Salinomycin promotes cell death via the activation of the ROS/NF-κB/NLRP3 pathway in cholangiocarcinoma.

BackgroundThymosin β10 (Tβ10) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of Tβ10 in liver fluke-associated cholangiocarcinoma (CCA) are not fully understood. In this study, we investigated the expression of Tβ10 in CCA tumor tissues and cell lines as well as molecular mechanisms of Tβ10 in tumor metastasis of CCA cell lines.MethodsTβ10 expression was determined by real time RT-PCR or immunocytochemistry. Tβ10 silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell migration was assessed using modified Boyden chamber and wound healing assay. The effect of silencing Tβ10 on CCA tumor metastasis was determined in nude mice. Phosphorylation of ERK1/2 and the expression of EGR1, Snail and matrix metalloproteinases (MMPs) were studied.ResultsTen pairs of CCA tissues (primary and metastatic tumors) and 5 CCA cell lines were studied. With real time RT-PCR and immunostaining analysis, Tβ10 was highly expressed in primary tumors of CCA; while it was relatively low in the metastatic tumors. Five CCA cell lines showed differential expression levels of Tβ10. Silence of Tβ10 significantly increased cell migration, invasion and wound healing of CCA cells in vitro; reversely, overexpression of Tβ10 reduced cell migration compared with control cells (P<0.05). In addition, silence of Tβ10 in CCA cells increased liver metastasis in a nude mouse model of CCA implantation into the spleen. Furthermore, silence of Tβ10 activated ERK1/2 and increased the expression of Snail and MMPs in CCA cell lines. Ras-GTPase inhibitor, FPT inhibitor III, effectively blocked Tβ10 silence-associated ERK1/2 activation, Snail expression and cell migration.ConclusionsLow expression of Tβ10 is associated with metastatic phenotype of CCA in vitro and in vivo, which may be mediated by the activation of Ras, ERK1/2 and upregulation of Snail and MMPs. This study suggests a new molecular pathway of CCA pathogenesis and a novel strategy to treat or prevent CCA metastasis.

ABSTRACTThis study was to investigate the biological function and underlying mechanisms of FENDRR in cholangiocarcinoma (CCA) cell proliferation, migration and invasion. FENDRR and survivin expression in CCA tissues or cell lines were measured by qRT-PCR. In QBC939 and HuCCTl cells, cell proliferation was detected by CCK-8, cell migration and invasion were using transwell assay. RNA pull-down and RIP assay were performed to determine whether FENDRR can combine with SETDB1 in CCA cell. The effect of SETDB1 on survivin and H3K9me1 expression in CCA cells were determined by western blotting. ChIP analysis was performed to analyze the combination of SETDB1 with survivin promoter in CCA cell. The effect of SETDB1 knockdown on survivin and H3K9me1 expression in CCA cells after transfection with FENDRR were determined by western blotting. The results showed that lncRNA FENDRR was downregulated in CCA tissues and cells, and was negatively correlated with survivin expression. Further investigation demonstrated that FENDRR represses CCA cell proliferation, migration and invasion through regulating survivin expression. FENDRR associated with SETDB1 and H3K9 to epigenetically silence survivin and then regulated cell proliferation, migration and invasion. These findings indicate an important role for FENDRR–survivin axis in CCA cell proliferation, migration and invasion, and reveal a novel epigenetic mechanism for survivin silencing. Our data indicated that FENDRR silences survivin via SETDB1-mediated H3K9 methylation, thereby represses CCA cell proliferation, migration and invasion.

