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RESEARCH PAPER
Cullin 4A/protein arginine methyltransferase 5 (CUL4A/PRMT5) promotes cell
malignant phenotypes and tumor growth in nasopharyngeal carcinoma
Xiuying Sun, Jinhui Zhou, and Zhicun Zhang
Department of Otolaryngology, The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University, Huai’an, Jiangsu, China
ABSTRACT
Targeted therapy is an important therapeutic strategy currently, however, the development
of targeted therapy for nasopharyngeal carcinoma (NPC) is relatively lagging. Cullin 4A
(CUL4A) was reported to be overexpressed in NPC; nevertheless, the specific role of CUL4A
remains unrevealed. NPC cells and tumor-bearing mice were cultivated to explore the role
and mechanism of CUL4A in NPC. After evaluating CUL4A levels in NPC cells, functional
experiments were carried out to investigate the effects of CUL4A knockdown and over-
expression on cell proliferative, invasive and migratory aptitude as well as NF-κB signaling.
Following the GeneMANIA database predicted that protein arginine methyltransferase 5
(PRMT5) was downstream of CUL4A, the mediated role of PRMT5 in the regulation of
CUL4A on cells was then determined. Moreover, the tumor volumes and weights of tumor-
bearing mice were recorded, and the levels of proliferation-, migration-, and NF-κB signaling-
related proteins in the tumor were determined. Herein, CUL4A was enhanced in NPC cells,
and its knockdown and overexpression separately suppressed and promoted cell prolifera-
tive, invasive, and migratory aptitude as well as NF-κB signal activation. Novelty, PRMT5
knockdown reversed the influences of CUL4A overexpression on these aspects. In addition,
its knockdown likewise reversed the facilitating impact of CUL4A expression on tumor
growth and declined the expression levels of proliferation-, migration-, and NF-κB signaling-
related protein in the tumor. Together, this paper indicated that CUL4A promoted the
proliferative, invasive, and migratory aptitude of NPC cells as well as tumor growth by
promoting PRMT5 to activate NF-κB signaling.
ARTICLE HISTORY
Received 29 January 2022
Revised 9 March 2022
Accepted 14 March 2022
KEYWORDS
Cullin 4A; protein arginine
methyltransferase 5; p65;
nasopharyngeal carcinoma;
C666-1 cell
CONTACT Xiuying Sun sunxiuy2107@163.com Department of Otolaryngology, The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical
University, 1 Huanghe West Road, Huai’an, Jiangsu 223300, China
BIOENGINEERED
2022, VOL. 13, NO. 4, 8712–8723
https://doi.org/10.1080/21655979.2022.2054756
© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
Introduction
Nasopharyngeal carcinoma (NPC) is an epithelial
malignant tumor that occurs in the nasopharynx.
It originates from the pharyngeal recesses and
easily invades adjacent tissues and lymph nodes
[1]. The degree of malignancy is relatively high
and distant metastasis can occur [2]. NPC was
divided into keratinizing, non-keratinizing, and
basaloid squamous cell carcinoma by World
Health Organization according to its histology
[3]. Numerous epidemiological surveys indicate
that Epstein-Barr virus (EBV) infection is the
most universal cause, meanwhile, environmental
and genetic factors may also be involved [4].
EBV infection is common in the non-keratinizing
type, taking up over 90% of cases; EBV is rare in
keratinizing type with a relatively favorable prog-
nosis [5]. Because NPC has an insidious onset and
is prone to regional lymph nodes and distant
hematogenous metastasis, the most patients are
in the middle and late stages when the disease is
clinically diagnosed [6]. Intensity-modulated
radiotherapy (IMRT) can significantly improve
the local control rate of NPC [7], however, distant
metastasis, especially to the lung, bone, and liver,
is still an important reason for failed treatment [2].
Molecular targeted therapy of tumors has become
a hot spot in the field of tumor research, and it is
also an important therapeutic strategy currently.
Compared with other tumors, the development of
targeted therapy of NPC is relatively lagging.
