Archives of Biochemistry and Biophysics 661 (2019) 196–202

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Archives of Biochemistry and Biophysics

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CircZFR promotes hepatocellular carcinoma progression through regulating T

miR-3619–5p/CTNNB1 axis and activating Wnt/β-catenin pathway
Aichun Tan, Qiongxuan Li, Lizhang Chen∗
Department of Epidemiology and Health Statistics, School of Public Health, Central South University, Changsha, Hunan, 410078, China

A R T I C LE I N FO A B S T R A C T

Keywords: Circular RNAs (circRNAs) have been discovered to exert essential roles in human cancers, including hepato-
circZFR cellular carcinoma. Although circZFR has been reported to facilitate the growth of papillary thyroid cancer, the
miR-3619–5p role of circZFR in hepatocellular carcinoma (HCC) are largely unknown. In this study, bioinformatics analysis
CTNNB1 showed that circZFR was closely related with hepatocellular carcinoma. We then detect the expression of circZFR
Wnt/β-catenin pathway
in HCC tissues using qRT-PCR. Furthermore, Kaplan-Meier method and log rank test revealed that high ex-
HCC
pression of circZFR was associated with the poor prognosis of patients with HCC. Subsequently, loss-of-function
assay indicated that circZFR knockdown significantly suppressed cell proliferation and epithelial-mesenchymal
transition (EMT) in HCC. In addition, microarray analysis was utilized to identify the differentially expressed
mRNAs in response to circZFR knockdown. Moreover, Gene Ontology (GO) analysis further showed that circZFR
might regulate Wnt/β-catenin signaling pathway. The results were further confirmed by luciferase reporter assay
and western blot assays. Then bioinformatics tools predicted that cicrZFR enhanced the CTNNB1 expression via
sponging miR-3619–5p. In summary, our findings indicated that circZFR may exert carcinogenic role in HCC
through regulating miR-3619–5p/CTNNB1 axis and activating Wnt/β-catenin pathway. These findings may
provide a novel perspective for the treatment of hepatocellular carcinoma.

1. Introduction
As one of the most common malignancies in the world, hepatocel-
lular carcinoma (HCC) with an increasing incidence rate annually
[1–4]. Despite the advancement of newly-developing therapeutic
methods, such as radiofrequency ablation, liver transplantation, and
surgical resection, the prognosis of HCC patients remain unfavorable,
and life expectancy of HCC patients with advanced stage is around six
months [5–7]. Therefore, it is essential to discover the novel diagnostic
biomarkers and therapeutic targets for the treatment of HCC.
In recent years, circular RNAs (circRNAs) have been highly regarded
in human cancers [8–10]. Previous reports indicate that circRNAs can
alter gene expression through sponging microRNAs (miRNAs) [11–13].
Previous study demonstrated that circZFR contributed to the progres-
sion of papillary thyroid cancer by sponging miR-1261/C8orf4 axis
[14]. However, the biological roles of circZFR in hepatocellular carci-
noma have not been fully studied. Our research was designed to in-
vestigate the biological role and underlying mechanisms of circZFR in
HCC.
In the current study, three HCC-related circRNAs were predicted in
accordance with the searching data of circRNA Disease. Then qRT-PCR
was used to detect the expression of the three circRNAs in HCC tissues
and normal tissues. CircZFR was picked to do the following investiga-
tions. The expression of circZFR was detected in HCC tissues and cell
lines. Subsequently, functional assays were conducted to determine the
role of si-circZFR in regulating HCC cell proliferation and epithelial-
mesenchymal transition (EMT). In mechanism, the microarray dataset
was utilized to screen out mRNAs which could be regulated by circZFR.
Moreover, circZFR was found to associate with Wnt/β-catenin pathway
according to GO analysis. The regulatory effect of circZFR on Wnt/β-
catenin signaling was further identified. Furthermore, bioinformatics
tools were used to explore the miRNAs which could bind with circZFR
and CTNNB1. Finally, rescue assays were performed to verify the effect
of circZFR-miR-3619-5p-CTNNB1 axis on HCC progression.
2. Materials and methods
2.1. Samples collection
80 pairs of HCC tissues and paired normal tissues were obtained

