A novel circular RNA, circ-CCAC1, contributes to CCA progression, induces angiogenesis, and

Accepted Article
disrupts vascular endothelial barriers

Yi Xu1,2,8,†, Kaiming Leng2,3,†, Yue Yao2,4,†, Pengcheng Kang1, Guanqun Liao5,7, Yi Han6, Guangjun

Shi3, Daolin Ji1,2, Peng Huang1,2, Wangyang Zheng1,2, Zhenglong Li1,2, Jinglin Li1,2, Lining Huang1,2,

Liang Yu1, Yongxu Zhou1, Xingming Jiang1, Hao Wang1, Chunlong Li1, Zhilei Su1, Sheng Tai1, Xiangyu

Zhong1, Zhidong Wang1, Yunfu Cui1,*

1Department of Hepatopancreatobiliary Surgery, Second Affiliated Hospital of Harbin Medical

University, Harbin, 150086, China

2The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin,

150086, China
3Department of Hepatobiliary Surgery, Qingdao Municipal Hospital, Qingdao University, Qingdao,

266071, China
4Department of Endocrinology and Metabolism, Second Affiliated Hospital of Harbin Medical

University, Harbin, 150086, China

5Department of Interventional Radiology, Stanford University School of Medicine, Stanford, CA, 94305,

USA
6Department for Visceral, Thoracic and Vascular Surgery at the University Hospital, Technical

University Dresden, Fetscherstraße 74, 01307, Dresden, Germany

7Department of Hepatobiliary surgery, Foshan Hospital Affiliated to Southern Medical University,

Foshan, 528000, China

8Young Scholar of General Surgery Climbing Program of China
†These authors contributed equally to this work.

*Corresponding author.

Keywords: Cholangiocarcinoma; Extracellular vesicles; Circular RNA; Circ-CCAC1; Vascular

permeability
This article has been accepted for publication and undergone full peer review but has not been
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differences between this version and the Version of Record. Please cite this article as doi:
10.1002/HEP.31493
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Accepted Article
Contact information: Yunfu Cui, M.D., Department of Hepatopancreatobiliary Surgery, Second

Affiliated Hospital of Harbin Medical University, #246 Xuefu Street, 150001, Harbin, Heilongjiang

Province, China. Phone number: +86-0451-86605113; Fax: +86-0451-86605356; E-mail:

yfcui777@hotmail.com.

Financial support: This work was supported by the National Key Research and Development

Program of China (Grant No. 2017YFC1308600); National Natural Science Foundation of China (Grant

No. 81902431, 81972724, 81170426); Outstanding Youth Project of Natural Science Foundation of

Heilongjiang (Grant No. YQ2019H007); National Natural Science Foundation of Heilongjiang (Grant No.

H2018025); Special Project of China Postdoctoral Science Foundation (Grant No. 2019T120279);

Special Project of Heilongjiang Postdoctoral Science Foundation (Grant No. LBH-TZ1016); China

Postdoctoral Science Foundation (Grant No. 2018M641849, 2018M640311); Heilongjiang Postdoctoral

Science Foundation (Grant No. LBH-Z18107, LBH-Z18112); The Fundamental Research Funds for the

Heilongjiang Provincial Universities; Postgraduate Innovative Research Project of Harbin Medical

University (Grant No. YJSCX2016-21HYD); Foundation of Key Laboratory of Myocardial Ischemia,

Ministry of Education (Grant No. KF201810); Chen Xiaoping Foundation for the Development of

Science and Technology of Hubei Province (Grant No. CXPJJH11800004-001,

CXPJJH11800004-003); and Qingdao Outstanding Health Professional Development Fund.

Conflicts of interest: The authors have no commercial or other associations that might pose a conflict

of interest.

List of abbreviations: AO/EB: acridine orange/ethidium bromide; CCA: cholangiocarcinoma; AUC:

area under the curve; CCK-8: cell counting kit-8; ceRNA: competitive endogenous RNA; ChIP:

chromatin immunoprecipitation; circ-CCAC1: cholangiocarcinoma-associated circular RNA 1; circRNAs:

circular RNAs; DAPI: 4′,6-diamidino-2-phenylindole; FBS: fetal bovine serum; FISH: fluorescence in

situ hybridization; FITC: fluorescein isothiocyanate; HIBEC: human intrahepatic biliary epithelial cells;

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 IF: immunofluorescence; ISH: in situ hybridization; LNM: lymph node metastasis; NTA: nanoparticle
Accepted Article
tracking analysis; PI: propidium iodide; RNase R: ribonuclease R; RIP: RNA immunoprecipitation;

shRNA: short hairpin RNA; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy

Electronic word count: Total word count: 5,964; Abstract word count: 264

Number of figures and tables: 8 figures

Abstract

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). CircRNA microarray was used to identify circRNA expression profiles in

CCA tissues and bile-derived EVs. Cholangiocarcinoma-associated circular RNA 1 (circ-CCAC1)

expression was measured by qRT-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

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

upregulate YY1. Meanwhile, YY1 directly bound to the promoter of CAMLG 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 EZH2 in the cytoplasm, thus elevating SH3GL2 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.

Conclusion: 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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Accepted Article
Introduction

Cholangiocarcinoma (CCA) is a highly metastatic and invasive malignancy, characterized by

diagnostic difficulty and high mortality(1). The incidence of CCA is higher in Asian countries than in the

West and has increased globally in recent years(2). Despite improvements in early diagnosis, the

postoperative recurrence of CCA remains high, showing a 5-year survival of only ~10%(3).

Understanding the process of CCA carcinogenesis and metastasis is important to facilitate the

discovery of novel biomarkers and improve prognosis.

Circular RNAs (circRNAs), widely present in mammalian cells, comprise a class of endogenous

non-coding RNAs with covalently closed loop structures and a limited capacity to encode proteins(4).

The dysregulation of circRNAs causes diseases such as cancer(5). Mechanistically, circRNAs exert

regulatory effects in multiple diseases by sponging certain miRNAs to inhibit their functions, including

cardiovascular disease(6) and Alzheimer's disease(7), among others. Such interactions between

circRNAs and miRNAs are known as the competitive endogenous RNA (ceRNA) theory(8). Additionally,

circRNAs directly interact with certain proteins to exert their biological functions in tumors.

Extracellular vesicles (EVs) are endocytic-oriented membrane vesicles 30–150 nm in size. They

are released by tumor cells and contain enzymes, lipids, and nucleic acids depending on the host

cell(9). EVs are pivotal in maintaining physiology and regulating cellular microenvironments by

transporting biological materials(10). Tumor cell-secreted EVs are involved in intercellular

communication and are responsible for tumor-induced inflammation, chemoresistance, and vascular

permeability(11,12). EVs are extensively distributed in body fluids and can be taken up by other cells(13).

Additionally, circRNAs are also enriched in EVs and thus might be important for intercellular

communication(14).

Herein, we identified a novel CCA-associated circRNA upregulated in cancerous bile EVs and

tissues, hsa_circRNA_102064, located on chr17:37880978-37882106 and looped by exons 23–26 of

ERBB2. The spliced variant of hsa_circRNA_102064 is 565 nucleotides long. Previous studies

indicated that circ-ERBB2 is spliced out from ERBB2 and functions as an oncogene in gallbladder and

gastric cancer(15,16). Circ-ERBB2 is spliced from exons 3–7 of ERBB2, differing from the circRNA

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 studied in this study. Therefore, hsa_circRNA_102064 was named CCA-associated circular RNA 1
Accepted Article
(circ-CCAC1) to distinguish it from circ-ERBB2. Here, we showed that circ-CCAC1 plays a vital role in

CCA tumorigenesis and metastasis and may be an important biomarker/therapeutic target for CCA.

Materials and methods

Patients and specimens

Intrahepatic CCA (iCCA; n = 236), perihilar CCA (pCCA; n = 40), and distal CCA (dCCA; n = 40)

specimens and their adjacent normal tissues were harvested from patients at the Second Affiliated

Hospital of Harbin Medical University. Bile samples from pCCA and dCCA patients were obtained by

percutaneous transhepatic cholangial drainage or endoscopic nasobiliary drainage preoperatively.

Patients with benign hepatobiliary disease (choledocholithiasis) underwent choledochotomy followed

by T-tube drainage; bile was collected the day before T-tube removal. One patient with bile duct injury

underwent end-to-end anastomosis with T-tube drainage. No patient had undergone

radio/chemotherapy before surgery. Informed consent was obtained from each participant. This study

was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University

(Approval No. KY2018-306 and KY2020-127). Detailed clinical data of iCCA, pCCA, and dCCA

patients are displayed in Table S1-3.