Being a bridging protein in the ubiquitin ligase
complex, Cullin 4A (CUL4A) is involved in the
ubiquitination degradation and ubiquitination mod-
ification of proteins in vivo and determines the
specificity of substrate proteins [8]. Previous studies
have revealed that CUL4A is involved in various
physiological activities of cells, including the regula-
tion of cell cycle, the transduction of signal, the
repair of damaged DNA, the methylation of histone,
the regulation of transcription as well as the activa-
tion of an oncogene [9]. Therefore, abnormal
expression of CUL4A protein may affect the phy-
siological activities of cells and lead to tumorigenesis
[10]. Overexpression of CUL4A protein was initially
found in primary breast cancer, suggesting that
CUL4A overexpression has closed relation to the
development of breast cancer [11]. Subsequent
studies found that CUL4A overexpression also
occurred in the prostate, colon, liver, as well as
lung cancer [
12–15
]. Of note, it was evidenced that
CUL4A was overexpressed in NPC [16], nonethe-
less, the role of CUL4A, as well as its mechanism,
has not been revealed.
Therefore, this study selected NPC cells and
constructed tumor-bearing mouse model to
explore the role and mechanism of CUL4A in
NPC. Exploring molecular therapeutic targets is
of great significance for enriching the comprehen-
sive treatment strategies for patients with NPC and
improving the therapeutic efficacy.
Materials and methods
Cell culture
Human nasopharyngeal epithelial NP69 cells
(Biobw, Beijing, China) and NPC cells C666-1,
5–8 F, 6–10B (Meisen, Hangzhou, China), and
SUNE-1 (EK-Bioscience, Shanghai, China) were
cultured in RPMI-1640 (Gibco; Thermo Fisher
Scientific) supplemented with 10% FBS (Gibco)
and 1% P/S in an incubator set at 37°C and 5%
CO
2
[17].
Cell transfection
CUL4A knockdown was achieved by transfection
with small interfering (si)RNA targeting CUL4A
(siRNA-CUL4A-1/2), and a non-targeted siRNA
was used as negative control (siRNA-NC).
CUL4A overexpression was achieved through the
transfection with pcDNA3.1 vector containing
CUL4A (Ov-CUL4A), and the empty vector was
used as Ov-NC. PRMT5 knockdown was achieved
by transfection with short hairpin RNA (shRNA)
targeting PRMT5 (shRNA-PRMT5-1/2), and
a scrambled shRNA was used as shRNA-NC. All
these plasmids were provided by GenePharma
(Shanghai, China). The transfection [18] was car-
ried out with the aid of Lipofectamine® 2000
(Invitrogen; Thermo Fisher Scientific).
Preparation of tumor transplants
All procedures followed the ethical guidelines of The
Affiliated Huai’an No. 1 People’s Hospital of Nanjing
Medical University and were approved by The
BIOENGINEERED 8713
Research and Education Animal Use and Care
Committee. Great efforts have been made to relieve
animal suffering. The experiments were carried out
using 18 adult male Balb/c nude mice [19] (age, 4–
6 weeks; weight, 16–18 g; Cyagen Biosciences, Jiangsu,
China) which were housed at a constant ambient
temperature of 21 ± 2°C and relative humidity of
55 ± 5%, with a 12-h light/dark cycle, and free access
to food and water. For each group (Ov-NC, Ov-
CUL4A, and Ov-CUL4A + shRNA-PRMT5), the
resuspension of cells (2 × 10
6
) was separately con-
ducted in a 0.2-ml volume and the cell suspension
was injected using a syringe. The weights of the mice
along with the size of the tumors were recorded every
3 days from the fourth day. A total of 28 days later,
after the nude mice were euthanized using an over-
dose of sodium pentobarbital (100 mg/kg, ip) followed
by cervical dislocation, the tumor tissue was taken out
for the subsequent assays.
