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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.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0747-x) contains supplementary material, which is available to authorized users.

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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.

Background : Ovarian cancer (OC) is the gynecologic malignant tumor with high mortality. Accumulating evidence indicates that M2-like tumor-associated macrophages (TAMs) can secret EGF to participate in ovarian cancer growth, migration, and metastasis. An EGF-downregulated lncRNA, LIMT (lncRNA inhibiting metastasis), was identified as a critical regulator of mammary cell migration and invasion. Nevertheless, whether EGF secreted from M2-like TAMs regulates LIMT expression in ovarian cancer progression remains largely unknown. Methods : The human OC cell lines OV90 and OVCA429 were recruited in this study. The differentiation of the human monocyte cell line THP-1 into M2-like TAMs was confirmed using flow cytometry within the application of phorbol 12-myristate 13-acetate (PMA). ELISA was performed to detect EGF concentration in co-culture system of M2-like TAMs and OC cell lines. Moreover, CCK-8, flow cytometry and immunofluorescence staining of Ki67 were performed to assess the capacity of cell proliferation. Besides, cell migration and invasion were determined by wound healing and transwell assays. Furthermore, the expression levels of epithelial-mesenchymal transition (EMT) markers and EGFR/ERK signals were analyzed by qRT-PCR and western blot. Female athymic nude mice (8–12 weeks of age; n = 8 for each group) were recruited for in vivo study. Results : In the present study, THP-1 cells exhibited the phenotype markers of M2-like TAMs with low proportion of CD14 + marker and high proportion of CD68 + , CD204 + , CD206 + markers within the application of PMA. After co-culturing with M2-like TAMs, EGF concentration in the supernatants was significantly increased in a time-dependent manner. Besides, OC cells presented better cell viability, higher cell proliferation, and stronger migration and invasion. The expression of EMT-related markers N-cadherin, Vimentin and EGFR/ERK signals were markedly up-regulated, while E-cadherin was significantly decreased. However, these effects induced by co-culture system were reversed by the application of AG1478 (an EGFR inhibitor) or LIMT overexpression. Furthermore, the endogenous expression of LIMT was decreased in OC cell lines compared with the control group. Also, the in vivo experiments verified that the inhibition of EGFR signaling by AG1478 or overexpression of LIMT effectively repressed the tumor growth. Conclusion : Taken together, we demonstrated that EGF secreted by M2-like TAMs might suppress LIMT expression via activating EGFR-ERK signaling pathway to promote the progression of OC.