Statistical analysis

Multiple groups were compared by one-way ANOVA and Tukey’s multiple comparison test;

two-group comparisons were done by the t-test using GraphPad Prism 8.30 (GraphPad, Inc., La Jolla,

CA, USA). Continuous variables not following normal distribution were compared using the Mann–

Whitney U-test. Pearson’s correlation was used to analyze the correlation between the expression of

circRNA, miRNA, and mRNA. Receiver operating characteristic (ROC) and area under the curve (AUC)

were used to evaluate the diagnostic value of biomarkers in EVs from bile or serum. A combined ROC

was calculated based on the logistic regression model. Fisher’s exact test determined the correlation

between circ-CCAC1 expression and clinical characteristics. Kaplan–Meier plots and Cox regression

analyses were performed with SPSS 22.0 (SPSS, Inc., Chicago, IL, USA) to determine overall survival

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 (OS) and disease-free survival (DFS); p < 0.05 was considered statistically significant.
Accepted Article
The detailed experimental procedures are described in Supplementary materials and methods.

Results

Screening and expression of circRNAs in bile EVs and CCA tissues

EVs were isolated from the bile of patients with eCCA or benign hepatobiliary diseases (four

patients with choledocholithiasis, one patient with bile duct injury) to determine the differentially

expressed circRNAs. TEM confirmed the exosomal morphology of the membrane-encapsulated bile

extracts (Fig. 1A). 100 μL of EVs (2.9–3.1 × 1011 EVs/mL) was isolated from 50 mL bile. For NTA

assay, the experiments were performed at 1:6000 dilution, leading to particle concentrations around

5.0 × 107 EVs/mL with a 92.1 ± 34.7 nm in size (Fig. 1A). The particles were positive for the exosomal

markers Alix, CD63, and TSG101 relative to the groups of unextracted bile and total cholangiocyte

lysates (Fig. 1A). CircRNA microarray analysis revealed 85 circRNAs (fold-change >2; p < 0.05) that

were differentially expressed between the bile-derived EVs of eCCA and healthy controls. Fig. 1B

shows the hierarchical clustering of the top 30 upregulated and downregulated circRNAs. We used

eCCA/adjacent normal tissues from the same five patients to study circRNA expression profiles using

rRNA-depleted RNA sequencing. Among the 140 differentially expressed circRNAs (fold-change >2; p

< 0.05), the top 30 upregulated and downregulated circRNAs are shown in Fig. 1B. Analysis of

differentially expressed circRNAs in EVs and tissues revealed three commonly dysregulated circRNAs

(Fig. S1A). qRT-PCR showed that, of these circRNAs (hsa_circRNA_102064, hsa_circRNA_100364,

and hsa_circRNA_101721), hsa_circRNA_102064 showed the most dysregulated expression in 20

pairs of CCA and normal tissues (5 eCCA and 15 iCCA; Fig. S1B). Hsa_circRNA_102064 is

synthesized from exons 23–26 of ERBB2 (Fig. S1C). To help distinguish this from the known

circ-ERBB2, we named this circRNA “circ-CCAC1.” Sanger sequencing validated the back-spliced

junction of circ-CCAC1 (Fig. S1D). Moreover, it harbored a loop structure resistant to RNase R (Fig.

S1E). Total RNA was extracted to detect the expression of circ-CCAC1 and linear ERBB2 after

treatment with actinomycin D at different time points. Linear ERBB2 showed a shorter half-life

compared to circ-CCAC1, highlighting the stability of circ-CCAC1 (Fig. S1F).

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Accepted Article
Clinical relevance of elevated circ-CCAC1 in CCA patient-derived EVs and tissues

Circ-CCAC1 levels were higher in CCA patient-derived serum EVs than in EVs isolated from

patients with benign hepatobiliary disease. Additionally, we also identified upregulation of circ-CCAC1

and hsa_circRNA_100364 in the bile-derived EVs of CCA patients (Fig. 1C). The AUCs for circ-CCAC1

in bile and serum EVs were 0.857 and 0.759, respectively. The diagnostic value of circ-CCAC1 (serum

EVs) was almost equal to that of serum CA19-9 (AUC = 0.757). However, hsa_circRNA_100364

expression in bile-derived EVs is not preferable for predicting eCCA (AUC = 0.619). Moreover, a

combined ROC was calculated based on the logistic regression model. The combination of bile- or

serum-derived EVs-circ-CCAC1 with serum CA19-9 both provided better results than either individually

(Fig. 1D). Circ-CCAC1 expression was higher in all the three subtypes of CCA tissues than in normal

tissues (Fig. 1E). We divided 236 iCCA patients into two groups based on the median cut-off to analyze

the clinical significance of circ-CCAC1 expression in tissues. Circ-CCAC1 was notably upregulated in

patients with more than one tumor compared to patients with only one tumor (p = 0.013). Circ-CCAC1

levels were higher in patients with positive lymph node metastasis (LNM) (p = 0.038) and advanced

TNM stages (p = 0.036; Table S5). For pCCA patients, clinicopathological characteristics, including

LNM (p = 0.031), vascular invasion (p = 0.014), and TNM stage (p = 0.010), were associated with

circ-CCAC1 expression (Table S6). Moreover, circ-CCAC1 overexpression was more frequently

observed in higher T classification (p = 0.025), positive LNM (p = 0.008), and advanced TNM stage (p =

0.019) in dCCA patients (Table S7). Kaplan–Meier survival curves showed a correlation between

circ-CCAC1 expression and poor prognosis/high postoperative recurrence of iCCA (Fig. 1F).

Multivariate analysis using the Cox regression model confirmed that >1 tumor (p = 0.022), large tumors

(p = 0.024), positive LNM (p = 0.028), positive serum CEA (p = 0.006), and high circ-CCAC1

expression (p = 0.001) were independent prognostic markers for iCCA (Table S8); circ-CCAC1

expression was an independent predictor for postoperative recurrence of iCCA (p = 0.002; Table S9).

Circ-CCAC1 expression was higher in EVs isolated from HCCC-9810, CCLP1, KMBC, and QBC939

cells compared to EVs from HIBECs (Fig. 1G). Comparison of circ-CCAC1 expression in multiple CCA

cell lines showed that the relative expression of circ-CCAC1 was elevated in CCA cells compared to

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 HIBECs (Fig. 1G).
Accepted Article
Circ-CCAC1 promotes CCA cell progression in vitro

Two shRNAs targeting the back-spliced junction of circ-CCAC1 were used to silence circ-CCAC1

expression. CCLP1 and QBC939 cells heavily expressing circ-CCAC1 were used for knockdown

experiments. Fig. 2A shows the transfection and knockdown efficiency of sh-circ-CCAC1-1 and

sh-circ-CCAC1-2 in CCLP1 and QBC939 cells. Expression of linear ERBB2 was unaffected in CCLP1

and QBC939 cells (Fig. S2A). Circ-CCAC1-shRNA-mediated downregulation of circ-CCAC1 resulted in

lower cell viability and Ki67-positive cells, indicating that circ-CCAC1 depletion suppresses CCLP1 and

QBC939 cell proliferation (Fig. 2B-C). Colony-forming assays showed that circ-CCAC1 silencing

reduced the number of colonies formed in both cell types (Fig. 2D). AO/EB staining and flow cytometry

showed that circ-CCAC1 depletion enhanced cell apoptosis (Fig. S2B-C). Wound-healing and

Transwell assays revealed that reducing the levels of circ-CCAC1 impaired the migratory potential and

invasiveness of CCA cells (Fig. 2E-F).

Circ-CCAC1 promotes CCA proliferation and invasiveness by sequestering miR-514a-5p that

regulates YY1

Circ-CCAC1 was primarily localized in the cytoplasm of CCA cells, as analyzed by RNA-FISH and

subcellular distribution assays, suggesting that circ-CCAC1 participates in post-transcriptional gene

regulation (Fig. 3A and S3A). We performed RNA-Seq and generated a heatmap for the 10 most

differentially expressed circRNAs in circ-CCAC1-depleted and control cells. YY1 was a common gene

target of circ-CCAC1 in CCLP1 and QBC939 cells (Fig. 3B). Starbase 2.0 and circBank predicted 14

and 58 miRNAs binding to circ-CCAC1, respectively (Fig. 3C). MiR-182-5p, miR-514a-5p,

miR-1343-3p, miR-3619-5p, and miR-6746-5p were identified as target miRNAs for circ-CCAC1 (Fig.

3C). Circ-CCAC1 was markedly enriched in the anti-Ago2 immunoprecipitated pool compared to the

anti-IgG pool; this phenotype was abrogated upon circ-CCAC1 depletion (Fig. 3D). Biotin-labeled

circ-CCAC1 probes were used to pull down circ-CCAC1 from CCA cells; circ-CCAC1 overexpression

enhanced the efficiency of pulldown using biotin-labeled circ-CCAC1 probes (Fig. S3B). We then

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 extracted miRNAs from the pulldown samples; only miR-514a-5p was enriched in the circ-CCAC1
Accepted Article
pulldowns from CCLP1 and QBC939 cells (Fig. 3E). A luciferase reporter gene containing the wt or mut

circ-CCAC1 sequences was constructed (Fig. 3F). MiR-514a-5p mimics reduced the fluorescence

signals from cells transfected with wt-circ-CCAC1, whereas the luciferase activity obtained from cells

transfected with mut-circ-CCAC1 was unchanged (Fig. 3F). TargetScan and miRmap were used to

determine binding potential between 45 miRNAs and the 3′-UTR of YY1, among which miR-514a-5p

was predicted (Fig. 3C). According to TCGA, YY1 was upregulated in CCA tissues compared to normal

samples (Fig. S3C). Patients heavily expressing YY1 showed poorer OS (Fig. S3D). qRT-PCR and

immunoblotting showed that YY1 was upregulated in CCA cells, particularly CCLP1 and QBC939 (Fig.

