Cholangiocarcinoma Migration Research Articles

The CLEC3B inhibits cellular proliferation and metastasis of cholangiocarcinoma through Wnt/β-catenin pathway

Purpose Cholangiocarcinoma (CCA) is a cancer of the biliary system, including intrahepatic and extrahepatic cholangiocarcinoma, and is highly aggressive. C-type lectins family member 3b (CLEC3B) is a Ca2+ binding transmembrane protein with different biological functions in a variety of cancers. The objective of this study was to explore the biological function of CLEC3B in CCA. Methods The CLEC3B gene was identified using the TCGA database and survival analysis of the cholangiocarcinoma clinical cohort. The expression CLEC3B cholangiocarcinoma and correlation with prognosis was investigated in our patient cohort. The effects of CLEC3B on proliferation, apoptosis, migration and invasion were verified in QBC939 and HUCCT1 cells. The effect of CLEC3B on the tumor formation was proved by xenograft tumor model in nude mice. The signaling pathway of CLEC3B in regulating CCA progression was further analyzed RNA sequencing and western blot. Results CLEC3B was decreased in the cholangiocarcinoma in the database. The mRNA and protein expression level of CLEC3B were significantly lower and correlated with poor overall survival in cholangiocarcinoma of our patient cohort. In vitro experiments proved that overexpression of CLEC3B can inhibit proliferation, migration and invasion in bile duct cancer cells. The CLEC3B was correlated with epithelial–mesenchymal transition and apoptosis. The calcium could promote the biological function of CLEC3B. The vivo study indicated that CLEC3B inhibited tumor formation. RNA sequencing indicating CLEC3B may transduce signal through e Wnt/β-catenin signaling pathway. Conclusions The CLEC3B inhibits cellular proliferation and migration of cholangiocarcinoma through the Wnt/β-catenin pathway.

TMEM16A partners with mTOR to influence pathways of cell survival, proliferation, and migration in cholangiocarcinoma.

Expression of transmembrane protein 16 A (TMEM16A), a calcium activated chloride channel, is elevated in some human cancers and impacts tumor cell proliferation, metastasis, and patient outcome. Evidence presented here uncovers a molecular synergy between TMEM16A and mechanistic/mammalian target of rapamycin (mTOR), a serine-threonine kinase that is known to promote cell survival and proliferation in cholangiocarcinoma (CCA), a lethal cancer of the secretory cells of bile ducts. Analysis of gene and protein expression in human CCA tissue and CCA cell line detected elevated TMEM16A expression and Cl- channel activity. The Cl- channel activity of TMEM16A impacted the actin cytoskeleton and the ability of cells to survive, proliferate, and migrate as revealed by pharmacological inhibition studies. The basal activity of mTOR, too, was elevated in the CCA cell line compared with the normal cholangiocytes. Molecular inhibition studies provided further evidence that TMEM16A and mTOR were each able to influence the regulation of the other's activity or expression respectively. Consistent with this reciprocal regulation, combined TMEM16A and mTOR inhibition produced a greater loss of CCA cell survival and migration than their individual inhibition alone. Together these data reveal that the aberrant TMEM16A expression and cooperation with mTOR contribute to a certain advantage in CCA.NEW & NOTEWORTHY This study points to the dysregulation of transmembrane protein 16 A (TMEM16A) expression and activity in cholangiocarcinoma (CCA), the inhibition of which has functional consequences. Dysregulated TMEM16A exerts an influence on the regulation of mechanistic/mammalian target of rapamycin (mTOR) activity. Moreover, the reciprocal regulation of TMEM16A by mTOR demonstrates a novel connection between these two protein families. These findings support a model in which TMEM16A intersects the mTOR pathway to regulate cell cytoskeleton, survival, proliferation, and migration in CCA.

Transmembrane protein 16A partners with mTOR to influence pathways of cell survival, proliferation and migration in cholangiocarcinoma

Transmembrane protein 16A partners with mTOR to influence pathways of cell survival, proliferation and migration in cholangiocarcinoma

Transmembrane protein 16A partners with mTOR to influence pathways of cell survival, proliferation and migration in cholangiocarcinoma

Transmembrane protein 16A partners with mTOR to influence pathways of cell survival, proliferation and migration in cholangiocarcinoma

MiRNA-196-5p Promotes Proliferation and Migration in Cholangiocarcinoma via HAND1/Wnt/β-Catenin Signaling Pathway.

