlas (TCGA) database and used to construct a weighted gene coexpression network. Module-trait correlations were calculated to identify the key module related to recurrence in iCCA patients. Genes in the key module were subjected to functional enrichment analysis, and candidate hub genes were filtered through coexpression and protein–protein interaction (PPI) network analysis. Validation studies were conducted to detect the “real” hub gene. Furthermore, the biological functions and the underlying mechanism of the real hub gene in iCCA tumorigenesis and progression were determined via in vitro experiments.ResultsA total of 1019 DEGs were filtered and used to construct four coexpression modules. The red module, which showed the highest correlations with the recurrence status, family history, and day to death of patients, was identified as the key module. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses demonstrated that genes in the red module were enriched in genes and pathways related to tumorigenesis and tumor progression. We performed validation studies and identified estrogen receptor 1 (ESR1), which significantly impacted the prognosis of iCCA patients, as the real hub gene related to the recurrence of iCCA. The in vitro experiments demonstrated that ESR1 overexpression significantly suppressed cell proliferation, migration, and invasion, whereas ESR1 knockdown elicited opposite effects. Further investigation into the mechanism demonstrated that ESR1 acts as a tumor suppressor by inhibiting the JAK/STAT3 signaling pathway.ConclusionsESR1 was identified as the real hub gene related to the recurrence of iCCA that plays a critical tumor suppressor role in iCCA progression. ESR1 significantly impacts the prognosis of iCCA patients and markedly suppresses cholangiocarcinoma cell proliferation, migration and invasion by inhibiting JAK/STAT3 signaling pathway.

MicroRNA-1182 and let-7a exert synergistic inhibition on invasion, migration and autophagy of cholangiocarcinoma cells through down-regulation of NUAK1

BackgroundCholangiocarcinoma (CCA) is the second most common primary liver malignancy worldwide. Several microRNAs (miRNAs) have been implicated as potential tumor suppressors in CCA. This study aims to explore the potential effects of miR-1182 and let-7a on CCA development.MethodsBioinformatics analysis was conducted to screen differentially expressed genes in CCA, Western blot analysis detected NUAK1 protein expression and RT-qPCR detected miR-1182, let-7a and NUAK1 expression in CCA tissues and cell lines. Dual luciferase reporter gene assay and RIP were applied to validate the relationship between miR-1182 and NUAK1 as well as between let-7a and NUAK1. Functional experiment was conducted to investigate the role of miR-1182, let-7a and NUAK1 in cell migration, proliferation and autophagy. Then, the CCA cells that received various treatments were implanted to mice to establish animal model, followed by tumor observation and HE staining to evaluate lung metastasis.ResultsCCA tissues and cells were observed to have a high expression of NUAK1 and poor expression of miR-1182 and let-7a. NUAK1 was indicated as a target gene of miR-1182 and let-7a. Importantly, upregulation of either miR-1182 or let-7a induced autophagy, and inhibited cell progression and in vivo tumor growth and lung metastasis; moreover, combined treatment of miR-1182 and let-7a overexpression presented with enhanced inhibitory effect on NUAK1 expression and CCA progression, but such synergistic effect could be reversed by overexpression of NUAK1.ConclusionTaken together, the findings suggest the presence of a synergistic antitumor effect of miR-1182 and let-7a on the development of CCA via the down-regulation of NUAK1, providing novel insight into the targeted therapy against CCA.

MiR-144-5p and miR-451a Inhibit the Growth of Cholangiocarcinoma Cells Through Decreasing the Expression of ST8SIA4.

Accumulating evidences indicate that non-coding RNAs play crucial roles in the progression of an extensive range of carcinomas. This study aimed to investigate the action mechanism of miR-144-5p and miR-451a in cholangiocarcinoma. We found that miR-144-5p and miR-451a were significantly decreased in cholangiocarcinoma patient samples compared to the adjacent normal bile duct samples. The downregulation of these two miRNAs was correlated with a more advanced disease state of cholangiocarcinoma patients. Overexpression of miR-144-5p and miR-451a suppressed the proliferation, invasion and migration of cholangiocarcinoma cells in vitro and inhibited xenograft tumor growth. Knockdown of these two miRNAs had the opposite effects. miR-144-5p and miR-451a regulated the expression of ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4 (ST8SIA4), and presented a correlation with ST8SIA4 in patient samples. Overexpression of ST8SIA4 promoted the proliferation, invasion and migration of cholangiocarcinoma cells, and the changes were reversed by upregulating the expression of miR-144-5p and miR-451a. Our findings indicated that miR-144-5p and miR-451a displayed a tumor suppressor role through decreasing the expression of ST8SIA4 in cholangiocarcinoma.