S3E-F). We also validated YY1 downregulation induced by sh-circ-CCAC1-1, which corresponds with

the trend in the microarray data (Fig. S3G). Pearson’s correlation analysis revealed a positive

correlation between circ-CCAC1 and YY1 expression and negative correlation between YY1 and

miR-514a-5p expression in CCA tissues (Fig. S4A-B). Moreover, miR-514a-5p expression in CCA cell

lines was generally downregulated (Fig. S4C). Increasing the levels of miR-514a-5p reduced YY1

expression, whereas inhibiting miR-514a-5p increased the expression of YY1 (Fig. S4D-E). To validate

the binding between the YY1 3′-UTR and miR-514a-5p, we generated constructs for wt-YY1 3′-UTR

(Luc wt) and mut-YY1 3′-UTR (Luc mut; Fig. 3G and Fig. S4F). MiR-514a-5p overexpression reduced

luciferase activity in cells with wt-YY1 3′-UTR compared to cells with mutated binding sites for

miR-514a-5p (Luc mut2 and mut3). Furthermore, simultaneously mutating sites 2 and 3 did not affect

luciferase activity (Fig. 3G).

We then co-transfected sh-circ-CCAC1-1 and miR-514a-5p inhibitor or YY1 vector in CCLP1 and

QBC939 cells, followed by immunoblotting. Circ-CCAC1 depletion decreased YY1 protein levels,

whereas cotransfection with miR-514a-5p inhibitor or YY1 vector increased YY1 protein levels (Fig.

S5A). CCK-8 and Transwell invasion assays showed that inhibiting miR-514a-5p or upregulating YY1

reversed the inhibition of CCA cell proliferation and invasion induced by sh-circ-CCAC1-1 (Fig. S5B-C).

Flow cytometry showed that circ-CCAC1 silencing enhanced apoptosis in CCLP1 and QBC939 cells;

this effect was partly rescued by inhibiting miR-514a-5p or overexpressing YY1 (Fig. S5D). All rescue

experiments indicated the involvement of a ceRNA-like mechanism in the circ-CCAC1-regulated

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 miR-514a-5p/YY1 axis.
Accepted Article
CAMLG functions as an oncogene activated by YY1 in CCA cells

YY1 is a transcription factor that activates the transcription of downstream targets in cancer cell

signaling pathways. We performed ChIP-seq to explore the changes in gene expression by YY1 in

CCLP1 cells. The following targets with a peak score >1,000 and motif score >15 were selected for

further experiments: PCIF1, GLTSCR2, UBA52, TAF5, MORF4L1, and CAMLG (Fig. 4A). YY1

overexpression increased the expression of CAMLG in CCLP1 and QBC939 cells, whereas the other

putative target expression levels were not affected (Fig. 4B). As expected, decreasing the levels of YY1

inhibited CAMLG expression (Fig. 4C). TCGA data revealed elevated levels of CAMLG in CCA

samples (Fig. S6A). Furthermore, we identified positive correlations among circ-CCAC1, YY1, and

CAMLG expression levels (Fig. 4D and S6B-C). However, data from TCGA failed to indicate a positive

correlation between YY1 and CAMLG (Fig. S6D). Immunoblots confirmed the positive correlation

between the expression of YY1 and CAMLG in CCA cells (Fig. 4E). YY1 was predicted to bind to the

promoter of CAMLG. ChIP-qPCR uncovered significant YY1-binding activity on the endogenous

CAMLG promoter region around −128 to −117 (Fig. 4F). Luciferase reporter assays also indicated the

binding ability between YY1 and CAMLG promoter at the predicted binding site (Fig. 4G).

CAMLG expression was inhibited in CCLP1 and QBC939 cells transfected with si-CAMLG as

compared to cells transfected with si-NC (Fig. S6E) Cell viability was significantly lower in CCLP1 and

QBC939 cells depleted of CAMLG (Fig. S6F). Moreover, silencing of CAMLG enhanced the rate of cell

apoptosis (Fig. S6G). Transwell assays revealed that the number of invasive cells was remarkably

decreased in CCLP1 and QBC939 cells transfected with si-CAMLG compared to cells transfected with

si-NC (Fig. S6H).

Circ-CCAC1 enters HUVECs via EVs to disrupt vascular endothelial barriers and induce

angiogenesis

EVs were isolated from cell-conditioned medium by differential ultracentrifugation and visualized

by TEM (Fig. S7A). NanoSight indicated that the average particle size of CCLP1-EVs was 88.6 ± 35.3

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 nm (Fig. S7B). The presence of specific exosomal markers was confirmed by immunoblotting (Fig.
Accepted Article
S7C). To investigate the target cell of exosomal circ-CCAC1, Huh-28 cells and HUVECs were treated

with DiI-stained EVs. After 6 h treatment, DiI rapidly entered the receptor cells and localized in the

cytoplasm. After 24 h, the labeled EVs were fully phagocytosed by Huh-28 cells and HUVECs (Fig.

5A). This indicated that circ-CCAC1 is transferred to Huh-28 cells and HUVECs via EVs. Fig. 5B shows

that EVs from HIBECs and CCLP1 cells could not regulate the expression of circ-CCAC1 in Huh-28

cells. However, after 12 and 24 h of treatment with CCLP1-EVs, circ-CCAC1 levels increased in

HUVECs (Fig. 5B). Compared to EVs from control cells, those isolated from

circ-CCAC1-overexpressing CCLP1 cells showed increased levels of circ-CCAC1 in HUVECs (Fig.

5C). However, EVs derived from circ-CCAC1-depleted CCLP1 cells did not increase circ-CCLP1 levels

in HUVECs (Fig. 5C). To determine the impact of exosomal circ-CCAC1 on vascular permeability,

rhodamine leakage and Huh-28 trans-endothelial invasion assays were performed. HUVEC

monolayers exhibited increased permeability to rhodamine and GFP+-Huh-28 cells after exposure to

EVs derived from CCLP/Vector cells compared to PBS-treated cells. More rhodamine and Huh-28 cells

passed through the Transwell chambers upon incubation with CCLP1-EVs/circ-CCAC1 compared to

those incubated with CCLP1-EVs/Vector. Furthermore, circ-CCAC1 silencing in EVs reduced HUVEC

permeability (Fig. 5D-E). We observed more tubes and vascular sprouts in cells/aortic rings incubated

with CCLP1-EVs/Vector than in control cells/aortic rings. CCLP1-EVs/circ-CCAC1 cells further

enhanced the number of tubes and vascular sprouts. As expected, a reduction in exosomal

circ-CCAC1 inhibited angiogenesis (Fig. 5F-G). Furthermore, we detected the relative expression of

circ-CCAC1 and its localization in 57 pairs of iCCA tissues/adjacent normal tissues by ISH. Higher

circ-CCAC1 expression in the cytoplasm of tumor cells and vascular endothelial cells was observed

(Fig. S8).

Circ-CCAC1 increases endothelial monolayer cell permeability by translocating EZH2 to

modulate SH3GL2/ZO-1/Occludin signaling

ZO-1 and Occludin expression in HUVECs was inversely proportional to treatment with exosomal

circ-CCLP1 (Fig. 6A-B). Next, we evaluated the change in YY1 and CAMLG expression by circ-CCAC1

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 in HUVECs. Interestingly, there was no significant modulation of YY1 and CAMLG expression after
Accepted Article
circ-CCAC1 silencing (Fig. S9A). RPISeq was used to predict the interacting RNA-binding proteins of

circ-CCAC1 and the data showed that circ-CCAC1 potentially bound to EZH2, DNMT1, and STAU1

(RF and SVM scores >0.5; Fig. S9B). There was a strong physical interaction between circ-CCAC1

and EZH2, as determined by RIP (Fig. 6C). Binding between U1 and SNRNP70 was used as a positive

control (Fig. S9C). EZH2 was pulled down by a circ-CCAC1 sense RNA probe but not by an antisense

RNA probe. (Fig. S9D). The circ-CCAC1-EZH2 interaction sequestered EZH2 in the cytoplasm,

thereby preventing its nuclear translocation (Fig. 6D). Circ-CCAC1 was primarily localized to the

cytoplasm, which was not influenced by the level of exosomal circ-CCAC1 (Fig. S9E). Increasing

exosomal circ-CCAC1 allowed more EZH2 to be sequestered in the cytoplasm (Fig. 6E). The overall

expression of EZH2 was not affected by circ-CCAC1 levels (Fig. 6E). SH3GL2 is a negative regulator

of ZO-1 and Occludin in endothelial monolayer cells(17,18). We found a positive correlation between

SH3GL2 expression in HUVECs and the levels of treated exosomal circ-CCAC1 (Fig. 6E). EZH2

silencing induced SH3GL2 upregulation (Fig. S9F-G). Moreover, EZH2 directly bound to the promoter

of SH3GL2 and induced H3K27 trimethylation. Increasing exosomal circ-CCAC1 in HUVECs impaired

this binding ability and inhibited trimethylation (Fig. 6F). A rescue assay was performed to evaluate

whether the alterations in ZO-1 and Occludin were EZH2/SH3GL2-dependent. SH3GL2 expression

was inversely correlated with ZO-1 and Occludin levels in HUVECs (Fig. 6G). Cells transfected with

EZH2 vector or si-SH3GL2 showed partial reversal of the reduced ZO-1 and Occludin levels caused by

exosomal circ-CCAC1 (Fig. 6G). For CCA cells, the distribution of circ-CCAC1 was not affected by

circ-CCAC1 levels (Fig. S9H). Overexpression/knockdown of circ-CCAC1 did not translocate EZH2 in

CCA cells, suggesting a cell-specific mechanism employed by circ-CCAC1 (Fig. S9I).