The study is designed to explore the regulatory network that MALAT1 competitively binds with miR-188-5p to up-regulate PSMD10 to facilitate cholangiocarcinoma cell migration and invasion and suppress apoptosis. qRT-PCR and fluorescence in situ hybridization (FISH) were used to examine the expression and positive signal of MALAT1 and miR-188-5p in cholangiocarcinoma tissues and HIBEC, HCCC-9810, RBE, and QBC939 cells. Western blot, qRT-PCR, and immunohistochemistry were selected to detect PSMD10 expression in cholangiocarcinoma tissues and cell lines. Dual luciferase reporter gene assay was adopted to verify that miR-188-5p targeted MALAT1 and PSMD10. qRT-PCR, pull down, and western blot were used to examine the regulation of MALAT1-miR-188-5p-PSMD10 axis. Transwell, wound healing assay, and Tunel cell apoptosis were adopted to respectively detect the regulatory abilities of MALAT1-miR-188-5p-PSMD10 axis on cell invasion, migration, and apoptosis. Western blot was used to detect the regulation mechanism of MALAT1 on Bax, Bcl-2, and caspase-3 proteins. Nude mice subcutaneous xenograft model of cholangiocarcinoma was established to examine the impacts of MALAT1 on subcutaneous tumor growth. Immunohistochemistry was adopted to examine the positive indicator of Ki67 antibodies and SMD10 antibodies in each group. MALAT1 and PSMD10 were highly expressed in cholangiocarcinoma tissues and cell lines, while miR-188-5p was lowly expressed. MALAT1 could competitively bind to miR-188-5p, and miR-188-5p could negatively regulate PSMD10. MALAT1, In-miR-188-5p, and PSMD10 could facilitate cell invasion and migration and inhibit apoptosis, while siMALAT1, miR-188-5p, and siPSMD10 produced an opposite result. MALAT1-miR-188-5p-PSMD10 axis could promote RBE cell invasion and migration and inhibit apoptosis, whereas siMALAT1-In-miR-188-5p-siPSMD10 axis showed an opposite result. On the other hand, it was verified that up-regulation/down-regulation of MALAT1 can inhibit/promote Bax and caspase-3 proteins and promote/inhibit the expression of Bcl-2 protein. MALAT1 could facilitate subcutaneous tumor growth and enhance cell proliferation and positive signal of PSMD10, while miR-188-5p worked in an opposite direction. MALAT1 competitively binds to miR-188-5p to up-regulate mRNA translation and protein expression of PSMD10, thereby facilitating cholangiocarcinoma cell invasion and migration and inhibiting its apoptosis. However, interfering MALAT1-miR-188-5p-PSMD10 axis could inhibit the occurrence and development of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a heterogeneous malignancy. The aim of the study was to investigate the regulatory role of long noncoding RNA LINC00844 in CCA progression, explore the underlying molecular mechanisms, and to analyze the potential prognostic value of LINC00844 in CCA patients. Expression of LINC00844 in CCA cell lines and tissues was examined by reverse transcription-quantitative PCR. Cell counting kit-8 assay was used to assess CCA cell proliferation, and the Transwell assay was used to evaluate tumor cell migration and invasion. miRNAs sponged by LINC00844 were predicted and confirmed using a luciferase reporter assay. Kaplan-Meier survival analysis was performed to evaluate the survival prognosis of CCA patients. The expression levels of LINC00844 were decreased in CCA tissues and cells. Overexpression of LINC00844 inhibited cell proliferation, migration and invasion in CCA cells. miR-19a-5p is directly targeted by LINC00844, mediating the inhibitory effects of LINC00844 on the proliferation, migration and invasion of CCA cells. LINC00844 and miR-19a-5p expression were associated with differentiation and tumor node metastasis stage in CCA patients. CCA patients with low LINC00844 expression or overexpression of miR-19a-5p had worse overall survival. The expression levels of LINC00844 were decreased in both CCA tissues and cells, and high LINC00844 inhibited CCA cell proliferation, migration and invasion through sponging miR-19a-5p. Low LINC00844 and high miR-19a-5p expression were associated with worse overall survival in CCA patients. All the data suggested that the LINC00844/miR-19a-5p axis may provide novel therapeutic targets and prognostic biomarkers for CCA patients.

Background/Aims:Cholangiocarcinoma (CCA) is a malignant and insidious tumor that is tricky to treat. Long non-coding RNA (LncRNA) LINC01123 is a biomolecule that influences cancer progression by regulating gene expression via influencing the regulatory function of microRNAs in gene expression. Therefore, this study investigated the connection between LINC01123 and CCA and explored the underlying mechanism. The objective of this study was to provide valuable information on the management of CCA.Materials and Methods:Tumor and normal paracancer tissue samples in this study were collected from 128 CCA patients. To measure LINC01123 and miR-641 expression characteristics, quantitative reverse transcription-polymerase chain reaction was used. To explore the biological functions of LINC01123 and miR-641 in CCA cells, cell counting Kit-8 (CCK-8) and transwell migration and invasion assays were used. The mechanism was investigated using dual luciferase reporter assays and rescue experiments.Results:This study found that the expression levels of LINC01123 were higher in CCA tissues and cells than in normal tissues and cells. LINC01123 promoted the proliferation, migration and invasion ability of CCA cells and consequently became an indicator of lymph node metastasis and advanced TNM stage in CCA. Moreover, the expression of miR-641 was negatively correlated with the expression of LINC01123. LINC01123 affected CCA progression by downregulating miR-641.Conclusion:There was an upregulation of LINC01123 in CCA tumor tissues and cells. LINC01123 promoted CCA aggravation by targeting miR-641. LINC01123 could be the target of future treatment for CCA.

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

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

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.