Shikonin Inhibits Cholangiocarcinoma Cell Line QBC939 by Regulating Apoptosis, Proliferation, and Invasion

This study was designed to clarify whether Shikonin causes proliferation, apoptosis, and invasion in cholangiocarcinoma cells and to investigate the mechanism of action. QBC939 cells were cultured with different doses of Shikonin, and then 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium assay was used to detect cell viability. Apoptosis of cells was detected using flow cytometry with Annexin V/propidium iodide (PI) assay after being stained with Hoechst 33242. The role of Shikonin on the invasive and metastasis ability was detected using Transwell invasion assay. Real-time polymerase chain reaction and Western blotting were used to detect the expression of caspase-3, caspase-8, epidermal growth factor receptor (EGFR), and matrix metalloproteinase (MMP)-9. Shikonin inhibited proliferation and invasive ability of QBC939 cells in a dose-dependent manner; at the same time, apoptosis of cells was also observed in a concentration-dependent fashion. Moreover, Annexin V/PI assay and Transwell invasion assay results indicated that Shikonin induced apoptosis and invasion inhibitory probably due to upregulation of caspase-3 and caspase-8 expression and downregulation of MMP-9 and EGFR expression in a concentration-dependent fashion. Shikonin could enhance apoptosis and inhibit proliferation and invasion of QBC939 cells; such biological behaviors mainly occurred via upregulating the expression of caspase-3 and caspase-8 and downregulating the expression of MMP-9 and EGFR.

Yin Yang 1-induced LINC00667 up-regulates pyruvate dehydrogenase kinase 1 to promote proliferation, migration and invasion of cholangiocarcinoma cells by sponging miR-200c-3p.

Cholangiocarcinoma (CCA) is one of the most aggressive and lethal malignancies. Long noncoding RNAs (lncRNAs) are being found to play crucial roles in CCA progression. This work aims to investigate the roles of long intergenic non-protein coding RNA 667 (LINC00667) in progression of CCA. RT-qPCR and western blot were applied to detect gene expression. Clinical correlation and survival were analyzed by statistical methods. Overexpression and RNA interference approaches were used to investigate the effects of LINC00667 on CCA cells. Tumor xenograft assay was performed to detect the function of LINC00667 in vivo. Transcriptional regulation and competing endogenous RNA (ceRNA) mechanism were predicted via bioinformatics analysis. ChIP, luciferase reporter, and Ago2 RIP assays further confirmed the predicted results. Our data indicated that LINC00667 was highly expressed in CCA tissues and cells, and transcription factor Yin Yang 1 (YY1) induced LINC00667 expression in CCA cells. Up-regulated LINC00667 was significantly associated with lymph node metastasis, advanced TNM stage, and poor prognosis. Knockdown of LINC00667 suppressed the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of CCA cells, while overexpression of LINC00667 acquired opposite effects. Moreover, knockdown of LINC00667 inhibited tumor growth in vivo. In addition, LINC00667 was demonstrated to function as a ceRNA for miR-200c-3p, and then LINC00667 up-regulated pyruvate dehydrogenase kinase 1 (PDK1) to promote CCA development by inhibiting miR-200c-3p. These findings identified a pivotal role of LINC00667 in tumorigenesis and development of CCA. Targeting the YY1/LINC00667/miR-200c-3p/PDK1 axis may provide a new therapeutic strategy for CCA treatment.

Ubiquitin-conjugating enzyme E2T regulates cell proliferation and migration in cholangiocarcinoma.

Ubiquitin-conjugating enzyme E2T (UBE2T) is overexpressed in several human cancer cells, but a role in cholangiocarcinoma (CAA) progression has not been investigated. We analyzed the expression of UBE2T in CAA tissues. Then, we generated UBE2T deregulation models in which it was overexpressed or silenced, and examined the effects on CAA malignant progression by flow cytometry, western blot, MTT assay, wound healing assay and transwell assay. We report the involvement of UBE2T in CAA malignant progression. UBE2T was found to be highly expressed in human CAA cells both in vitro and in vivo. Overexpression of UBE2T significantly enhanced epithelial-to-mesenchymal transition, proliferation, migration and invasion of CAA cells in vitro, while silencing UBE2T had opposing effects. Furthermore, UBE2T appears to exert its effects via the mammalian target of rapamycin (mTOR) pathway as the cellular effects caused by UBE2T overexpression are inhibited by the mTOR inhibitor rapamycin. Our findings suggest that UBE2T may have potential as a new therapeutic target for the prevention or treatment of CAA.

Huaier Restrains Cholangiocarcinoma Progression in vitro and in vivo Through Modulating lncRNA TP73-AS1 and Inducing Oxidative Stress.

PurposeHuaier, the fruiting body of Trametes robiniophila Murr, is a kind of traditional Chinese medicine. Recently, many studies have confirmed that Huaier has antitumor effects on various malignancies. Moreover, studies have demonstrated that long noncoding RNAs play an important regulatory role in the occurrence and progression of malignancies. Our present study was to explore whether Huaier has a potential antitumor effect in cholangiocarcinoma and reveal the relationship between lncRNAs and Huaier-induced tumor inhibition.MethodsMicroarray assay was performed to identify the candidate lncRNAs regulated by Huaier. Quantitative real-time PCR was applied to assess the effect of Huaier on TP73-AS1 expression. The effect of Huaier on the cell viability, proliferation, migration and invasion was evaluated by CCK-8, colony formation, wound healing and Transwell assays, respectively. The ratio of cell apoptosis was determined using AO/EB, Hoechst 33342 and flow cytometry. The effect of Huaier on oxidative stress was revealed using DCFH-DA, mito-SOX, JC-1 probes and Western blotting. In addition, the effect of Huaier on tumor growth and metastasis was explored using subcutaneous tumor model and lung metastatic tumor model in nude mice.ResultsIn vitro, Huaier inhibited the proliferation, migration and invasion of cholangiocarcinoma cells by down-regulating TP73-AS1 and induced apoptosis through mitochondrial apoptotic pathway. In vivo, Huaier suppressed the growth and metastasis of cholangiocarcinoma by modulating the expression of proliferation and EMT-associated proteins.ConclusionHuaier could inhibit cell proliferation, invasion and metastasis by modulating the expression of TP73-AS1, meanwhile promote apoptosis of CCA cells through disturbing mitochondrial function, inducing oxidative stress and activating caspases in vitro. In addition, Huaier could suppress tumor growth and metastasis by regulating the expression of proliferation and EMT-related proteins. In the meantime, Huaier prolonged the survival of nude mice in lung metastatic model with acceptable drug safety.

Transcription factor HIF1α promotes proliferation, migration, and invasion of cholangiocarcinoma via long noncoding RNA H19/microRNA-612/Bcl-2 axis

Cholangiocarcinoma, which is the most common invasive malignant tumor of the biliary tract, has poor prognosis. There is evidence suggesting that hypoxia-inducible factor 1α (HIF1α) plays an important role in cholangiocarcinoma. Also, microRNA-612 (miR-612) is another key regulator of cholangiocarcinoma. In this study, we investigate the scantly documented interaction of HIF1α and miR-612 in cholangiocarcinoma. We first undertook microarray-based cholangiocarcinoma gene expression profiles to screen out the differentially expressed long noncoding RNAs (lncRNAs) and genes. We used reverse transcription quantitative polymerase chain reaction to detect the expression of HIF1α in normal bile duct and cholangiocarcinoma tissues, and in corresponding cells lines. Cell counting kit 8, scratch, and Transwell assays were used to detect the proliferation, migration and invasion of cholangiocarcinoma cells. Cell cycle distribution and apoptosis were detected by flow cytometry. ChIP, dual luciferase reporter gene assay, RNA pull-down, and RNA immunoprecipitation were used to verify relationship between HIF1α and lncRNA H19, and lncRNA H19 and miR-612. We also monitored tumor formation in nude mice to verify the effect of HIF1α on cholangiocarcinoma. HIF1α expression was elevated in cholangiocarcinoma tissues and cells. Silencing HIF1α reduced proliferation, migration, and invasion of cholangiocarcinoma cells. HIF1α transcriptionally activated the expression of lncRNA H19. Overexpression of miR-612 could rescue the proliferation, migration and invasion of cholangiocarcinoma cells caused by lncRNA H19 overexpression. Taken together, HIF1α activated lncRNA H19-mediated miR-612/Bcl-2 pathway to promote cholangiocarcinoma, suggesting a promising therapeutic target for cholangiocarcinoma.

Phenformin inhibits proliferation, invasion, and angiogenesis of cholangiocarcinoma cells via AMPK-mTOR and HIF-1A pathways.

Phenformin (Phen), a potent activator of AMPK, is effective against some resistant cancers. This study evaluated the inhibition of proliferation, migration, invasion, and angiogenesis by Phen in aggressive cancer cells and investigated the underlying mechanism of the inhibition. Cholangiocarcinoma (CCA) KKU-156 and KKU-452 cells were used in this study. The results showed that Phen suppressed cell proliferation and induced apoptosis in both cells. Phen suppressed migration and invasion of cancer cells in wound healing and transwell chamber assays, respectively. The effects were associated with depletions of glutathione (GSH) and decreased glutathione redox ratio which represents cellular redox state. The redox stress was linked with the loss of mitochondrial transmembrane potential, as evaluated by JC-1 assay. The effect of Phen on angiogenesis was performed using HUVEC cultured cells. Phen alone did not affect tube formation of HUVEC cells. However, conditioned media from CCA cell cultures treated with Phen suppressed the tube-like structure formation. The antitumor effect of Phen was associated with AMPK activation and suppression of mTOR phosphorylation, HIF-1A, and VEGF protein expression. In conclusion, Phen inhibits cell proliferation, migration, invasion, and angiogenesis probably through AMPK-mTOR and HIF-1A-VEGF pathways. Phen may be repurposed as chemoprevention of cancer.

Down-regulation of USP8 Inhibits Cholangiocarcinoma Cell Proliferation and Invasion.

ObjectiveCholangiocarcinoma is the second most common primary hepatobiliary malignancy with high incidence and recurrence rate. Ubiquitin-specific protease 8 (USP8) is recently reported to be involved in tumor progression. Herein, we aimed to investigate the effects of USP8 on the growth and metastasis abilities of cholangiocarcinoma cells.MethodsThe siRNA interference was used to knock down USP8 in cholangiocarcinoma cell lines QBC939 and RBE; Hucct-1 cells were transfected with pcDNA3.1-USP8 to up-regulate its expression. The effects of USP8 on cholangiocarcinoma were detected by cell function assays. We analyzed the expressions of USP8, Bcl2, Bax, cleaved caspase-3, cleaved caspase-9, Akt, p-Akt, Cyclin D1 and P70S6K by Western blot analysis.ResultsWe demonstrated that knockdown of USP8 significantly inhibited the proliferation, migration and invasion of QBC939 and RBE cells in vitro, while USP8 overexpression showed significant promoting effects on Hucct-1 cells. Moreover, silencing of USP8 also promoted apoptosis in cholangiocarcinoma cells by regulating the Bcl-2/Bax axis and Caspase cascade; up-regulation of USP8 decreased apoptosis in Hucct-1 cells. Importantly, knockdown of USP8 inhibited activation of the Akt signaling pathway by decreasing the phosphorylation level of Akt and up-regulated p53 expression, while USP8 overexpression increased activation of the Akt signaling pathway in Hucct-1 cells. Further, IGF-1 could reverse the inhibitory effects of USP8 knockdown on the Akt signaling pathway and the proliferation of QBC939 and RBE cells.ConclusionTaken together, our findings suggest that USP8 exerts an oncogenic role in the progression of cholangiocarcinoma and may be a potential therapeutic target for cholangiocarcinoma treatment.

Overexpression of microRNA-96 is associated with poor prognosis and promotes proliferation, migration and invasion in cholangiocarcinoma cells via MTSS1.

MicroRNA-96 (miR-96) has been revealed serve an oncogenic role in various types of cancer. However, the role of miR-96 in cholangiocarcinoma (CCA) development and progression is yet to be elucidated. Thus, the aim of the present study was to investigate the role of miR-96 in CCA. The expression pattern of miR-96 in CCA tissues and cell lines was evaluated using reverse transcription-quantitative PCR analysis. Kaplan-Meier curves and Cox regression analyses were conducted to investigate the prognostic significance of miR-96 in CCA. Cell Counting Kit-8 and Transwell assays were performed to identify the functions of miR-96. The association between miR-96 and metastasis suppressor-1 (MTSS1) was verified using a dual-luciferase assay. The results demonstrated that miR-96 expression levels were increased in CCA tissues and cell lines compared with those in adjacent normal tissues and normal human intrahepatic biliary epithelial cell lines, respectively. High expression levels of miR-96 were significantly associated with lymph node metastasis, differentiation and TNM stage. In addition, upregulated expression of miR-96 was associated with a poorer prognosis and was predicted to be a prognostic factor in patients with CCA. Overexpression of miR-96 in vitro promoted CCA cell proliferation, migration and invasion. Additionally, MTSS1 was identified as a direct target of miR-96. The results of the present study indicated the clinical and biological importance of miR-96 as an oncogene in CCA. miR-96 may represent an independent prognostic biomarker and may promote CCA cell proliferation, migration and invasion by targeting MTSS1.

MicroRNA‑137 suppresses the proliferation, migration and invasion of cholangiocarcinoma cells by targeting WNT2B.

It is widely known that abnormal regulation of microRNAs (miRNAs/miRs) may contribute to the occurrence or development of tumors. The objective of the present study was to elucidate the function and underlying mechanism of miR-137 in the progression of cholangiocarcinoma (CCA). The expression levels of miR-137 in CCA tissues and cell lines were measured using reverse transcription-quantitative PCR. The role of miR-137 in the proliferation of CCA cells was assessed using the Cell Counting Kit-8 assay, colony formation assay and cell cycle distribution analysis, while its effects on the migration and invasion of CCA cells were evaluated using Transwell assays. The function of miR-137 on CCA growth in vivo was also investigated using a xenograft mouse model. Furthermore, the association between miR-137 and Wnt family member 2B (WNT2B) was analyzed using bioinformatics, double luciferase assay and western blotting. It was verified that the expression of miR-137 was low in CCA tissues and cell lines, whereas increased expression of miR-137 significantly suppressed cell proliferation, decreased colony formation ability and induced G1 phase arrest. miR-137 overexpression suppressed the migration and invasion ability of TFK-1 and HuCCT1 cells. Furthermore, the results of the xenograft mouse model assays revealed that miR-137 overexpression decreased tumor growth in vivo. The results of bioinformatics analysis and dual luciferase reporter assays demonstrated that WNT2B is directly regulated by miR-137. The expression of WNT2B and Wnt-pathway-related proteins was decreased when miR-137 was overexpressed. Restoring the expression of WNT2B notably reversed the inhibitory effect of miR-137 on CCA cells. Therefore, the findings of the present study demonstrated that miR-137 acts as a suppressor in CCA and inhibits CCA cell proliferation, migration and invasion through suppressing the expression of WNT2B.

Circ-0000284 arouses malignant phenotype of cholangiocarcinoma cells and regulates the biological functions of peripheral cells through cellular communication.

Circular RNAs (circRNAs) play a vital role in cancers. Accumulated evidences showed that the physiological condition of cells can be reflected by the circRNAs in the exosomes they secrete, and these exosomal circRNAs can be captured by the receptor cells, thereby inducing a series of cellular responses. We performed qRT-PCR to detect the expression level of circ-0000284 in cholangiocarcinoma cell lines, tissues and plasma exosomes. Then the direct interaction between circ-0000284 and miR-637 was investigated through dual-luciferase reporter assay, RNA binding protein immunoprecipitation (RIP) assay and Fluorescent in situ hybridization (FISH) assay. Subsequently, EdU (5-ethynyl-2'-deoxyuridine), migration, invasion assay, flow cytometry and nude mouse tumorigenicity assay were adopted to evaluate the effect of circ-0000284 on migration, invasion, proliferation and apoptosis of cholangiocarcinoma cells. Additionally, TEM was conducted to investigate the shape and size of exosomes from cholangiocarcioma and 293T cell lines. Circ-0000284 was evidently elevated in cholangiocarcinoma cell lines, tumor tissues and plasma exosomes. Meanwhile, the high expression of circ-0000284 enhanced the migration, invasion and proliferation abilities of cholangiocarcinoma cells in vivo and in vitro Besides, the levels of circ-0000284 were increased in cholangiocarcinoma cells and exosomes from them. Moreover, exosomes from cholangiocarcinoma cells enhanced circ-0000284 expression and stimulated migration and proliferation of the surrounding normal cells. Our findings suggest that on the one hand circ-0000284 functions as a competitive endogenous RNA to promote cholangiocarcinoma progression, and on the other hand, circ-0000284 can be directly transferred from cholangiocarcinoma cells to surrounding normal cells via exosomes and in this way regulate the biological functions of surrounding normal cells.