Higher levels of circ-CCAC1 in circulating EVs and cells accelerate CCA tumorigenesis and

metastasis in vivo

We generated mouse xenograft and lung and liver metastases models to understand the role of

circ-CCAC1 in vivo. PBS or indicated EVs (10 μg) were injected into nude mice via the tail vein on

alternate days for 2 weeks. Next, CCLP1 cells stably transfected with sh-NC or sh-circ-CCAC1-1 were

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 implanted subcutaneously into mice. The xenografts were harvested 3 weeks after recording (Fig. 7A).
Accepted Article
The tumor volume/weight of sh-circ-CCAC1-1+PBS mice was significantly reduced compared to

sh-NC+PBS mice. Mice pre-treated with EVs from CCLP1/Vector or CCLP1/circ-CCAC1 exhibited a

partial reversal in their decrease in tumor volume/weight (Fig. 7B-C). Ki67 antibody staining revealed a

similar trend in cell growth (Fig. 7D). At 5 weeks post-inoculation, the mice were imaged to visualize the

lung metastases. Reduced fluorescence intensity and lung metastases were observed in mice injected

with circ-CCAC1-depleted CCLP1 cells; this phenotype was partially reversed in EVs pre-treated mice

(Fig. 7E-F). In the liver metastasis assay, circ-CCAC1-depleted cells significantly attenuated liver

metastasis. Moreover, fluorescence in the liver/spleen and sites of metastasis was positively correlated

with the level of pre-treated exosomal circ-CCAC1 (Fig. 7G-H). We further evaluated whether

exosomal circ-CCAC1 regulates vascular permeability in vivo. A rhodamine leakage in vivo test

showed that increased exosomal circ-CCAC1 enhanced vascular permeability in the lungs and livers of

pre-treated mice (Fig. S10A-B).

Discussion

Emerging evidence indicates that tumor-secreted EVs in the cancer microenvironment mediate

intercellular communication by transferring multiple classes of cargo to recipient cells(9). Here, we

identified the overexpression of a novel circRNA, circ-CCAC1, in both CCA tissues and bile-resident

EVs. Bile-resident exosomal circ-CCAC1 functioned better as a diagnostic biomarker than

serum-derived EVs from CCA patients. Elevated circ-CCAC1 levels correlated with adverse

clinicopathological characteristics in all three subtypes of CCA tissues, indicating that circ-CCAC1

upregulation is a global event during cholangiocarcinogenesis. Multivariate Cox regression analysis

identified circ-CCAC1 as an independent prognostic/recurrent predictor for iCCA.

Functional experiments indicated that downregulated circ-CCAC1 inhibited CCA cell growth,

migration, and invasiveness and triggered apoptosis. RNA-Seq of circ-CCAC1-depleted CCLP1 and

QBC939 cells revealed YY1 as a target of circ-CCAC1. The localization of circRNAs suggests how

they exert their functions. Circ-CCAC1 primarily localized to the cytoplasm rather than the nucleus,

suggesting its mechanism in post-transcriptional gene regulation. The ceRNA hypothesis states that

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 circRNA, acting as a ceRNA sponge, interacts with miRNAs to suppress the biological functions of
Accepted Article
miRNAs(20) and circRNA-miRNA regulatory networks are important in cancer development, including

CCA(8,16). RIP and luciferase reporter assays confirmed that the regulatory network of circ-CCAC1 to

YY1 was mediated by miR-514a-5p. MiR-514a-5p has been reported to act as a tumor suppressor in

several malignancies. For example, miR-514a-5p inhibits cell progression by targeting the ATP-binding

cassette subfamily in ovarian cancer(21). In this study, rescue assays further demonstrated that

miR-514a-5p/YY1 signaling was modulated by circ-CCAC1. YY1 belongs to the GLI-Kruppel family,

which activates or inactivates gene expression, depending on interacting partners, promoter context,

and chromatin structure. YY1 might be involved in the transcriptional control of ~10% of all mammalian

genes(22). YY1 is upregulated in various malignancies and correlates with poor prognoses in several

malignancies(23). This is the first study on the oncogenic role of YY1 in CCA. ChIP-seq was used to

investigate the transcriptions that might be regulated by YY1. CAMLG was identified as a putative

target of YY1, and significant YY1-binding activity on the endogenous CAMLG promoter region was

observed. CAMLG was originally identified as a binding protein that interacts with cyclophilin B to

facilitate calcium-dependent activation of NF-AT in lymphocytes(24). Structurally, the C-terminus of

CAMLG is embedded in the cell membrane, with the majority facing the cytoplasm and binding to

membrane-bound receptors, including TACI, EGFR, mucin 1, and GABA receptor, thereby facilitating

cancer progression(25). For instance, CAMLG promotes the growth of breast cancer cells by regulating

prolactin receptor signaling(26). In this study, YY1-activated CAMLG was found to play an important role

in facilitating CCA progression, clarifying the YY1 mechanisms in human cancers. Collectively,

circ-CCAC1 promotes CCA progression by sponging miR-514a-5p to elevate YY1 expression, further

activating CAMLG expression, thereby emphasizing the importance of the

circ-CCAC1/miR-514a-5p/YY1/CAMLG axis in CCA (Fig. 8).

On one hand, several studies have demonstrated that highly invasive cells transfer this property to

cells with low invasiveness via EVs, thereby promoting metastasis to distant organs(9,10). On the other

hand, tumor-secreted EVs facilitate the formation of a premetastatic niche to offer a supportive

microenvironment to disseminate cancer cells by inducing immunosuppression, angiogenesis, or

vascular leakage(27). To determine the target cell of exosomal circ-CCAC1, Huh-28 cells (with low

This article is protected by copyright. All rights reserved

 circ-CCAC1 expression) and HUVECs were co-incubated with EVs derived from HIBECs or CCLP1
Accepted Article
cells. Interestingly, circ-CCAC1 expression was only increased in HUVECs treated with

CCLP1-derived EVs, suggesting a vascular endothelial cell remodeling function of circ-CCAC1. Further

in vitro experiments indicated that circ-CCAC1 enters endothelial cells via EVs to destroy vascular

endothelial barriers and induce angiogenesis. Although a premetastatic niche is important in

tumorigenesis and cell metastasis(28,29), mechanisms that induce niche formation are still largely

unclear. A previous study demonstrated that colorectal cancer cell-released exosomal miR-25-3p leads

to vascular permeability to facilitate metastasis to distant organs(30). Here, we found that exosomal

circ-CCAC1 promotes cell progression by inducing angiogenesis and increasing endothelial monolayer

permeability. Therefore, targeting the “vascular niche” microenvironment might be a promising strategy

to combat tumorigenesis and cell metastasis.

In HUVECs, we found that circ-CCAC1 remarkably attenuated the expression of ZO-1 and

Occludin, which are intercellular junction proteins controlling endothelial cell permeability(31).

Interestingly, YY1 and CAMLG levels were not significantly affected by circ-CCAC1 in HUVECs,

suggesting a cell-specific mechanism of circ-CCAC1. To clarify the specific mechanism of circ-CCAC1

in regulating ZO-1 and Occludin, the interaction between circ-CCAC1 and RNA-binding proteins was

predicted by RPISeq. We found that circ-CCAC1 strongly bound to EZH2, thereby restricting its nuclear

localization in HUVECs. SH3GL2, positively regulated by circ-CCAC1, is a negative regulator of ZO-1

and Occludin that regulates blood–brain barrier permeability(17,18). We previously reported that EZH2, a

core subunit of PRC2, binds to the promoter region of KLF2 and LATS2 and mediates H3K27

trimethylation modification, thereby inhibiting their transcription in CCA(19). EZH2 regulated SH3GL2

expression by this regulation pattern; it directly bound to the SH3GL2 promoter and induced H3K27

trimethylation to suppress SH3GL2 expression in HUVECs. Collectively, circ-CCAC1 was transferred

to vascular endothelial cells via CCA-secreted EVs. Circ-CCAC1 sequestered EZH2 in the cytoplasm

without affecting its overall expression. SH3GL2 expression increased without EZH2-mediated

promoter H3K27 trimethylation, thereby inhibiting ZO-1 and Occludin expression to disrupt the integrity

of vascular endothelial barriers (Fig. 8). Tumor growth can be affected by cancer cell characteristics,

tumor microenvironment, nutritional factors, etc. The results of the animal study showed that the tumor

This article is protected by copyright. All rights reserved

 growth/metastasis inhibition role of decreased circ-CCAC1 in CCA cells could not be totally reversed
Accepted Article
by exosomal circ-CCAC1. This phenomenon indicated that upregulated circ-CCAC1 in CCA cells is

crucial in tumor growth, and circ-CCAC1 transferred to vascular endothelial cells executes a

collaborative function in the tumor microenvironment to promote tumor progression.