Western blotting
Extracted proteins from tumor tissue or cells by
RIPA lysis buffer (Solarbio, Beijing, China) were
quantified with the adoption of a BCA assay
(Solarbio). After the separation with 10% polyacry-
lamide gel, the transfer of proteins (25 μg) was car-
ried out into PVDF membranes (Millipore). Prior to
the incubation with primary antibodies at 4°C, the
blocking of membranes with skimmed milk was
operated. Thereafter, membranes were cut in strips,
HRP-conjugated secondary antibody was applied to
incubate the strips. Taking advantage of an ECL
detection reagent (Millipore), the blots were tracked
and ImageJ version 1.52 software was utilized to
analyze the obtained data [20]. The details of the
antibodies are presented in Table 1.
Reverse transcription-quantitative PCR (RT-qPCR)
The synthesization of RNA isolated by TRIzol®
reagent (Invitrogen) into cDNA was operated with
PrimeScript™ RT Master Mix (Takara, Beijing,
China). qPCR was carried out with the application
of the QuantiTect SYBR-Green PCR kit (Qiagen).
The data were quantified with the ∆∆Cq method
[21] following normalization against GAPDH. The
following were the applied primers: CUL4A forward,
5’-AGGCACAGATCCTTCCGTTT-3’ and reverse,
5’-TCCTGCCAGCACGTGTTAAT-3’; PRMT5 for-
ward, 5’-CTGTCTTCCATCCGCGTTTCA-3’ and
reverse, 5’-GCAGTAGGTCTGATCGTGTCTG-3’;
and GAPDH forward, 5’- GACTCATGACCA
CAGTCCATGC-3’ and reverse, 5’-AGAGGCA
GGGATGATGTTCTG-3’.
Cell Counting Kit-8 (CCK-8) assay
Cells (5 × 10
3
cells/well) that plated into a 96-well
plate were cultured for 24, 48, and 72 h. For each
time point, the CCK-8 solution (Dojindo
Molecular Technologies) was added to each well
[22]. Absorbance was then tracked under the
application of a microplate reader (λ = 450 nm,
Bio-Rad Laboratories).
Co-Immunoprecipitation (Co-IP)
The lysis of cells was operated on ice for 10 min,
following which was the centrifugation at 13,000 x g
for 10 min and the collection of supernatants.
Subsequently, the lysate (500 µg/ IP) was supplemen-
ted with the 2.5 µg CUL4A or PRMT5 antibody and
10 µl protein A + G magnetic beads (Beyotime,
Shanghai, China) followed by gentle rotation at
room temperature for 2 h. The supernatant was
removed by magnetic force and the magnetic beads
with the added 1X SDS sample buffer were boiled for
5 min for western blotting [23].
Wound healing assay
Cells (5 × 10
5
cells/well) in six-well plates were
cultured until ~90% confluence. A 200-μl sterile
pipette tip was applied to create a wound in the
Table 1. Antibodies used for western blotting.
Antibody
Catalog
number Host
Dilution
ratio Company
CUL4A GTX33129 Rabbit 1:1,000 GeneTex
PRMT5 GTX116004 Rabbit 1:1,000 GeneTex
MMP12 ab52897 Rabbit 1:5,000 Abcam
MMP9 ab283575 Rabbit 1:1,000 Abcam
Ki67 ab16667 Rabbit 1:1,000 Abcam
PCNA ab29 Rabbit 1:1,000 Abcam
Phospho-p65 AF5875 Rabbit 1:1,000 Beyotime
p65 AF0246 Rabbit 1:1,000 Beyotime
IKBα ab97783 Rabbit 1:5,000 Abcam
Histone3 Gtx122148 Rabbit 1:5,000 GeneTex
HRP anti-rabbit lgG A0208 goat 1:1,000 Beyotime
8714 X. SUN ET AL.
cell monolayer. Following the rinse with PBS, the
cells were starved for 24 h [24]. Under an inverted
microscope (x100; Olympus Corporation), the
images were visualized at 0 and 24 h.
Transwell assay
Cells (3 × 10
4
cells) were suspended in 1 ml
serum-free medium. The upper chamber was pre-
coated with Matrigel (Sigma-Aldrich) and cells
were then cultivated in the upper chamber with
0.1 ml cell suspension in each well. RPMI-1640
which contains 20% FBS was applied to perfuse
the lower chamber. Following 24 h of incubation,
the fixation and staining with 4% paraformalde-
hyde (Biobw) and 0.5% crystal violet solution
(Yeasen, Shanghai, China) were operated [25].