∗
Corresponding author. Department of Epidemiology and Health Statistics, School of Public Health, Central South University, No. 238, Mayuanling Lane, Xiangya
Road, Kaifu District, Changsha, Hunan, 410078, China.
E-mail address: LizhangChen667_CZL@163.com (L. Chen).

https://doi.org/10.1016/j.abb.2018.11.020
Received 30 August 2018; Received in revised form 15 November 2018; Accepted 19 November 2018
Available online 20 November 2018
0003-9861/ © 2018 Elsevier Inc. All rights reserved.
A. Tan et al.
from patients diagnosed with HCC at Xiangya Hospital of Central South
University. And another 30 pairs of HCC tissues and paired normal
tissues were also received from Xiangya Hospital of Central South
University. No interferential treatments were performed before the
samples collection. All the samples received immediate dissection and
were put on ice, snap-frozen by the use of liquid nitrogen, and were in
store at −80 °C. The obtaining of all tissue samples were with the
confirmation of histopathological examination. All patients had pro-
vided the written informed consent.
2.2. Cell culture
Four HCC cell lines (MHCC97H, Hep3B, Huh7 and Bel-7402) were
purchased from American Type Culture Collection (Manassas, VA,
USA), and one human normal hepatocyte cell line (L-02) were obtained
from the Chinese Academy of Sciences Cell Bank (Shanghai, China).
Then the cell lines were cultured in Dulbecco's modified Eagle's medium
(DMEM, Gibco BRL Co. Ltd., USA), containing 10% fetal bovine serum,
penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37 °C with 5%
CO2.
2.3. RNA transfection
Small interfering RNAs (siRNAs) targeting the circRNAZFR (si-
circZFR) was designed and synthesized by Shanghai Integrated Biotech
Solutions Co., Ltd. (Shanghai, China). And the sequence of was 5′CAA
ATTTATGCCCAGCCGGCT3′. For si-ZFR, the sequence of the functional
siRNA was TCTCAAGTCTCAAGTCGTCGT. All transfections were con-
ducted by the use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA,
USA) based on the manufacturer's advice. The medium was replaced
with normal medium following transfection for 6 h.
2.4. RNA extraction and qRT-PCR analysis
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad,
CA, USA), and reverse transcription was carried out with PrimeScript
RT reagent Kit (Takara, Japan) in accordance with the manufacturer's
advices. GAPDH was regarded as the endogenous control for circRNA
and mRNA. The relative expression was determined by the 2−ΔΔCt
method.
2.5. CCK-8 assay
Cell viability was observed by Cell Counting Kit-8 (CCK-8; Dojindo
Molecular Technologies, Inc., Kumamoto, Japan) assay. U87MG and
U118 cells were seeded in DMEM with 10% FBS at a density of
5 × 103 cells per well and then cultivated in 96-well plates. 10 μl CCK-8
solution was added into each well after the transfection. The cells were
then cultivated at 37 °C for 2 h. The optical concentration values were
measured at 450 nm using Multiskan Go spectrophotometer (Thermo
Fisher Scientific, Inc.).
2.6. Colony formation assay
After being transfected for 72 h, all cells were digested and then
seeded into 6-well plates at 200 cells in each well. The cells were then
cultured at 37 °C in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) which
Table 1
HCC-related circRNAs from circRNA Disease.
