RESUMO
Cholangiocarcinoma is an extremely malignant cancer with poor prognosis. Finding efficient diagnosis and treatment is the indispensable way to improve the prognosis of CCA patients. Therefore, exploring molecular abnormalities in CCA development is urgently needed. DLEU1 is a potential tumor-related lncRNA and abnormally expressed in multiple cancers. In this study, TCGA data analysis showed upregulation of DLEU1 expression in CCA. Furthermore, we confirmed that DLEU1 expression was increased in CCA tissues and cells compared with corresponding controls. Upregulated DLEU1 was related to poor clinicopathological characteristics. Functionally, silencing DLEU1 inhibited CCA proliferation, invasion, stemness maintenance and chemo-resistance, whereas amplifying DLEU1 promoted malignant biological behavior of CCA cells. Mechanistically, DLEU1 expression was transcriptionally facilitated by transcription factor YY1. Moreover, DLEU1 promoted oncogene YAP1 expression by functioning as a sponge to competitively bind to miR-149-5p. YAP1 promoted CCA proliferation, invasion and stemness maintenance, whereas miR-149-5p inhibited malignant biological behavior of CCA. Rescue experiments confirmed that the cancer-promoting effect of DLEU1 was saved by interfering miR-149-5p or YAP1. Furthermore, YAP1 promoted tumor stemness maintenance partly by acting as a transcriptional coactivator to promote TEAD2-induced SOX2 expression. These findings indicated that YY1-induced DLEU1 played a crucial role in CCA progression via miR-149-5p/YAP1/TEAD2/SOX2 axis.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , MicroRNAs , RNA Longo não Codificante , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Colangiocarcinoma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , Fatores de Transcrição SOXB1 , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP , Fator de Transcrição YY1/genéticaRESUMO
BACKGROUND AND AIMS: Circular RNAs (circRNAs) and extracellular vesicles (EVs) are involved in various malignancies. We aimed to clarify the functions and mechanisms of dysregulated circRNAs in the cells and EVs of cholangiocarcinoma (CCA). APPROACH AND RESULTS: CircRNA microarray was used to identify circRNA expression profiles in CCA tissues and bile-derived EVs (BEVs). CCA-associated circRNA 1 (circ-CCAC1) expression was measured by quantitative real-time PCR. The clinical importance of circ-CCAC1 was analyzed by receiver operating characteristic curves, Fisher's exact test, Kaplan-Meier plots, and Cox regression model. The functions of circ-CCAC1 and exosomal circ-CCAC1 were explored in CCA cells and human umbilical vein endothelial cells (HUVECs), respectively. Different animal models were used to verify the in vitro results. RNA sequencing, bioinformatics, RNA immunoprecipitation, RNA pulldown, chromatin immunoprecipitation followed by sequencing, and luciferase reporter assays were used to determine the regulatory networks of circ-CCAC1 in CCA cells and HUVECs. Circ-CCAC1 levels were increased in cancerous bile-resident EVs and tissues. The diagnostic and prognostic values of circ-CCAC1 were identified in patients with CCA. For CCA cells, circ-CCAC1 increased cell progression by sponging miR-514a-5p to up-regulate Yin Yang 1 (YY1). Meanwhile, YY1 directly bound to the promoter of calcium modulating ligand to activate its transcription. Moreover, circ-CCAC1 from CCA-derived EVs was transferred to endothelial monolayer cells, disrupting endothelial barrier integrity and inducing angiogenesis. Mechanistically, circ-CCAC1 increased cell leakiness by sequestering enhancer of zeste homolog 2 in the cytoplasm, thus elevating SH3 domain-containing GRB2-like protein 2 expression to reduce the levels of intercellular junction proteins. In vivo studies further showed that increased circ-CCAC1 levels in circulating EVs and cells accelerated both CCA tumorigenesis and metastasis. CONCLUSIONS: Circ-CCAC1 plays a vital role in CCA tumorigenesis and metastasis and may be an important biomarker/therapeutic target for CCA.
Assuntos
Neoplasias dos Ductos Biliares/sangue , Carcinogênese/metabolismo , Colangiocarcinoma/sangue , Endotélio Vascular/metabolismo , Neovascularização Patológica/metabolismo , RNA Circular/sangue , RNA Circular/genética , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Coledocolitíase/sangue , Coledocolitíase/genética , Coledocolitíase/patologia , Vesículas Extracelulares/metabolismo , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Huaier, 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. METHODS: Microarray 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. RESULTS: In 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. CONCLUSION: Huaier 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.