Finally, the stained cells were totaled under an
inverted microscope (x100).
Colony formation assay
Cells were inoculated into culture dishes at a density
of 500 cells/dish. After discarding the supernatants,
the cells were fixed with 4% paraformaldehyde
(Biobw). The fixative solution was then replaced
with crystal violet (Yeasen) to stain the cells.
A cluster of >50 cells was considered a colony [26].
Bioinformatics and statistical analysis
GeneMANIA (http://genemania.org/) online data-
bases were used to search for the association
between known proteins [27]. Data that demon-
strated as the mean ± SD were analyzed with
Prism 8.0 software (GraphPad Software). To
show differences among multiple groups, one-
way ANOVA followed by Tukey’s post hoc test
was employed. P less than 0.05 is judged to be of
statistical significance.
Results
This study used NPC cells and tumor-bearing
mouse models to explore the role and mechanism
of CUL4A in NPC. CUL4A expression was
enhanced in NPC cells, and its knockdown and
overexpression separately suppressed and pro-
moted cell proliferative, invasive, and migratory
aptitude as well as NF-κB signal activation.
Novelty, PRMT5 knockdown reversed the influ-
ences of CUL4A overexpression on these aspects.
Moreover, its knockdown also reversed the facil-
itating impact of CUL4A expression on tumor
growth and declined the expression levels of pro-
liferation-, migration-, and NF-κB signaling-
related protein in the tumor. Together, these
results indicated that CUL4A promoted the pro-
liferative, invasive, and migratory aptitude of NPC
cells as well as tumor growth by promoting
PRMT5 to activate NF-κB signaling.
CUL4A expression level impacts NPC cell
proliferation
The levels of CUL4A in NP69, C666-1, 5–8 F, 6–
10B, and SUNE-1 cells were assessed by RT-
qPCR along with western blotting (Figure 1a-b).
Higher expression levels were found in NPC cells
compared with NP69 cells. To highlight the prob-
able effects of CUL4A level on NPC cells, we
adopted C666-1 cells for ensuing experiments.
The C666-1 cells were transfected with siRNAs
or plasmids to knock down or elevate the expres-
sion of CUL4A, respectively. The transfection
efficacy was assessed using RT-qPCR and western
blotting (Figure 1c-f). The level of CUL4A was
effectively down-regulated in the siRNA-CUL4A
-1/2 groups, and the siRNA-CUL4A-1 group was
selected for the following assays due to the rela-
tively lower level. In addition, CUL4A was effec-
tively overexpressed in the Ov-CUL4A group.
Cell proliferation in the knockdown and overex-
pression groups was determined using the CCK-8
assay (Figure 1g). During the period of 24, 48,
96 h, the proliferative ability of cells in the
siRNA-CUL4A group slowed down, whereas the
proliferation in the Ov-CUL4A group accelerated.
Moreover, the levels of Ki67 and PCNA were
evaluated by western blotting (Figure 1h). Their
levels were declined in the siRNA-CUL4A group
and elevated in the Ov-CUL4A group. Colony-
forming efficiency was likewise assessed by plate
colony formation assay (Figure 1i). The colony-
forming efficiency in the siRNA-CUL4A group
was obviously decreased, while that in the Ov-
CUL4A group was improved.
BIOENGINEERED 8715
Figure 1. CUL4A expression level impacts NPC cell proliferation (a) The expression levels of CUL4A in NP69, C666-1, 5–8 F,
6–10B, and SUNE-1 cells were determined using RT-qPCR and (b) western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs.
NP69. (c) The efficacy of CUL4A knockdown was assessed using RT-qPCR and (d) western blotting. ***P < 0.001 vs. siRNA-NC.
(e) The efficacy of CUL4A overexpression was assessed using RT-qPCR and (f) western blotting. ***P < 0.001 vs. Ov-NC. (g) Cell
proliferation in each group was determined using the CCK-8 assay. (h) The expression level of Ki67 and PCNA was determined
using western blotting. (i) Colony-forming efficiency was assessed by colony formation assay. **P < 0.01, ***P < 0.001 vs.
siRNA-NC;
##
P < 0.01,
###
P < 0.001 vs. Ov-NC.
8716 X. SUN ET AL.
CUL4A expression level impacts NPC cell
migration and invasion, and NF-κB signaling
The cell migratory and invasive aptitude were
assessed employing wound healing along with
Transwell assay (Figure 2a-b). The migration rate
of cells in the siRNA-CUL4A group was slightly
decreased and significantly increased in the Ov-
CUL4A group. The invasion rate of cells was sig-
nificantly decreased in the siRNA-CUL4A group
and increased in the Ov-CUL4A group, respec-
tively. In addition, with the adoption of western
blotting, the MMP12 and MMP9 levels were
detected (Figure 2c). Their levels were markedly
declined in the siRNA-CUL4A group and
increased in the Ov-CUL4A group. Furthermore,
as Figure 2d depicted, the levels of nuclear p65 and
IκBα were conspicuously descended in the siRNA-
CUL4A group and elevated in the Ov-CUL4A
group.
CUL4A regulates PRMT5 to impact NF-κB
signaling
To further figure out the mechanism of CUL4A,
the GeneMANIA database was applied to search
proteins associated with CUL4A (Figure 3a).
Figure 2. CUL4A expression level impacts NPC cell migration and invasion, and NF-κB signaling (a) Cell migration was
determined using wound healing assay. (b) Cell invasion was determined using Transwell assay. (c) The expression levels of MMP12
and MMP9 were determined using western blotting. (d) The expression level of nuclear p65, total p65, and IκBα were determined
using western blotting. **P < 0.01, ***P < 0.001 vs. siRNA-NC;
#
P < 0.05,
##
P < 0.01,
###
P < 0.001 vs. Ov-NC.
BIOENGINEERED 8717
PRMT5 was found to be co-expressed with
CUL4A. Hence, the levels of PRMT5 in NP69,
C666-1, 5–8 F, 6–10B, and SUNE-1 cells were
assessed with RT-qPCR and western blotting
(Figure 3b-c). The level of PRMT5 was enhanced
in the NPC cells. Moreover, the PRMT5 level was
markedly declined in the siRNA-CUL4A group
and elevated in the Ov-CUL4A group (Figure 3d-
e). Afterward, the association between CUL4A and
PRMT5 was verified using the Co-IP assay (figure
3f). The results indicated that CUL4A and PRMT5
could bind to each other and co-precipitate.
Figure 3. CUL4A regulates PRMT5 to impact NF-κB signaling (a) The GeneMANIA database was applied to search proteins
associated with CUL4A. (b) The expression level of PRMT5 in NP69, C666-1, 5–8 F, 6–10B, and SUNE-1 cells were determined using
RT-qPCR and (c) western blotting. ***P < 0.001 vs. NP69. (d) The expression level of PRMT5 in the transfected cells was determined
using RT-qPCR and (e) western blotting. ***P < 0.001 vs. siRNA-NC;
###
P < 0.001 vs. Ov-NC. (f) The association between CUL4A and
PRMT5 was verified using the Co-IP assay. (g) The efficacy of PRMT5 knockdown was confirmed with RT-qPCR and (h) western
blotting. ***P < 0.001 vs. shRNA-NC. (i) The expression level of nuclear p65, total p65, and IκBα were determined using western
blotting. ***P < 0.001 vs. Ov-NC;
#
P < 0.05,
##
P < 0.01 vs. Ov-CUL4A + shRNA-NC.
8718 X. SUN ET AL.
Following confirming the efficacy of shRNA-
PRMT5 with RT-qPCR along with western blot-
ting (Figure 3g-h), shRNA-PRMT5-1 and Ov-
CUL4A were co-transfected into C666-1 cells.
The levels of nuclear p65, total p65, and IκBα
were evaluated with the application of western
blotting (Figure 3i). PRMT5 knockdown reversed
the elevated expression levels of nuclear p65 and
IκBα caused by CUL4A overexpression.