Accumulating evidence has indicated the crucial role of microRNA-196 in mediating tumor progression, while its significance in cholangiocarcinoma (CCA) remains unclear. In this study, we provided the first evidence that the expression level of miR-196-5p is elevated in both CCA cell lines and clinic specimen. MiR-196-5p inhibition notably suppressed cell proliferation as well as metastasis in CCA cell line HuCCT1. Furthermore, the interaction between miR-196-5p and its downstream molecule HAND1 was verified. Moreover, a series of rescue assay verified that both HAND1 and β-catenin silencing could reverse the abnormal elevated cell proliferation and migration brought by miR-196-5p elevation, indicating that HAND1/Wnt/β-catenin signaling pathway activation is essential for miR-196-5p to exert its roles. In summary, we successfully depict the oncogenic role of miR-196-5p in promoting cell proliferation and migration in CCA via HAND1/Wnt/β-catenin axis.

Licochalcone A Induces Cholangiocarcinoma Cell Death Via Suppression of Nrf2 and NF-κB Signaling Pathways.

Objective:To investigate the anti-tumor effect of licochalcone A (LCA) on proliferation and migration in cholangiocarcinoma (CCA) cells and to elucidate their underlying mechanisms. Methods:Human CCA cells, KKU-100, KKU-213, KKU-214, KKU-156, and KKU-452 were used to study effect of LCA on proliferation and migration by a cytotoxicity assay, wound healing assay. Reactive oxygen species levels were evaluated using DHE-fluorescent probes. Proteins associated with cancer survival and progression were analyzed by immune blotting assay. Results:LCA suppressed proliferation and induced cell death in CCA cells including KKU-100, KKU-213, KKU-214, KKU-156, and KKU-452. The CCAs cells were suppressed in association with LCA-induced accumulation of intracellular reactive oxygen species (ROS). Increased formation of ROS was causally related with suppression of Nrf2 and its down-stream antioxidant and cytoprotective enzymes. These effects may lead to the expression of Bax and release of cytochrome c and ensuring cell death. Interestingly, LCA could also inhibit cell migration and cell cycle arrest at low concentrations. These effects were associated with down-regulation of NF-kB, STAT3 and their down-stream proteins, cyclin D1, VEGF, and ICAM-1. Conclusions:These results suggest that LCA has potential therapeutic activity in suppression of CCA cells.

Tumor-associated macrophages promote cholangiocarcinoma progression via exosomal Circ_0020256

This study investigated the exosomal circular RNAs (CircRNAs) produced by tumor-associated macrophages and delivered into the microenvironment of cholangiocarcinoma cells in order to use them as molecular targets for clinical therapy. Tumor-associated M2 macrophages (TAMs) were induced from THP-1 cells and identified by flow cytometry. The TAM-secreted exosomes were isolated from conditioned medium and a CircRNA microarray assay was performed to identify CircRNAs that were uniquely expressed in the isolated exosomes. Circ_0020256 was especially identified based on having the highest differential expression level among all of the CircRNA candidates. In vitro and in vivo experiments were performed to assess the effects of TAMs, exosomes, and Circ_0020256 on the growth and migration of cholangiocarcinoma (CCA) cells. The induced TAMs promoted the proliferation, migration, and invasion of CCA cells and those effects were mediated by exosomes secreted by the TAMs. In CCA cells (RBE and HCCC-9810), Circ_0020256 significantly promoted cellular activity by interacting with its intra-cellular microRNA target, miR-432-5p. In contrast, overexpression of transcription factor E2F3 in CCA cells restored the CCA cellular activities that were inhibited by miR-432-5p. On the other hand, treatment with small interference RNA (siRNA) for Circ_0020256 inhibited CCA cell proliferation, migration, and invasion both in vitro and in vivo. In conclusion, Circ_0020256 in TAM-secreted exosomes promoted the proliferation, migration, and invasion of CCA cells, and that promotional activity was regulated via a Circ_0020256/miR-432-5p/E2F3 axis.

Alendronate blocks human cholangiocarcinoma cell proliferation and migration

Purpose: To explore the effect of alendronate on cell death and migration of cholangiocarcinoma (CCA).
 Methods: Migration and cell death of CCA cells were determined using sulforhodamine B (SRB), colony formation, wound healing, and gelatin zymography assays. The mechanism of action of alendronate was studied with reverse-transcriptase polymerase reaction (RT-PCR) for gene expression and by Western blotting analysis for protein expression.
 Results: Alendronate stimulated KKU-100 cell death in dose- and time-dependent manner, with low IC50 value, and significantly inhbited colony formation at doses of 5 - 100 µM. Moreover, alendronate at doses of 250 - 1000 µM significantly stimulated CCA apoptosis via reactive oxygen species (ROS) generation, and enhanced caspase 3 activity at a dose of 1000 µM. Moreover, at a dose of 250 µM, it significantly inhibited cell growth through induction of caspase 3 and p53, and reduction of protein expression levels of NF-ĸB. Furthemore, alendronate altered mevalonate (MVA) pathway via downregulation of Rac1 protein expression. In contrast, it significantly inhibited CCA cell migration, and reduced MMP 2 and MMP 9 levels at doses of 25 - 100 µM.
 Conclusion: Alendronate may be useful as a novel drug for prevention and chemotherapy of CCA.