In conclusion, we identified upregulation of circ-CCAC1 in CCA tissues and bile-resident EVs.

Circ-CCAC1 promoted CCA progression through miR-514a-5p/YY1/CAMLG signaling. Additionally,

CCA-delivered exosomal circ-CCAC1 was transferred to endothelial cells to disrupt endothelial barriers

and induce angiogenesis. Mechanistically, circ-CCAC1 increased cell permeability by sequestering

EZH2 in the cytoplasm, thereby increasing SH3GL2 expression to suppress intercellular junction

proteins. We propose that circ-CCAC1 might play a vital role in CCA progression; therefore,

suppressing circ-CCAC1 expression or blocking the transmission of exosomal circ-CCAC1 might be a

novel therapeutic strategy for CCA.

References

1. Razumilava N, Gores GJ. Cholangiocarcinoma. Lancet 2014;383:2168-2179.

2. Patel T. New insights into the molecular pathogenesis of intrahepatic cholangiocarcinoma. J

Gastroenterol 2014;49:165-172.

3. Skipworth JR, Olde Damink SW, Imber C, Bridgewater J, Pereira SP, Malagó M. Review article:

surgical, neo-adjuvant and adjuvant management strategies in biliary tract cancer. Aliment Pharmacol

Ther 2011;34:1063-1078.

4. Meng S, Zhou H, Feng Z, Xu Z, Tang Y, Li P, Wu M. CircRNA: functions and properties of a novel

potential biomarker for cancer. Mol Cancer 2017;16:94.

5. Xu Y, Yao Y, Zhong X, Leng K, Qin W, Qu L, Cui Y, et al. Downregulated circular RNA

hsa_circ_0001649 regulates proliferation, migration and invasion in cholangiocarcinoma cells.

Biochem Biophys Res Commun 2018;496:455-461.

6. Tan WL, Lim BT, Anene-Nzelu CG, Ackers-Johnson M, Dashi A, See K, Tiang Z, et al. A landscape

of circular RNA expression in the human heart. Cardiovasc Res 2017;113:298-309.

7. Zhao Y, Alexandrov PN, Jaber V, Lukiw WJ. Deficiency in the ubiquitin conjugating enzyme UBE2A

This article is protected by copyright. All rights reserved

 in Alzheimer’s disease (AD) is linked to deficits in a natural circular miRNA-7 sponge (circRNA; ciRS-7).
Accepted Article
Genes (Basel) 2016;7.

8. Li LJ, Zhao W, Tao SS, Leng RX, Fan YG, Pan HF, Ye DQ. Competitive endogenous RNA network:

potential implication for systemic lupus erythematosus. Expert Opin Ther Targets 2017;21:639-648.

9. James R. Edgar. Q&A: What are exosomes, exactly? BMC Biology 2016;14:46.

10. Liu Y, Gu Y, Cao X. The exosomes in tumor immunity. Oncoimmunology 2015;4:e1027472.

11. Zhang H, Jiang L, Hou J, Zhong S, Zhu LP, Wang DD, Zhou SY, et al. Exosome: a novel mediator

in drug resistance of cancer cells. Epigenomics 2018;10:1499-1509.

12. Umezu T, Ohyashiki K, Kuroda M, Ohyashiki JH. Leukemia cell to endothelial cell communication

via exosomal miRNAs. Oncogene 2013;32:2747-2755.

13. Cappello F, Logozzi M, Campanella C, Bavisotto CC, Marcilla A, Properzi F, Fais S. Exosome

levels in human body fluids: a tumor marker by themselves? Eur J Pharm Sci 2017;96:93-98.

14. Bao C, Lyu D, Huang S. Circular RNA expands its territory. Mol Cell Oncol 2016;3:e1084443.

15. Huang X, He M, Huang S, Lin R, Zhan M, Yang D, et al. Circular RNA circERBB2 promotes

gallbladder cancer progression by regulating PA2G4-dependent rDNA transcription. Mol Cancer

2019;18:166.

16. Li X, He M, Guo J, Cao T. Upregulation of circular RNA circ-ERBB2 predicts unfavorable prognosis

and facilitates the progression of gastric cancer via miR-503/CACUL1 and miR-637/MMP-19 signaling.

Biochem Biophys Res Commun 2019;511:926-930.

17. Liu W, Wang P, Shang C, Chen L, Cai H, Ma J, et al. Endophilin-1 regulates blood-brain barrier

permeability by controlling ZO-1 and occludin expression via the EGFR-ERK1/2 pathway. Brain Res

2014;1573:17-26.

18. Zhu L, Lin M, Ma J, Liu W, Gao L, Wei S, et al. The role of LINC00094/miR-224-5p

(miR-497-5p)/Endophilin-1 axis in Memantine mediated protective effects on blood-brain barrier in AD

microenvironment. J Cell Mol Med 2019;23:3280-3292.

19. Xu Y, Yao Y, Jiang X, Zhong X, Wang Z, Li C, Kang P, et al. SP1-induced upregulation of lncRNA

SPRY4-IT1 exerts oncogenic properties by scaffolding EZH2/LSD1/DNMT1 and sponging miR-101-3p

in cholangiocarcinoma. J Exp Clin Cancer Res 2018;37:81.

This article is protected by copyright. All rights reserved

 20. Cui X, Wang J, Guo Z, Li M, Li M, Liu S, et al. Emerging function and potential diagnostic value of
Accepted Article
circular RNAs in cancer. Mol Cancer 2018;17:123.

21. Xiao S, Zhang M, Liu C, Wang D. MiR-514 attenuates proliferation and increases chemoresistance

by targeting ATP binding cassette subfamily in ovarian cancer. Mol Genet Genomics

2018;293:1159-1167.

22. Shi Y, Lee JS, Galvin KM. Everything you have ever wanted to know about Yin Yang 1. Biochim

Biophys Acta 1997;1332:F49-66.

23. Zaravinos A, Spandidos DA. Yin yang 1 expression in human tumors. Cell Cycle 2010;9:512-522.

24. Bram RJ, Crabtree GR. Calcium signalling in T cells stimulated by a cyclophilin B-binding protein.

Nature 1994;371:355-358.

25. Tran DD, Russell HR, Sutor SL, van Deursen J, Bram RJ. CAML is required for efficient EGF

receptor recycling. Dev Cell 2003;5:245-256.

26. Lim JH, Kim TY, Kim WH, Park JW. CAML promotes prolactin-dependent proliferation of breast

cancer cells by facilitating prolactin receptor signaling pathways. Breast Cancer Res Treat

2011;130:19-27.

27. Zhang H, Deng T, Liu R, Bai M, Zhou L, Wang X, Li S, et al. Exosome-delivered EGFR regulates

liver microenvironment to promote gastric cancer liver metastasis. Nat Commun 2017;8:15016.

28. Sleeman JP. The lymph node pre-metastatic niche. J Mol Med (Berl) 2015;93:1173-1184.

29. Fong MY, Zhou W, Liu L, Alontaga AY, Chandra M, Ashby J, Chow A, et al. Breast-cancer-secreted

miR-122 reprograms glucose metabolism in premetastatic niche to promote metastasis. Nat Cell Biol

2015;17:183-194.

30. Zeng Z, Li Y, Pan Y, Lan X, Song F, Sun J, Zhou K, et al. Cancer-derived exosomal miR-25-3p

promotes pre-metastatic niche formation by inducing vascular permeability and angiogenesis. Nat

Commun 2018;9:5395.

31. Tsukita S, Furuse M, Itoh M. Molecular architecture of tight junctions: occludin and ZO-1. Soc Gen

Physiol Ser 1997;52:69-76.

Figure legends

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 Fig. 1. Screening and expression of circRNAs in bile EVs and tissues and clinical relevance of
Accepted Article
circ-CCAC1 in CCA patients.

(A) TEM of bile-derived EVs (BEV). Bar = 200 nm; NTA showing the diameter of the particles;

immunoblotting for Alix, TSG101, and CD63 in BEV, total cholangiocyte lysates (TCL), and unextracted

bile (UB). (B) Clustered heatmap showing EV-/tissue-specific circRNAs. (C) qRT-PCR for circ-CCAC1

expression in BEV from eCCA/benign hepatobiliary diseases. (Mann–Whitney U-test). (D) The

diagnostic value of bile/serum-derived exosomal circ-CCAC1, bile-derived exosomal

hsa_circRNA_100364, and serum CA19-9. (E) qRT-PCR for circ-CCAC1 expression in

iCCA/pCCA/dCCA cancerous and adjacent normal tissues (Mann–Whitney U-test). (F) Kaplan–Meier

analysis of OS/DFS in CCA patients according to circ-CCAC1 expression. (G) qRT-PCR for exosomal

circ-CCAC1 expression in CCA cells and HIBECs; qRT-PCR for circ-CCAC1 expression in CCA cells

and HIBECs. *p < 0.05, **p < 0.01.