CUL4A regulates PRMT5 to impact NPC cell
malignant phenotypes
The influences of PRMT5 knockdown on the reg-
ulatory roles of CUL4A in cell malignant pheno-
types were evaluated. Cell proliferative ability was
firstly tracked by CCK-8 assay (Figure 4a), western
blotting (Figure 4b), as well as colony formation
assay (Figure 4c). PRMT5 knockdown reversed the
promotion of cell proliferation resulting from
CUL4A overexpression and simultaneously
reduced the expression levels of Ki67 and PCNA.
Moreover, the colony-forming efficiency in the
Ov-CUL4A + shRNA-PRMT5 group was declined
compared with the Ov-CUL4A + shRNA-NC
group. Next, the capacity of cells to migrate and
invade was determined using wound healing
(Figure 4d), Transwell assay (Figure 4e), and wes-
tern blotting (figure 4f). PRMT5 knockdown like-
wise reversed the promoted migration and
invasion caused by CUL4A overexpression, mean-
while, it reduced the expression levels of MMP12
and MMP9.
CUL4A regulates PRMT5 to promote tumor
growth
Based on cellular experiments, we performed
in vivo study using tumor-bearing mouse models.
The body weights of mice (Figure 5a-b) and the
volumes of tumors (Figure 5c-d) were recorded
over 28 days. Overall, the body weights presented
fluctuating upward trends. Compared with the
Ov-NC group, the weight of the mice in the Ov-
CUL4A group was lower for the first 13 days and
surpassed in the following days. Compared with
the Ov-CUL4A group, the weight of mice in the
Ov-CUL4A + shRNA-PRMT5 group was higher in
the first 20 days, and lower in the remaining days.
The tumor volume in the Ov-CUL4A group
increased sharply and was significantly larger
than that in the Ov-NC group on the day of strip-
ping. The tumor volume in the Ov-CUL4A +
shRNA-PRMT5 group increased slowly and was
tremendously smaller than that in the Ov-CUL4A
group on the day of stripping. The tumor weight
in the Ov-CUL4A group was also greatly larger
than that in the Ov-NC group, and PRMT5 knock-
down greatly inhibited the growth of tumor weight
(Figure 5e). The levels of Ki67, PCNA, MMP12,
and MMP9 in the tumor were assessed by western
blotting (figure 5f). Their levels in the tumor of the
Ov-CUL4A group were elevated and partly
reversed in the Ov-CUL4A + shRNA-PRMT5
group. Furthermore, the levels of CUL4A,
PRMT5, nuclear p65, total p65, and IκBα in the
tumor were assessed by western blotting
(Figure 5g). CUL4A overexpression promoted
intratumoral PRMT5 levels, as well as nuclear
p65 and IκBα levels, whereas PRMT5 knockdown
reduced their levels.
Discussion
The incidence of NPC has unique geographical
distribution characteristics, with the highest inci-
dence in China and Southeast Asian countries,
followed by North Africa, and the least in
Europe, America, and Oceania [28]. This distribu-
tion is thought to be relevant to racial genetics,
consumption of pickled foods, high levels of nickel
in soil and water, and climate suitable for EBV
survival. EBV is best suited to survive in hot and
humid conditions [4,29]. As aforementioned, its
infection is intimately associated with the occur-
rence and advancement of NPC. EBV DNA level
has been identified as markers of tumor burden,
recurrence monitoring, and prognosis in NPC
[30]. The C666-1 cells, majorly studied in this
paper, are EBV-positive cells [31], and the expres-
sion level of CUL4A is higher in them compared
with other cells. As NPC is prone to metastasis,
this study evaluated the effects of low and high
CUL4A expression on C666-1 cell proliferative,
invasive and migratory aptitude, respectively. The
results indicated that down-regulated and up-
regulated CUL4A expression separately inhibited
and promoted cell proliferation, invasion, and
BIOENGINEERED 8719
Figure 4. CUL4A regulates PRMT5 to impact NPC cell malignant phenotypes (a) Cell proliferation level was determined with the
CCK-8 assay. (b) The expression level of Ki67 and PCNA was determined using western blotting. (c) Colony-forming efficiency was
assessed by colony formation assay. (d) Cell migration was determined using a wound healing assay. (e) Cell invasion was
determined using Transwell assay. (f) The expression levels of MMP12 and MMP9 were determined using western blotting.