circRNA id circRNA name disease host gene
hsa circ 0072088 circZFR Liver cancer ZFR
hsa circ 0003028 circFUT8 Liver cancer FUT8
hsa circ 0007915 circIPO11 Liver cancer IPO11
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contained 10% fetal bovine serum (FBS; Gibco; Thermo Fisher
Scientific, Inc.) for 21 days. The medium was replace as needed.
Colonies then were fixed with the application of methanol and stained
with 0.5% crystal violet (Sigma-Aldrich). Visible colonies were counted
manually.
2.7. Western blot assay
Total proteins were extracted with the application of RIPA lysis
buffer (Beyotime Biotechnology, China) containing protease inhibitors
(Roche, China), and separated by 10% SDS-PAGE and then transferred
onto a PVDF membrane (Millipore, USA). To block nonspecific binding,
the membranes were incubated with 5% skim milk powder at room
temperature for 1 h. The primary antibodies were prepared in 5%
blocking buffer which had a dilution of 1:1000 and later went through
incubation for 2 h at 4 °C with the membrane. The primary antibodies
were listed as below: anti-β-catenin, anti-MMP7, anti-MMP9 and
GAPDH (Abcam, Cambridge, UK); anti-Vimentin, anti-E-cadherin, anti-
N-cadherin, and anti-C-Myc (Cell Signaling Technology, Mass, USA).
After that, the membranes were cultivated for 2 h at room temperature
with secondary antibody (1:1000) that was marked by horseradish
peroxidase. Protein bands were visualized by the ECL detecting system
(Applygen, Beijing).
2.8. Luciferase reporter assay
TCF wild-type (TOP Flash) or mutated control (FOP Flash) luci-
ferase reporter plasmids were purchased from Upstate Biotechnology
(Lake Placid, NY, USA). Transfection was performed with
Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the man-
ufacturer's protocol. Luciferase activity was measured with the Dual
Luciferase Reporter Assay System (Promega, WI, USA) according to the
manufacturer's manual. The results were normalized to the renilla ac-
tivity.
2.9. Statistical analysis
Statistical analyses were performed using SPSS 18.0 software (SPSS,
Inc., Chicago, IL, USA). Data were presented as the mean ± SD from
over three independent experiments. Student's t-test was used to ana-
lyze the differences between two groups. Multiple comparisons were
analyzed by one-way analysis of variance (ANOVA). Kaplan-Meier
method and log-rank test were utilized to analyze the relation between
the overall survival rate of HCC patients and circZFR expression.
Expression correlations were determined using Spearman's correlation
analysis. P < 0.05 indicated data were statistically significant.
3. Results
3.1. CircZFR was upregulated in HCC tissues and associated with poor
prognosis of HCC patients
Three HCC-related circRNAs (circZFR, circFUT8 and circIPO11)
were found from circRNA Disease (Table 1). Then the expression of
circZFR, circIPO11 and circFUT8 was detected in 30 pairs of HCC tis-
sues and normal tissues using qRT-PCR. Results displayed that only
circZFR was upregulated in HCC tissues (Fig. 1A). Then we further
method expression pattern pubmed
qRT-PCR, microarray up-regulated 28727484
qRT-PCR, microarray up-regulated 28727484
qRT-PCR, microarray up-regulated 28727484
A. Tan et al. Archives of Biochemistry and Biophysics 661 (2019) 196–202

Fig. 1. CircZFR was upregulated in HCC tissues and associated with poor prognosis of HCC patients. (A) The expression of circZFR, circFUT8 and circIPO11
were detected in 30 pairs of HCC tissues and normal tissues using qRT-PCR. (B) The expression of circZFR was detected in 80 pairs of HCC tissues and normal tissues
using qRT-PCR. (C) The expression of circZFR in four HCC cell lines (Hep3B, Huh7, MHCC97H and Bel-7402) and normal cell line (L-02) was detected by qRT-PCR.
(D) Kaplan-Meier method was used to analyze the relationship between circZFR expression and the overall survival of HCC patients. ∗P < 0.05, ∗∗P < 0.01 vs
control group. NS: no significance.

Fig. 2. Knockdown of circZFR suppressed cell proliferation and EMT process in HCC (A). The transfection efficiency was determined in Hep3B and Huh7 cell
lines transfected with si-circZFR or si-ZFR. (B) CCK-8 assay and (C) Colony formation assay were performed to investigate the effect of si-circZFR on the proliferative
ability of Hep3B and Huh7 cell lines. (D) Western blot assay was conducted to detect the levels of EMT-related proteins in Hep3B and Huh7 cell lines transfected with
si-circZFR. ∗P < 0.05, ∗∗P < 0.01 vs control group.

detected the expression of circZFR in another 80 pairs of HCC tissues
and normal tissues. qRT-PCR assay showed that circZFR was sig-
nificantly upregulated in HCC tissues (Fig. 1B). Moreover, we examined
the expression of circZFR in four HCC cell lines (Hep3B, Huh7,
MHCC97H and Bel-7402) and one normal liver cell line (L-02).
Similarly, qRT-PCR indicated that circZFR was highly expressed in HCC
cell lines, especially in Hep3B and Huh7 cells (Fig. 1C). Then, total 80
HCC tissues were divided into two group according to the mean ex-
pression of circZFR. Kaplan-Meier analysis demonstrated that high ex-
pression of circZFR was associated with low overall survival rate of HCC

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Fig. 3. Downregulation of circZFR inhibited the Wnt/β-catenin signaling in HCC (A) Cluster heat map showed the differentially expressed mRNAs over two fold
change in HCC samples after circZFR knockdown. (B) GO analysis revealed the enriched pathways and biological processes. (C) qRT-PCR was used to detect the
expression of downstream target genes of Wnt/β-catenin pathway (c-Myc, β-catenin, MMP-7 and MMP-9). (D) The protein level of c-Myc, β-catenin, MMP-7 and
MMP-9 were detected by western blot assay in Hep3B and Huh7 cell lines transfected with si-circZFR. GAPDH was used as negative control. (E) The luciferase
activities of TOP flash and FOP flash were measured in Hep3B and Huh7 cells transfected with si-circZFR. ∗∗P < 0.01 vs control group.

patients (Fig. 1D). In summary, all these results indicated that circZFR
was upregulated in HCC tissues and associated with the poor prognosis
of HCC patients.
3.2. Knockdown of circZFR suppressed cell proliferation and EMT process
in HCC
To determine the biological roles of circZFR in HCC, small inter-
fering RNA against circZFR (si-circZFR) and negative control siRNA (si-
NC) were transfected into Hep3B and Huh7 cell lines. qRT-PCR assay
showed that si-circZFR significantly decreased the relative expression
level of circZFR but not ZFR (Fig. 2A). Similarly, si-ZFR efficiently
decreased the expression level of ZFR but not circZFR. Subsequently,
CCK-8 and colony formation assay were conducted to observe the cell
proliferation in Hep3B and Huh7 cell lines transfected with si-circZFR
or si-NC. Results suggested that downregulation of circZFR efficiently
inhibited Hep3B and Huh7 cell proliferation (Fig. 2B and C). Moreover,
we investigated whether cicrZFR affected the EMT process. The results
of western blot assay showed that knockdown of cicrZFR increased the
protein level of epithelial markers (E-cadherin and β-catenin), while
decreased the levels of mesenchymal markers (N-cadherin and Vi-
mentin) (Fig. 2D). Taken together, cicrZFR might exert carcinogenic
roles in HCC.
3.3. Downregulation of circZFR inhibited the Wnt/β-catenin signaling in
HCC cells
To further investigate the underlying mechanism of circZFR in HCC,
hierarchical clustering analysis was used to screen out the mRNAs
which could be regulated by circZFR. As the heat map showed that 217
mRNAs were upregulated and 246 mRNAs were downregulated in
samples. CTNNB1 and some Wnt/β-catenin pathway-related genes were
downregulated in these samples (Fig. 3A). GO analysis further showed
that circZFR might regulate Wnt/β-catenin signaling pathway (Fig. 3B).
To confirm this hypothesis, qRT-PCR and western blot assay were
performed to detect the mRNA level and protein level of MMP-7, MMP-
9, C-Myc and β-catenin. Results showed that circZFR knockdown sig-
nificantly decreased the mRNA levels and protein levels of MMP-7,
MMP-9, C-Myc and β-catenin (Fig. 3C and D). Moreover, luciferase
reporter assay was performed to further confirm the effect of circZFR on
the Wnt/β-catenin pathway. Results showed that downregulation of
cirZFR impaired the activity of Wnt/β-catenin signaling in Hep3B and
Huh7 cell lines (Fig. 3E). These findings indicated that cirZFR acted as
an oncogene through regulating Wnt/β-catenin signaling in HCC.
3.4. CircZFR functioned as a molecular sponge of miR-3619–5p in HCC
Previous studies have discovered that circRNAs could regulate
miRNAs. To determine whether circZFR could exert functions in HCC

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Fig. 4. CircZFR functioned as a molecular sponge of miR-3619-5p in HCC (A) Bioinformatics analysis was used to find the miRNAs which could bind with both
circZFR and CTNNB1. (B) qRT-PCR was utilized to explore the expression levels of the seven miRNAs after the transfection of si-circZFR. (C) The expression of miR-
3619–5p, miR-214–3p and miR-200a-3p were detected in 30 pairs of HCC tissues and normal tissues using qRT-PCR. (D) The expression levels of miR-3619–5p and
CTNNB1 were detected in 80 HCC tissues and adjacent normal tissues. (E) Spearman's correlation analysis was used to analyze the expression correlations among
circZFR, miR-3619–5p and CTNNB1. ∗P < 0.05, ∗∗P < 0.01 vs control group. NS: no significance.

via miRNA-mRNA axis, we found potential miRNAs which may interact
with circZFR and CTNNB1 from CSCD (http://gb.whu.edu.cn/CSCD/)
and starBase (http://starbase.sysu.edu.cn/browseNcRNA.php). Seven
miRNAs (miR-141–3p, miR-200a-3p, miR-214–3p, miR-761, miR-
3619–5p, miR-28–5p, miR-708–5p) were found out (Fig. 4A). Then we
detected the expression of the seven miRNAs in response to the
knockdown of circZFR. Results showed that miR-3619–5p and miR-
214–3p as well as miR-200a-3p exhibited significant fold changes
(Fig. 4B). Among which, miR-3619–5p was significantly downregulated
in 30 HCC tissues. Thus we chose miR-3619–5p for the following re-
search (Fig. 4C). Consistently, the expression of miR-3619–5p was
downregulated in 80 HCC tissues. Whereas, the expression of CTNNB1
was significantly upregulated in 80 HCC tissues (Fig. 4D). Spearman's
correlation analysis revealed that the expression of miR-3619–5p was
negatively correlated with that of circZFR and CTNNB1 (Fig. 4E).
Whereas, the expression of circZFR was positively related with that of
CTNNB1. These data suggested that circZFR might regulate HCC pro-
gression via miR-3619–5p/CTNNB1 axis in HCC.