Fig. 2. Circ-CCAC1 contributes to CCA cell progression in vitro.

(A) Two circ-CCAC1 shRNAs and sh-NC were designed; qRT-PCR for circ-CCAC1 expression after

silencing of circ-CCAC1 in CCLP1 and QBC939 cells. (B) Cell viability detected by CCK-8 after

circ-CCAC1 silencing in CCLP1 and QBC939 cells. (C) Location and expression of Ki67 detected by IF

after silencing of circ-CCAC1 in CCLP1 and QBC939 cells. Bar = 50 μm. (D) Colony-forming ability was

detected by clonogenic assay after silencing of circ-CCAC1 in CCLP1 and QBC939 cells. (E) Cell

migration was detected by wound-healing assay after silencing of circ-CCAC1 in CCLP1 and QBC939

cells. Bar = 200 μm. (F) Cell migration and invasion were detected by Transwell assay after silencing of

circ-CCAC1 in CCLP1 and QBC939 cells. Bar = 100 μm. *p < 0.05, **p < 0.01.

Fig. 3. Circ-CCAC1 sponges miR-514a-5p to upregulate YY1 expression in CCA cells.

(A) RNA-FISH of circ-CCAC1 localization in CCLP1 and QBC939 cells. Bar = 20 μm. (B) Clustered

heatmap showing circ-CCAC1-regulated mRNAs in CCLP1 and QBC939 cells. (C) Venn diagram

showing the number of overlapping miRNAs and predicted binding sites of miRNAs and circ-CCAC1.

(D) Ago2-RNA RIP assay for circ-CCAC1 levels in CCLP1 and QBC939 cells after transfection. (E)

This article is protected by copyright. All rights reserved

 qRT-PCR for the expression of five miRNAs in CCLP1 and QBC939 lysates. (F) Schematic illustration
Accepted Article
of circ-CCAC1-wt and circ-CCAC1-mut luciferase reporter vectors; the binding ability between

circ-CCAC1 and miR-514a-5p was measured by dual-luciferase reporter assay in CCLP1 and QBC939

cells. (G) Schematic illustration showing the YY1 3′-UTR of luciferase reporters; reporter assays

showing the luciferase activity of luc-wt/Luc mut1-4 in CCLP1 and QBC939 cells. *p < 0.05, **p < 0.01.

Fig. 4. YY1 activates CAMLG transcription.

(A) Venn diagram showing the overlapping targets with peak score >1000 and motif score >15. (B-C)

qRT-PCR for target gene expression after YY1 upregulation/downregulation in CCLP1 and QBC939

cells. (D) 3D plot showing correlations of circ-CCAC1, YY1, and CAMLG expression in 25 CCA tissues.

(E) Immunoblotting for CAMLG expression after YY1 upregulation/downregulation in CCLP1 and

QBC939 cells. (F) YY1 binding site prediction in the promoter region of CAMLG; ChIP-qPCR analysis

of YY1 occupancy in the CAMLG promoter in CCLP1 and QBC939 cells. (G) The binding ability

between YY1 and CAMLG promoter was measured by a reporter assay in CCLP1 and QBC939 cells.

*p < 0.05, **p < 0.01.

Fig. 5. Circ-CCAC1 enters HUVECs via EVs to destroy vascular endothelial barriers and induce

angiogenesis.

(A) DiI-labeled EVs co-cultured with Huh-28 and HUVECs. Bar = 20 μm. (B) qRT-PCR for circ-CCAC1

expression in Huh-28 and HUVECs incubated with EVs from HIBECs or CCLP1 cells. (C) qRT-PCR for

circ-CCAC1 expression in HUVECs incubated with indicated EVs. (D-E) Permeability of the HUVECs

to rhodamine and GFP+Huh-28 after exposure to PBS or indicated EVs. Bar = 200 μm. (F-G)

Angiogenesis was detected after incubation with PBS or indicated EVs by tube formation and aortic

ring sprouting assays. Bar = 200 μm. *p < 0.05, **p < 0.01.

Fig. 6. Circ-CCAC1 increases the permeability of endothelial monolayer cells by sequestering

EZH2 in cell cytoplasm to modulate SH3GL2/ZO-1/Occludin signaling.

(A-B) IF and immunoblotting for ZO-1 and Occludin localization and expression in HUVECs incubated

This article is protected by copyright. All rights reserved

 with PBS or indicated EVs. Bar = 100 μm. (C) Circ-CCAC1 levels in immunoprecipitates were
Accepted Article
determined by qRT-PCR. (D) EZH2 distribution was determined after treatment with PBS or

CCLP1-EVs circ-CCAC1 in HUVECs. Bar = 20 μm. (E) Total/nuclear/cytoplasmic EZH2 and total

SH3GL2 expression was detected by immunoblotting after treatment with PBS or indicated EVs in

HUVECs. (F) ChIP-qPCR for EZH2 and H3K27me3 occupancy on the SH3GL2 promoter region after

treatment with PBS or indicated EVs. (G) Immunoblotting for SH3GL2, ZO-1, and Occludin expression

after transfection in HUVECs. *p < 0.05, **p < 0.01.

Fig. 7. Higher circ-CCAC1 levels in circulating EVs and cells accelerate CCA tumorigenesis and

metastasis in vivo.

(A) The xenografts were removed 21 days after injection. (B) Growth curves of subcutaneous tumors.

(C) Tumors were weighed. (D) Ki67 detection by IHC. Bar = 100 μm. (E) Bioluminescence imaging of in

vivo metastatic activity and collected lungs. (F) H&E staining showing metastatic tumors in lungs. Bar =

100 μm. (G) Bioluminescence imaging of in vivo metastatic activity and collected livers and spleens.

(H) H&E staining showing metastatic tumors in livers. Bar = 100 μm. *p < 0.05, **p < 0.01.

Fig. 8. Summary of the regulatory network and mechanisms of circ-CCAC1 in CCA and

HUVECs.

This article is protected by copyright. All rights reserved

A Bile EVs B Bile EVs Tissue samples
eCCA Normal eCCA Normal

7E+6

6E+6
Particles/ml

5E+6

4E+6

3E+6

2E+6 High

1E+6

0E+0
1 10 100 1000 10000
Low
Diameter (nm)
C D E
1.0
Relative circ-CCAC1/hsa_circRNA

circ-CCAC1 circ_100364 n=236

Relative circ-CCAC1 expression

120 60
0.8
100 50
_100364 expression

40
80
CCA Normal 60
30
20 n=40

Sensitivity%
60 Bile EVs-circ-CCAC1 20
BEV TCL UB BEV TCL UB 0.6
AUC=0.857 n=40
Serum EVs-circ-CCAC1 15
Alix 40 AUC=0.759
Serum CA19-9 10
0.4
CD63 20
AUC=0.757
Bile EVs-hsa_circRNA_100364 5
AUC=0.619
TSG101 0
0.2
Bile EVs-circ-CCAC1+Serum CA19-9 0
AUC=0.914
s

A
al

al

al
EV

EV

EV

EV

EV

EV

Serum EVs-circ-CCAC1+Serum CA19-9

C
m

m
iC

pC

dC
or

or

or
AUC=0.862
m

le

le

le

le

N
bi

bi

bi

bi
ru

ru

Reference line
se

se

A
al

al
C

0.0
m

m
A
al

eC

eC

0.0 0.2 0.4 0.6 0.8 1.0

or

or
C
m

eC

N
or

1-Specificity%
F G
N

1.0 1.0
CCA cell EVs CCA cells
Relative circ-CCAC1 expression

Relative circ-CCAC1 expression

Disease-free survival

0.8 0.8 iCCA eCCA

Overall survival

50 iCCA eCCA 12
40 10
0.6 0.6
Low circ-CCAC1 expression
30
30 8
Low circ-CCAC1 expression 25
0.4 0.4
6
20
High circ-CCAC1 expression
15 4
High circ-CCAC1 expression
0.2 0.2 10
2
5
0 0
0.0 p<0.001 0.0 p<0.001
EC

T1

EC

T1

9
-2

81

LP

93
B

-2

81

LP

93
B

B
C

C
IB

R
uh

IB

R
uh

M
-9

-9

C
C
uC

C
uC
.00 20.00 40.00 60.00 80.00 100.00 120.00
K

.00 20.00 40.00 60.00 80.00 100.00 120.00

H

K
H

B
C

C
H

C
H
Q
H

Q
C

C
Months Months
C

C
H

H
 CCLP1 CCLP1

F
A

D
C
QBC939 CCLP1
sh-circRNA-2 sh-circRNA-1 sh-NC
Number of migrated cells
per field sh-circ-CCAC1-1

sh-NC

0
30
60
90
120
150
sh-NC
sh-NC
sh
-c sh
irc -N

DAPI
sh -C C
C
-c A
circ-CCAC1

irc C
-C 1-
C 1
A
sh-NC

CCLP1
1-
2
sh
sh-circ-CCAC1-2

-c sh
irc -N

Ki67
sh -C C
C
CCLP1
-c A Relative circ-CCAC1 expression
irc C
-C 1-
C 1
0.0
0.2
0.4
0.6
0.8
1.0
1.2

A M
C

QBC939
1- sh oc
2 -c k
sh
irc
sh -C -NC
-c C
irc AC
-C 1

sh-circRNA-1 sh-circRNA-2
sh-circRNA-1 sh-circRNA-2
C -1
Merge
CCLP1

A
QBC939 QBC939 C
1-
2

sh-circRNA-1 sh-circRNA-2
M
sh oc
-c k

Cell invasion
Numbers of invaded cells s

Cell migration
ir h
per field sh c-C -NC
C
-c
Relative colony numbers (%)

sh-NC
sh-NC
irc AC
1

0
25
50
75
100
-C
DAPI

sh
C -1
-c sh A
QBC939

0
30
60
90
120
irc -N sh C
sh -C C -c sh 1-
2
C irc -N
-c A
irc C sh -C C
-C 1- C
1 -c A
C irc C
A 1-
C -C

CCLP1
C 1
B

1-
2 A
C Cell viability (OD450)
CCLP1
1-
2
Ki67

sh
0.0
0.4
0.8
1.2
1.6

-c sh
0

QBC939

irc -N sh
sh -C C -c sh
C irc -N
-c A
irc C sh -C C
-C 1- C
1 -c A
C irc C
A
24

1-
C -C 1

QBC939
C
sh-NC

1-
2 A
C
QBC939

1-
2
Merge
48

sh-circRNA-1 sh-circRNA-2
sh-circRNA-1 sh-circRNA-2
sh-circ-CCAC1-2
sh-circ-CCAC1-1

E
Time (h)
CCLP1

QBC939 CCLP1
72

48h
0h
32h
0h

Wound closure area Ki67 positive cells (%)

96

(relative to sh-NC)
0
20
40
60
80

sh

0.0
0.3
0.6
0.9
1.2
sh -c sh
irc -N
sh-NC

-c sh -C C Cell viability (OD450)

irc -N sh C
-C C -c A
C
0.0
0.4
0.8
1.2
1.6

sh C irc
-c A 1-
0

C -C 1
irc
1- C
-C 1 A
C C
CCLP1

A 1-
2
C CCLP1
1-
2
24

sh
-c sh
sh-NC

sh irc -N
-c sh -C C
irc -N sh C
-C C -c A
sh C irc C
-c A 1-
48

C -C 1
irc
1- C
-C 1 A
sh-circ-CCAC1-2
sh-circ-CCAC1-1

C C
Time (h)

A 1-
QBC939

C 2
QBC939

QBC939

1-
2
72
96

sh-circRNA-1sh-circRNA-2
ACCLP1 DAPI circ-CCAC1 Merge B CCLP1 QBC939

PAGR1 METTL3
LRRC41 PLA2G6
RPL13A SCYL3
YY1 LRDSAM1
CCDC58 FEM1A
CPEB4 TTLL12
DPAGT1 PSMD8
QBC939

TXNDC15 YY1
CRADD MED25
STAMBP KLF5
TMEM17 ZNF274
NARF SLC25A44
TSC1 TOPORS
GNB2L1 NDUFB5
C ZBED5
USE1
ZBTB17
MAP1LC3B
starbase 2.0 TargetScan EMG1 WDR7 High

SNAP25 SLC39A7
CNPY4 DCAF6

miR-182
HOOK2 USP31
mi
Low
0 152
R- NC KD NC KD
36
D E oligo probe
mi

-5p
R- 19
5 13 -5p Anti-IgG
4a circ-CCAC1 probe
43 -5p
2 35 -3p 51

miRNA expression
R- Anti-Ago2
mi 10 6 CCLP1 QBC939
29 56 circ-CCAC1 CCLP1 QBC939
1 3

Fold Enrichment
8 5
mi

4
p

R-

1 6
6-5

36

11 1 3
19
4
-67

6 1
a-5

4
miR

2
p

Relative
2
circBank miRmap 1

0 0

m 514 5p
m 134 5p
m 361 p
-6 -5p

m -18 p
m 514 p
m -13 5p
m 361 3p
-6 -5p

5p
F

1-

1-
-N

-N

-3

iR -5

-5
-
-

-
-

6-
C

iR 2
iR a
iR 3
iR 9
m 746

iR 2
iR a
iR 43
iR 9
sh

sh
A

m -18

74
C

C
hRluc hluc+

-C

-C
circ-CCAC1

iR

-
-
-

-
irc

irc

m
-c

-c
sh

sh
G Position
Luc wt
Luc mut1
Luc mut2
Luc mut3
Luc mut4
Luc mut2+3
Relative luciferase activity

1.5 CCLP1 QBC939

CCLP1 QBC939
mimics-NC mimics-NC
1.0 miR-514a-5p mimics miR-514a-5p mimics
Relative luciferase activity
Relative luciferase activity

1.5 1.5
0.5

1.0 1.0
0.0

circ-CCAC1 wt 0.5 0.5

circ-CCAC1 mut
miR-514a-5p 0.0
0.0
mimics-NC wt mut1 mut2 mut3 mut4 mut2+mut3 wt mut1 mut2 mut3 mut4 mut2+mut3
A Peak Score > 1000 ChIP-seq B CCLP1
Vector
YY1 vector
Position ID Sequence Motif Name Strand Peak Score Motif Score Gene

Relative mRNA expression

20 QBC939

140 peaks525 CAAGATGGCGGC YY1 + 4621 15.393 PCIF1 15

peaks430 CAAGATGGCGGC YY1 + 3750 15.393 GLTSCR2 10

6 peaks408 CAAGATGGCGGC YY1 + 2931 15.393 UBA52

6
peaks132 CAAGATGGCGGC YY1 + 2135 15.393 TAF5 4
48 peaks252 CAAGATGGCGGC YY1 + 1249 15.393 MORF4L1 2

peaks628 CAAGATGGCGGC YY1 + 1217 15.393 CAMLG 0

G PC Y1
SC F1
B 2
M T 52
R 5
A 1
LG
G PC Y1
S F1
B 2
M T 52
R 5
A 1
LG
U R

O AF
C 4L

U CR

O AF
C 4L
Motif Score > 15

A
LT I

LT I
Y

Y
M

M
F

F
C D E

or
ct

1
ve
r

YY
YY1

to

N
1
c

Relative YY1/CAMLG expression

sh-NC

-
CAMLG

YY
Ve

sh

sh
Relative mRNA expression

1.5 sh-YY1 4
YY1 CCLP1 QBC939
CCLP1 QBC939

CCLP1
3
1.0 CAMLG
2
GAPDH
0.5
YY1 1

QBC939
0.0 CAMLG 0
G PC Y1
SC 1
B 2
M T 52
R 5
A 1
LG
G PC Y1
S 1
B 2
M T 52
R 5
A 1
LG

- C
1 ec 1

- C
1 ec 1

-Y C
YY V Y1

-Y C
Y1
s ct r
shh-Nor

s ct r
shh-Nor

s ct r
shh-Nor

s ct r
shh-Nor
LT IF
U R

O AF
C F4L

LT IF
U CR

O AF
C 4L

ve to

ve to

ve to

ve to
YY V YY

YY V YY
A

A
Y

Y
M

1 ec

1 ec
F

GAPDH

YY V
F IgG G Vector

Relative luciferase activity

5 CCLP1 YY1
Percentage of input (%)

25 YY1
YY1 QBC939
20 4

15 3

10 2
-128 to -117
5 1
Site CAMLG
0 0
CCLP1 QBC939 wt mut wt mut
CAAGATGGCGGC
A CCLP1
Huh-28 Huh-28 HUVEC
DAPI Dil Merge DAPI Dil Merge
Dil

HUVEC 6h

B
Relative circ-CCAC1 expression

24h

Relative circ-CCAC1 expression Relative circ-CCAC1 expression

2.0 PBS 4 PBS
HIBEC-EVs HIBEC-EVs
in Huh-28 cells

n.s.

in HUVEC cells
1.5 CCLP1-EVs n.s. 3 CCLP1-EVs
200 HUVEC monolayer
D

Fluorescence (OD590)
0min
1.0 2
5min
150
10min
0.5 1
30min
100 60min
0.0 0
0h 3h 6h 12h 24h 0h 3h 6h 12h 24h Rhodamine-dectran(~70kDa) n.s.
50