***P < 0.001 vs. Ov-NC;
#
P < 0.05,
##
P < 0.01,
###
P < 0.001 vs. Ov-CUL4A + shRNA-NC.
8720 X. SUN ET AL.
migration. Although we found that CUL4A has
a regulatory effect on cells as above-mentioned,
the pathway mediated by it remains unknown,
hence, we continued to investigate the
downstream.
EBV can establish latent or lytic infection in
host cells, both of which are involved in the carci-
nogenesis of NPC. Among them, EBV infection of
NPC is dominantly based on latent infection [32].
EBV-encoded latent membrane protein 1 is the
primary oncogenic protein of NPC, and many of
its downstream events are mediated through acti-
vation of NF-κB [33]. Moreover, ubiquitination
plays a very important role in the activation of
the NF-κB signaling pathway and participates in
the degradation of IκB protein [34]. Hence, we
Figure 5. CUL4A regulates PRMT5 to promote tumor growth (a) Photo of tumor-bearing mouse models. (b) The body weights of
mice and (c) the volumes of tumors were recorded over 28 days. (d) Photo of the dissected tumor. (e) The weights of dissected
tumors were recorded. (f) The expression levels of Ki67, PCNA, MMP12, and MMP9 in the tumor were determined using western
blotting. (g) The expression level of CUL4A, PRMT5, nuclear p65, total p65, and IκBα in the tumor was determined using western
blotting. **P < 0.01, ***P < 0.001 vs. Ov-NC;
#
P < 0.05,
##
P < 0.01,
###
P < 0.001 vs. Ov-CUL4A.
BIOENGINEERED 8721
speculated that CUL4A could regulate NPC cells
through the activation of the NF-κB signaling
pathway. The results of this study display that
CUL4A can promote the activation of NF-κB sig-
naling in NPC cells, and inhibit its activity when
its expression is knocked down. Activated NF-κB
is an integral part of inducing tumor formation,
and it has been suggested that NF-κB signaling is
involved in the malignant behavior of NPC
cells [35].
Afterward, we screened the GeneMANIA data-
base and found that CUL4A can co-express with
PRMT5. PRMT5 has been testified to be enhanced
in NPC tissues and cells, and its knockdown can
enhance the radiosensitivity [36]. We co-
transfected CUL4A overexpression plasmid and
PRMT5 interference plasmid into C666-1 cells
and injected the cells subcutaneously into nude
mice. We found that PRMT5 knockdown inhib-
ited cell migration and invasion and slowed
tumor growth in nude mice. PRMT5 is an emer-
ging epigenetic enzyme that regulates the tran-
scription of target genes through di-methylation
of histone residues [37]. At present, there are
highly selective PRMT5 inhibitors developed
with PRMT5 as the target, which are used for
the treatment of malignant solid tumors and
hematological tumors, and have been approved
for clinical trials [38]. This suggests that the use
of PRMT5 as a target for the treatment of NPC is
of great practical significance.
Conclusion
Taken together, this paper indicates that CUL4A
promotes the proliferation, migration, invasion of
NPC cells, and tumor growth by promoting
PRMT5 to activate NF-κB signaling. This finding
revealed the mechanism of CUL4A/PRMT5 in
NPC and laid a theoretical foundation for the
selection of therapeutic targets. It is hoped that
this research will advance the development of tar-
geted drugs for the treatment of NPC.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
Nanjing Medical University Science and Technology
Development Fund NMUB2020149
Ethics approval
All procedures followed the ethical guidelines of The
Affiliated Huai’an No. 1 People’s Hospital of Nanjing
Medical University and were approved by The Research and
Education Animal Use and Care Committee of our hospital.
Competing interests
The authors declare that they have no competing interests.
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