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3.5. CircZFR regulated the progression of HCC through regulating miR-
3619–5p/CTNNB1 axis
To detect the effect of circZFR-miR-3619-5p-CTNNB1 axis on cell
proliferation and migration in HCC, rescue assays were performed.
CCK-8 assay indicated that the inhibitory effect on cell proliferation
induced by si-circZFR was partly reversed by miR-3619–5p inhibitor or
pcDNA-CTNNB1 (Fig. 5A). Western blot assay was conducted to ob-
serve the effect on the EMT process. Result showed that the protein
levels of E-cadherin and β-catenin increased by si-circZFR were reduced
by co-transfection of pcDNA-CTNNB1 or miR-3619–5p inhibitor
(Fig. 5B). However, the decreased protein levels of N-cadherin and
Vimentin caused by si-circZFR were recovered by pcDNA-CTNNB1 or
miR-3619–5p inhibitor. Taken together, we confirmed that circZFR
regulated the progression of HCC through regulating miR-3619–5p/
CTNNB1 axis.
4. Discussion
Although great advancement has been achieved in treating HCC, the
prognosis of HCC patients remain poor [15–17]. Thus it is beneficial to
explore the more effective therapeutic targets for HCC. Mounting evi-
dence has indicated that circRNAs are dysfunctional in human cancers
through miRNA-mRNA axis [18–20]. What's more, circRNAs have the
potential to be the biomarkers or prognostic factors in human cancers
[21–23]. However, the biological roles and mechanisms of circRNAs in
HCC have not been fully discovered.
In our current research, we found circZFR, circFUT8 and circIPO11
were closely associated with HCC via circRNA Disease website. CircZFR
was found to significantly upregulated in HCC tissues and cell lines
compared with two other circRNAs. Then, Kaplan-Meier analysis re-
vealed that high circZFR expression was associated with the low overall
survival rate of HCC patients, indicating that high circZFR expression
might lead to the poor prognosis of HCC patients. Therefore, we hy-
pothesized that circZFR might involve in the HCC progression. To de-
termine the role of circZFR in regulating HCC cellular processes, loss-of-
function assays were carried out. It was found that knockdown of
circZFR obviously suppressed Hep3B and Huh7 cell proliferation and
reversed the EMT progress. These findings suggested that circZFR ex-
erted oncogenic role in HCC.
In mechanism, microarray analysis revealed that a total of 463
mRNAs were differentially expressed after circZFR knockdown, among
which 217 mRNAs was found to be upregulated and 246 to be down-
regulated. CTNNB1 was found to have the most obvious fold change
among all upregulated mRNAs. GO analysis further indicated the po-
tential correlation between circZFR and Wnt/β-catenin signaling
pathway. Then, qRT-PCR and western blot assay were conducted to
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Fig. 5. CircZFR regulated the progression
of HCC through regulating miR-3619-5p/
CTNNB1 axis (A) CCK-8 assay was used to
detect cell viability in cells transfected with
si-circZFR, si-circZFR and pcDNA-CTNNB1
or miR-3619–5p inhibitor. (B) Western blot
assay was performed to observe the levels of
EMT-related proteins in cells transfected
with si-circZFR, si-circZFR and pcDNA-
CTNNB1 or miR-3619–5p inhibitor.
∗
P < 0.05, ∗P < 0.01 vs control group.
detect the expression levels of the core factors of Wnt/β-catenin
pathway. It was found that the levels of these core factors were effi-
ciently decreased by the knockdown of circZFR. According to the result
of luciferase reporter assay, we founs that knockdown of circZFR in-
hibited the activity of Wnt/β-catenin signaling pathway.
In addition, circRNAs can upregulate mRNA expression by inter-
acting with miRNAs [24–26]. Then we used bioinformatics tools to find
out miRNAs which could bind with both circZFR and CTNNB1. All
seven miRNAs were subjected to qRT-PCR and the result suggested that
miR-3619–5p was obviously downregulated in HCC tissues. The ex-
pression correlation among circZFR, miR-3619–5p and CTNNB1 was
analyzed. These data demonstrated that circZFR can regulate miR-
3619–5p/CTNNB1 axis in HCC. Finally, rescue assays were performed
to observe the effect of circZFR-miR-3619-5p-CTNNB1 axis on HCC. We
found that cell proliferation and EMT progress suppressed by si-circZFR
were partially reversed by co-transfection with miR-3619–5p inhibitor
or pcDNA-CTNNB1. These results indicated that miR-3619–5p and
CTNNB1 could involve in circZFR-mediated HCC progression. In con-
clusion, we confirmed that circZFR accelerated HCC progression
through regulating miR-3619–5p/CTNNB1 axis and activating Wnt/β-
catenin pathway. All findings in this study may contribute to finding
novel therapeutic targets for HCC.
Conflicts of interest
The authors declare that there are no competing interests associated
with this study.
Acknowledgements
The authors appreciate all laboratory members.
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