C
Relative circ-CCAC1 expression

0
14 CCLP1-EVs sh-NC 4 CCLP1-EVs sh-NC HUVECs

ll

Vs

1
r
CCLP1-EVs sh-circ-CCAC1-1 CCLP1-EVs sh-circ-CCAC1-1

to
ce

EV

1-
PB

-N
12

-E

A
c

C
in HUVEC cells

sh
1-

Ve
in HUVEC cells

A
EC
N
3

-C
LP

C
s
10

s
IB

-C
rc

EV
EV
C
H

irc
ci
C

1-
1-
8

-c
LP
LP

EV
2

sh
C
C

1-
6

C
C

s
LP

EV
C

1-
4

C
1

LP
C
2

C
0 0 PBS
0h 3h 6h 12h 24h 0h 3h 6h 12h 24h
CCLP1-EVs Vector

E CCLP1-EVs CCLP1-EVs CCLP1-EVs CCLP1-EVs CCLP1-EVs circ-CCAC1-1

PBS CCLP1-EVs sh-NC
Vector circ-CCAC1 sh-NC sh-circ-CCAC1-1
CCLP1-EVs GFP-Huh-28 CCLP1-EVs sh-circ-CCAC1-1
50

Cell numbers per field

Huh-28

HUVECs
40
Serum-free
30
10%FBS
20
PBS
CCLP1-EVs Vector 10
CCLP1-EVs circ-CCAC1-1
F CCLP1-EVs CCLP1-EVs CCLP1-EVs CCLP1-EVs
CCLP1-EVs sh-NC
CCLP1-EVs sh-circ-CCAC1-1
0
Huh-28
PBS
Relative length of tubes

Vector circ-CCAC1 sh-NC sh-circ-CCAC1-1 5

HUVEC

2 PBS
CCLP1-EVs Vector
1 CCLP1-EVs circ-CCAC1-1
CCLP1-EVs sh-NC
0 CCLP1-EVs sh-circ-CCAC1-1
Tube formation

G
Number of microvessels
CCLP1-EVs CCLP1-EVs CCLP1-EVs CCLP1-EVs 30
PBS Vector circ-CCAC1 sh-NC sh-circ-CCAC1-1
20
Aortic ring

10

0
Microvessels sprouts
 E
A

C
Relative circ-CCAC1 RIP
levels vs IgG RIP PBS

GAPDH
SH3GL2
EZH2
LaminB1
GAPDH
EZH2
GAPDH
LaminB1
EZH2
sh-NC
Vector

0
5
10
50
100
100
150
200
250
circ-CCAC1
Ig

CCLP1-EVs
CCLP1-EVs
CCLP1-EVs
CCLP1-EVs
G

sh-circ-CCAC1-1
PBS
EZ
H
CCLP1-EVs 2
Vector D
N
DAPI

M
T1
CCLP1-EVs ST
circ-CCAC1 A
U
1
CCLP1-EVs In
ZO-1

sh-NC pu
t
CCLP1-EVs
sh-circ-CCAC1-1

D
Total Cytoplasmic Nuclear
CCLP1-EVs PBS
MERGE

C circ-CCAC1
C C C
C LP C Relative EZH2/SH3GL2 expression
LP1 L

F
1- C-E P1-
EV C Vs EV

0.0
0.5
1.0
2
4
6
Percentage of input (%) s LP ci s P
sh 1 rc Ve B
-c -E -C ct S

0
10
20
30
C irc Vs CA or
DAPI

C C C
DAPI

-C sh C
C L C C -N 1
LP P1 LP A C
1- C-E 1- C
1-
EV C Vs EV 1

IgG
s LP ci s P
sh 1 rc Ve B

PBS
-c -E -C ct S
C irc Vs CA or
C C C -C sh C
C L C C -N 1
LP P1 LP A C
C
EZH2

1- C-E 1- 1-
EV C Vs EV 1
L
Occludin

s P ci s P

EZH2
sh 1 rc Ve B

CCLP1-EVs sh-NC
CCLP1-EVs Vector
-c -E -C ct S
C i r Vs C o
c- s A r
C C C

CCLP1-EVs circ-CCAC1
C L C C h C1
C -N
LP P1 LP A C
C

CCLP1-EVs sh-circ-CCAC1-1
1- C-E 1- 1-
EV C Vs EV 1
s LP ci s P
sh 1 rc Ve B
-c -E -C ct S

H3K27me3
MERGE
MERGE

irc Vs CA or
Total EZH2

-C sh C
C -N 1
Total SH3GL2

A C
Nuclear EZH2

C
1-
C 1
C
Cytoplasmic EZH2

LP
C 1-
C E
B

LP V C
G

1- s c CL Relative ZO-1/Occludin/SH3GL2 Relative ZO-1/Occludin

EV irc P
expression
GAPDH
Occludin
ZO-1

s -C 1-E expression C
ci C V C CC
C r c A s LP L
C -C C1 ci C
C 1- P1

0.0
0.5
1.0
2
3
4
0.0
0.5
1.0
1.5

LP EV -E
C 1- C +E rc- P LP V P PBS
GAPDH
SH3GL2
Occludin
ZO-1

C E A Z C B
LP V C C H C S 1- CC s c s BS
s 1+ 2 A EV L ir Ve
1- c CL si ve C1 s P1 c-C cto
EV irc P -S c
s -C -E 1 H to sh -E C r
c 3G r PBS -c Vs AC CCLP1-EVs
C irc CA Vs irc s 1
C L2
ZO-1

LP -C C1 ci -C h- Vector
1 C + E
rc P C NC
A
C - A Z -C B C C C
SH3GL2

C E C
Occludin

C C 1-
LP V C 1+ H2 CA S CCLP1-EVs C
LP L 1 CCLP1-EVs
1- s c CL si ve C1
C 1 P1
EV irc P -S c
H to
circ-CCAC1 -E -E
V V P
s -C 1-E 3G r
LP
1- CC s c s BS
circ-CCAC1
ci C V EV L ir Ve
rc AC s L2
-C 1 ci s P1 c-C cto
C +E rc- P
A Z C B
CCLP1-EVs sh -E C r CCLP1-EVs
C H C S
1+ 2 A circ-CCAC1 -c Vs AC
irc s 1 sh-NC
si ve C1
-S c
EZH2 vector -C h-
C NC
H to A
C
ZO-1

3G r 1-
L2 CCLP1-EVs 1 CCLP1-EVs
Occludin

circ-CCAC1 sh-circ-CCAC1-1
si-SH3GL2
 A B 1000
sh-NC+PBS
sh-circ-CCAC1-1+PBS D Ki67

Tumor volume (mm3)

sh-circ-CCAC1-1 +EVs Vector
800 sh-circ-CCAC1-1+EVs circ-CCAC1

+PBS
sh-NC
CCLP1 subcutaneous injection

sh-NC 600

+PBS 400

200

0
sh-circ-CCAC1-1

+PBS 0 3 6 9 12 15 18 21

+PBS
Days
sh-NC+PBS
sh-circ-CCAC1-1+PBS
+EVs Vector sh-circ-CCAC1-1 +EVs Vector
sh-circ-CCAC1-1+EVs circ-CCAC1

sh-circ-CCAC1-1
1200

Tumor weight (mg)

+EVs Vector +EVs circ-CCAC1

1000
+EVs circ-CCAC1
800

E
600

sh-NC sh-circ-CCAC1-1 400

CCLP1 tail vein injection

+PBS +PBS +EVs Vector +EVs circ-CCAC1 200

Lung metastasis

sh-NC+PBS 0
Xenograft
sh-circ-CCAC1-1+PBS
sh-circ-CCAC1-1 +EVs Vector

Lung metastasis nodules

sh-circ-CCAC1-1+EVs circ-CCAC1

(Normalized to sh-NC+PBS)
Bioluminescence intensity
120 50

100 40

80
30
60
20
40
Lung

10
20

0 0
Lung Lung

F sh-NC sh-circ-CCAC1-1

G sh-NC sh-circ-CCAC1-1 +PBS +PBS +EVs Vector +EVs circ-CCAC1

CCLP1 intra-spleen injection

+PBS +PBS +EVs Vector +EVs circ-CCAC1

Liver metastasis

sh-NC+PBS
sh-circ-CCAC1-1+PBS
sh-circ-CCAC1-1 +EVs Vector
sh-circ-CCAC1-1+EVs circ-CCAC1

Liver metastasis nodules

(Normalized to sh-NC+PBS)
Bioluminescence intensity

120 25

100 20

80
15
60
10
40
5
20
Liver

0 0
Liver Liver

H sh-NC
+PBS +PBS
sh-circ-CCAC1-1
+EVs Vector +EVs circ-CCAC1
Spleen
 CCA HUVECs
Cytoplasm Cytoplasm
miR-514a-5p
EZH2
MVB ZO-1
Occludin

EVs EZ
H2 circ-CCAC1
EZ
A H2
N
mR
Y Y1
YY1

CAMLG SH3GL2
circ-CCAC1
EZH2 H3K27
YY1 me3
CAMLG SH3GL2

chr17:37880978-37882106

Exon 23 Exon 24 Exon 25 Exon 26

Nucleus Nucleus
Ex
23

on

circ-CCAC1
on

26
Ex

circ-CCAC1
Ex

565nt
on
25

Exon 24

Cell proliferation Cell apoptosis Cell metastasis Cell permeability