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

Ficolin 3 (FCN3) is a pattern recognition molecule that activates the complement system via the lectin pathway. While its immunological roles are known, the specific mechanisms by which FCN3 affects cholangiocarcinoma (CCA) pathogenesis remain unclear. In this study, we investigated FCN3 expression in CCA and benign cells, as well as tumor versus non-tumor tissues, using RT-qPCR and Western blotting analyses. The effects of FCN3 on CCA cell proliferation, migration, and invasion were analyzed through CCK-8, EdU, transwell, and wound-healing assays, with invivo studies supporting these findings. The complement-mediated cytotoxicity of CCA cells was assessed using human serum with or without heat inactivation and an anti-C6 blocking antibody. Immunocytochemical staining was used to examine membrane attack complex (MAC) deposition, and an immunoprecipitation assay was adopted to evaluate the interaction between FCN3 and MASP family members. The role of FCN3 in inducing necroptosis was explored through transmission electron microscopy (TEM) and Western blotting analysis, focusing on the RIPK1/RIPK3/MLKL pathway. The results of the study demonstrate that FCN3 expression was significantly lower in CCA cells and tissues. Overexpressing FCN3 suppressed cell proliferation and migration, enhanced complement-mediated cytotoxicity via MASP2 binding, and increased MAC deposition. FCN3 also induced necroptosis through activating the RIPK1/RIPK3/MLKL pathway. These results highlight FCN3 as a tumor suppressor in CCA and suggest its potential as a therapeutic target for this malignancy.

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

BackgroundCholangiocarcinoma (CCA) is a biliary epithelial malignant tumor with an increasing incidence worldwide. Therefore, further understanding of the molecular mechanisms of CCA progression is required to identify new therapeutic targets.MethodsThe expression of RPL35A in CCA and para-carcinoma tissues was detected by immunohistochemical staining. IP-MS combined with Co-IP identified downstream proteins regulated by RPL35A. Western blot and Co-IP of CHX or MG-132 treated CCA cells were used to verify the regulation of HSPA8 protein by RPL35A. Cell experiments and subcutaneous tumorigenesis experiments in nude mice were performed to evaluate the effects of RPL35A and HSPA8 on the proliferation, apoptosis, cell cycle, migration of CCA cells and tumor growth in vivo.ResultsRPL35A was significantly upregulated in CCA tissues and cells. RPL35A knockdown inhibited the proliferation and migration of HCCC-9810 and HUCCT1 cells, induced apoptosis, and arrested the cell cycle in G1 phase. HSPA8 was a downstream protein of RPL35A and overexpressed in CCA. RPL35A knockdown impaired HSPA8 protein stability and increased HSPA8 protein ubiquitination levels. RPL35A overexpression promoted CCA cell proliferation and migration. HSPA8 knockdown inhibited CCA cell proliferation and migration, and reversed the promoting effect of RPL35A. Furthermore, RPL35A promoted tumor growth in vivo. In contrast, HSPA8 knockdown suppressed tumor growth, while was able to restore the effects of RPL35A overexpression.ConclusionRPL35A was upregulated in CCA tissues and promoted the progression of CCA by mediating HSPA8 ubiquitination.

Tumor-associated angiogenesis plays a critical role in the pathogenesis of cholangiocarcinoma (CCA). In this study, we examined the biological effects and molecular mechanisms of transcription factor 21 (TCF21) on CCA-associated angiogenesis. TCF21 expression was compared between 15 pairs of peritumor normal tissues and CCA tissues and also between normal bile duct epithelial cells and two CCA cell lines (QBC-939 and TFK-1) using real-time PCR and Western blot. With the use of both CCA cell lines as the model system, we stably expressed TCF21 by lentiviral transduction (Lv-TCF21). In vivo, we monitored xenograft growth from different CCA cells, measured tumor-associated angiogenesis by histological analysis, and determined the expressions and circulatory levels of VEGFA and PDGF-BB by immunohistochemistry and ELISA, respectively. In vitro, we assessed the effects of conditioned medium collected from different CCA cells on the viability, migration, and tube formation of endothelial cells and explored the significance of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), as well as ERK1/2 signaling in this process. TCF21 was significantly downregulated in CCA tissues or cell lines. Ectopic expression of TCF21 in CCA cells inhibited xenograft growth or tumor-associated angiogenesis in vivo and targeted the expression and secretion of proangiogenic factors, VEGFA and PDGF-BB. In vitro, the conditioned medium collected from Lv-TCF21 CCA cells significantly reduced the viability, migration, and tube formation of endothelial cells. On the molecular level, the targeting of PI3K/Akt and ERK1/2 signaling mediated the anti-angiogenic activity of TCF21. TCF21 presents growth-inhibitory and anti-angiogenic activities, and thus the elevation of TCF21 expression may provide therapeutic benefits for CCA. NEW & NOTEWORTHY Transcription factor 21 (TCF21) is downregulated in cholangiocarcinoma (CCA) tissues or cells. TCF21 inhibits the growth of xenografts derived from CCA cells. TCF21 suppresses in vivo tumor-associated angiogenesis. TCF21 targets expression and production of proangiogenic factors from CCA cells. The targeting of phosphatidylinositol 3-kinase/protein kinase B and ERK1/2 signaling mediates the anti-angiogenesis of TCF21.

Activation of the PI3K-AKT signaling pathway by SPARC contributes to the malignant phenotype of cholangiocarcinoma cells

HSPB8 promoted intrahepatic cholangiocarcinoma progression by enhancing epithelial-mesenchymal transition and autophagy