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.

LINC01410 promotes cell proliferation and migration of cholangiocarcinoma through modulating miR-124-3p/SMAD5 axis.

Cholangiocarcinoma (CCA) is generally associated with high incidence and poor prognosis. Nowadays, increasing experimental data demonstrate that long non-coding RNA (lncRNA) plays an indispensable role in tumor occurrence. Nevertheless, the specific mechanism of lncRNA is not clear in CCA. The relative expressions of lncRNAs, miRNAs, and mRNAs were detected by real-time quantitative PCR (RT-qPCR). CCK8 and colony formation assays were applied to examine cell proliferation ability in CCA. Transwell assay was conducted to measure the migration and invasion capabilities of CCA cells. Nuclear and cytoplasmic separation assay was implemented to figure out the location of LINC01410. Luciferase reporter assay, RIP and RNA pull-down assays were applied to certify the molecular bindings. Western blot was applied to detect the protein level. The high expression of LINC01410 was proved in CCA tissues and CCA cell lines. Also, CCA patients with high LINC01410 level presented poor prognosis. LINC01410 deficiency impeded cell proliferation, migration and invasion in HuCCT1 and RBE cell lines. What's more, LINC01410 interacted with miR-124-3p. Meanwhile, SMAD5 targeted and inhibited by miR-124-3p. SMAD5 expression was enhanced by LINC01410. LINC01410 facilitates cell proliferation, migration and invasion through miR-124-3p/SMAD5 axis.

High level of interleukin-33 in cancer cells and cancer-associated fibroblasts correlates with good prognosis and suppressed migration in cholangiocarcinoma.

Interleukin 33 (IL-33) promotes cholangiocarcinoma (CCA) genesis in a mouse model, however, its function in human CCA has not been clearly understood. This study was aimed to investigate IL-33 level in CCA tissues and its clinicopathological correlations. The results revealed that IL-33 was found in both cancer cells and stromal cancer-associated fibroblast (CAFs) staining patterns which were divided into high (CH) and low level (CL) in cancer cells; and presence (FP) and absence (FA) in CAFs. Kaplan-Meier analysis showed that patients in the CL group were significantly correlated with a short 2-year survival time (P = 0.027). The CL/FP group had a shorter survival time compared to the other groups with statistical significance for 2-year (P = 0.030) and 5-year (P = 0.023) survivals. In contrast, CH/FP patients had significantly greater 2-year (P = 0.003) and 5-year (P = 0.003) survivals. Univariate and multivariate analysis confirmed that CL/FP was a significantly independent risk factor whereas CH/FP was a significant protective factor in CCA patients. High IL-33 expressing CCA cells had low migration, but they showed increased migration when IL-33 expression was knocked down. The low level of recombinant human IL-33 (rhIL-33) (0.002 - 2 ng/ml) could promote CCA cell migration, in contrast to the suppressive effect at a high dose (20 - 200 ng/ml). In conclusion, the combination of high IL-33 level in cancer cells and CAFs is a potentially good prognosis marker in CCA patients. The in vitro migration suppressive effect of IL-33 may be the potential mechanism supporting its role as a good prognostic marker in CCA patients. The obtained results strengthen IL-33 as a promising predictor and therapeutic target for CCA.

Antitumor activity of celastrol by inhibition of proliferation, invasion, and migration in cholangiocarcinoma via PTEN/PI3K/Akt pathway.

AimCholangiocarcinoma is a malignant tumor originating from bile duct epithelium. Currently, the treatment strategy is very limited and the prognosis is poor. Recent studies reported celastrol exhibits antigrowth and antimetastasis properties in many tumors. Our study aimed to assess the anti‐CCA effects of cholangiocarcinoma (CCA) and the mechanisms involved in it.MethodsIn this study, the long‐term and short‐term antiproliferation effects was determined using colony formation and Cell Counting Kit‐8 (CCK‐8) assays, respectively. Flow cytometry was performed to quantify apoptosis. Furthermore, wound healing and transwell assays were performed to determine the cell migration and invasion capabilities, respectively. To further find the mechanism involved in the celastrol‐induced biological functions, LY204002, a PI3K/Akt signaling inhibitor, and an Akt‐1 overexpression plasmid were employed to find whether PI3K/Akt pathway was involved in the celastrol‐induced CCA cell inhibition. Additionally, short interfering RNA (siRNA) was also used to investigate the mechanism involved in the celastrol‐induced PI3K/Akt signaling inhibition. Western blotting and immunofluorescence assays were also performed to detect the degree of relative proteins. Moreover, we validated the antiproliferation and antimetastasis effects of celastrol in vivo by constructing subcutaneous and lung metastasis nude mice models.ResultsWe discovered that celastrol effectively induced apoptotic cell death and inhibited the capacity of migration and invasion in CCA cells. Further mechanistic study identified that celastrol regulated the PI3K/Akt signaling pathway, and the antitumor efficacy was likely due to the upregulation of PTEN, a negative regulator of PI3K/Akt. Blockage of PTEN abolished the celastrol‐induced PI3K/Akt signaling inhibition. Additionally, in vivo experiments conformed celastrol inhibited the tumor growth and lung metastasis with no serious side effects.ConclusionsOverall, our study elucidated a mechanistic framework for the anti‐CCA effects of celastrol via PTEN/PI3K/Akt pathway.

