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.

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Akirin2 is a key regulator of embryonic development and the innate immunity response. However, this regulator’s role in tumorigenesis especially in cholangiocarcinoma (CCA) development has not been thoroughly elucidated to date. In the current work, we used RT-qPCR, western blot analysis, and immunohistochemistry (IHC) to explore the expression level of Akirin2, and the relationship between Akirin2 levels and clinicopathological characteristics was evaluated. The biological functions of Akirin2 were examined in vitro and in vivo by using a lentiviral vector system. Luciferase reporter assays were applied to detect the direct binding relationship between the 3′-UTR of Akirin2 mRNA and miR-490-3p. The results showed that Akirin2 was overexpressed in CCA and this upregulation was associated with a shorter overall survival. Silencing or overexpressing Akirin2 by lentiviral approaches significantly influenced CCA cell proliferation, migration, invasion, and angiogenesis. An in vivo tumor model further validated the oncogenic effect of Akirin2 on CCA cell growth, metastasis, and angiogenesis. Mechanistic studies demonstrated that Akirin2 induced angiogenesis by increasing the expression of VEGFA by activating the IL-6/STAT3 signaling pathway. Akirin2 promoted cell migratory and invasive potential by affecting the epithelial–mesenchymal transition (EMT) process. In addition, Akirin2 expression was negatively controlled by miR-490-3p in CCA cells, and miR-490-3p attenuated cell migration and angiogenesis in CCA cells by silencing Akirin2. Taken together, the data indicated that Akirin2 could be regulated by miR-490-3p at the posttranscriptional level and facilitate CCA cell progression via the IL-6/STAT3/VEGFA signaling pathway. The present study may expedite the development of novel therapeutic strategies for CCA.

The purpose of this article is to explore the function and mechanism of HOXD-AS1 in cholangiocarcinoma. TCGA, StarBase and JASPAR were applied to predict the differential expression and molecular mechanism. The qRT-PCR was conducted to detect molecular expression. The effect of HOXD-AS1 on tumor proliferation, metastasis and stemness was measured through corresponding experiments. ChIP, luciferase reporter and RIP assays were implemented to explore the regulatory mechanism of HOXD-AS1 in CCA. In this study, HOXD-AS1 expression was significantly upregulated in CCA tissues and cells compared with control groups, respectively. Increased HOXD-AS1 was markedly correlated with lymph node invasion, advanced TNM stage and poor survival of CCA patients. Moreover, HOXD-AS1 was confirmed to be an unfavorable independent prognostic factor for CCA patients. Functionally, gain- and loss-of-function experiments demonstrated that HOXD-AS1 facilitated tumor proliferation, migration, invasion, EMT, stemness and drug resistance in vitro and in vivo . For the mechanism, transcription factor SP1-induced HOXD-AS1 upregulated oncogene MYCN through competitively binding to miR-520c-3p. Furthermore, HOXD-AS1-induced malignant phenotypes were rescued by interfering miR-520c-3p and MYCN, respectively. SP1/HOXD-AS1/miR-520c-3p/MYCN plays a vital role in initiation and progression of CCA, and HOXD-AS1 is expected to be an efficient biomarker and therapeutic target.

Double minute chromosomes (DMs) are extrachromosomal cytogenetic structures found in tumour cells. As hallmarks of gene amplification, DMs often carry oncogenes and drug-resistance genes and play important roles in malignant tumour progression and drug resistance. The mitogen-activated protein kinase (MAPK) signalling pathway is frequently dysregulated in human malignant tumours, which induces genomic instability, but it remains unclear whether a close relationship exists between MAPK signalling and DMs. In the present study, we focused on three major components of MAPK signalling, ERK1/2, JNK1/2/3 and p38, to investigate the relationship between MAPK and DM production in tumour cells. We found that the constitutive phosphorylation of ERK1/2, but not JNK1/2/3 and p38, was closely associated with DMs in tumour cells. Inhibition of ERK1/2 activation in DM-containing and ERK1/2 constitutively phosphorylated tumour cells was able to markedly decrease the number of DMs, as well as the degree of amplification and expression of DM-carried genes. The mechanism was found to be an increasing tendency of DM DNA to break, become enveloped into micronuclei (MNs) and excluded from the tumour cells during the S/G2 phases of the cell cycle, events that accompanied the reversion of malignant behaviour. Our study reveals a linkage between ERK1/2 activation and DM stability in tumour cells.