A Circular RNA, Cholangiocarcinoma‐Associated Circular RNA 1, Contributes to Cholangiocarcinoma Progression, Induces Angiogenesis, and Disrupts Vascular Endothelial Barriers

Abstract

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.

<p>Identification of a Functional ceRNA Network to Explore Potential Biomarkers for Hepatocellular Carcinoma</p>

“…Besides, it promotes the occurrence and metastasis of cholangiocarcinoma, and might be an important organism markers for the treatment of cholangiocarcinoma. 26 Based on qRT-PCR results, circ_0130911 was relatively and highly expressed in HCC tissues and was closely related to the poor prognosis of HCC. Importantly, the biological function of circ_0130911 in tumors is largely understudied.…”

Roles of Bile-Derived Exosomes in Hepatobiliary Disease

“…circ-CCAC1 can be incorporated into the exosomes and subsequently is ingested by vascular endothelial cells. This circRNA enters the cytoplasm of the cells and binds to EZH2 to reduce its intranuclear transport, decrease the formation of H3K27me3, and promote the transcription of SH3GL2 to increase the level of SH3GL2, which can negatively regulate ZO-1/occludin; these events eventually destroy the endothelial barrier function and promote tumour angiogenesis [ 30 ]( Figure 4 ). Rafal et al [ 31 ] have confirmed that bile duct ligation (BDL) increased release of Hh-containing exosome-enriched microparticles into plasma and bile, and all microparticles induced similar Hh-dependent changes in SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.…”

Exosomal circRNAs: Sorting Mechanisms, Roles and Clinical Applications in Tumors

“…Chromatin Immunoprecipitation (ChIP) experiments showed that EZH2 could directly bind to the promoter of SH3GL2 and promote its H3K27me3 methylation level, and thus inhibit the expression of SH3GL2. Circ-CCAC1 derived from EVs could reduce the efficiency of EZH2 binding to the SH3GL2 promoter, thereby promoting the expression of SH3GL2, which further inhibited the expression of ZO-1/Occludin (Xu et al, 2020).…”