Long non-coding RNA MIR22HG inhibits cell proliferation and migration in cholangiocarcinoma by negatively regulating the Wnt/β-catenin signaling pathway.

Cholangiocarcinoma (CCA) is one of the most common primary biliary malignant tumors with a high mortality. MIR22HG has been reported to act as a tumor-suppressor gene in several types of cancers. However, the role and molecular regulatory mechanism of MIR22HG in CCA still remains unclear. The present study aimed to investigate the role and underlying mechanism of MIR22HG in CCA. The expression of MIR22HG was detected by RT-qPCR assayin CCA tissues and cells. CCK-8, colony formation and transwell assays were performed to study the biological function of MIR22HG in CCA. Western blot and immunofluorescence assays were performed to detect the expression ofWnt/β-catenin signaling pathway markers. In vivo assays were conducted to explore the biological role of MIR22HG. We first found that MIR22HG expression was significantly down-regulated in CCA tissues and cell lines. Moreover, MIR22HG expression was related to TNM stage and bore prognostic significance in CCA patients. Function experiments demonstrated that overexpression of MIR22HG inhibited cell proliferation, migration and invasion in CCA, whereas knockdown of MIR22HG caused the opposite result. It was found that MIR22HG negatively regulated mRNA and the expression levels of proteins in the Wnt/β-catenin signaling pathway (β-catenin, cyclin D1 and c-myc). The effect of MIR22HG overexpression on CCA progression could be partly rescued by activating the Wnt/β-catenin signaling pathway. MIR22HG suppressed CCA tumorigenesis in vivo. In summary, the results of the present study show that MIR22HG repressed cell proliferation, migration and invasion in CCA by negatively regulating the Wnt/β-catenin signaling pathway. MIR22HG may be a novel target for diagnosis and therapy in CCA.

Upregulation of transferrin receptor-1 induces cholangiocarcinoma progression via induction of labile iron pool.

Labile iron pool is a cellular source of ions available for Fenton reactions resulting in oxidative stress. Living organisms avoid an excess of free irons by a tight control of iron homeostasis. We investigated the altered expression of iron regulatory proteins and iron discrimination in the development of liver fluke-associated cholangiocarcinoma. Additionally, the levels of labile iron pool and the functions of transferrin receptor-1 on cholangiocarcinoma development were also identified. Iron deposition was determined using the Prussian blue staining method in human cholangiocarcinoma tissues. We investigated the alteration of iron regulatory proteins including transferrin, transferrin receptor-1, ferritin, ferroportin, hepcidin, and divalent metal transporter-1 in cholangiocarcinoma tissues using immunohistochemistry. The clinicopathological data of cholangiocarcinoma patients and the expressions of proteins were analyzed. Moreover, the level of intracellular labile iron pool in cholangiocarcinoma cell lines was identified by the RhoNox-1 staining method. We further demonstrated transferrin receptor-1 functions on cell proliferation and migration upon small interfering RNA for human transferrin receptor 1 transfection. Results show that Iron was strongly stained in tumor tissues, whereas negative staining was observed in normal bile ducts of healthy donors. Interestingly, high iron accumulation was significantly correlated with poor prognosis of cholangiocarcinoma patients. The expressions of iron regulatory proteins in human cholangiocarcinoma tissues and normal liver from cadaveric donors revealed that transferrin receptor-1 expression was increased in the cancer cells of cholangiocarcinoma tissues when compared with the adjacent normal bile ducts and was significantly correlated with cholangiocarcinoma metastasis. Labile iron pool level and transferrin receptor-1 expression were significantly increased in KKU-214 and KKU-213 when compared with cholangiocyte cells (MMNK1). Additionally, the suppression of transferrin receptor-1 expression significantly decreased intracellular labile iron pool, cholangiocarcinoma migration, and cell proliferation when compared with control media and control small interfering RNA. In Conclusion, high expression of transferrin receptor-1 resulting in iron uptake contributes to increase in the labile iron pool which plays roles in cholangiocarcinoma progression with aggressive clinical outcomes.

Effects of thymidine phosphorylase on tumor aggressiveness and 5-fluorouracil sensitivity in cholangiocarcinoma

To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. TP mRNA and protein expression were decreased by 87.1% + or - 0.49% and 72.5% + or - 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy.