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ARTICLE OPEN
Long non-coding RNA NRSN2-AS1 promotes ovarian cancer
progression through targeting PTK2/β-catenin pathway
Yi-Bo Wu
1,8
, Shen-Yi Li
1,2,8
, Jin-Yan Liu
3,8
, Jia-Jia Xue
4,8
, Jin-Fu Xu
5
, Ting Chen
6
, Tian-Yue Cao
6
, Hui Zhou
1
, Tian-Tian Wu
5
,
Chun-Lin Dong
1
, Wei-Feng Qian
3
, Long-Wei Qiao
7
✉, Shun-Yu Hou
6
✉, Ting Wang
7
✉and Cong Shen
7
✉
© The Author(s) 2023
As a common malignant tumor among women, ovarian cancer poses a serious threat to their health. This study demonstrates that
long non-coding RNA NRSN2-AS1 is over-expressed in ovarian cancer tissues using patient sample and tissue microarrays. In
addition, NRSN2-AS1 is shown to promote ovarian cancer cell proliferation and metastasis both in vitro and in vivo. Mechanistically,
NRSN2-AS1 stabilizes protein tyrosine kinase 2 (PTK2) to activate the β-catenin pathway via repressing MG-53-mediated
ubiquitinated degradation of PTK2, thereby facilitating ovarian cancer progression. Rescue experiments verify the function of the
NRSN2-AS1/PTK2/β-catenin axis and the effects of MG53 on this axis in ovarian cancer cells. In conclusion, this study demonstrates
the key role of the NRSN2-AS1/PTK2/β-catenin axis for the first time and explores its potential clinical applications in ovarian cancer.
Cell Death and Disease (2023) 14:696 ; https://doi.org/10.1038/s41419-023-06214-z
INTRODUCTION
Ovarian cancer (OC) is the most malignant gynecological cancer
and seriously jeopardizes women’s health and quality of life [1,2].
Owing to its nontypical symptoms in the early stages and its high
invasiveness, OC has a 5-year patient survival rate of 20–40% [3,4].
Identifying suitable biomarkers for diagnosing and treating OC is
urgent right now.
Long-coding RNAs (lncRNAs), which are functional RNA
molecules longer than 200 nucleotides without protein-coding
ability, play a vital role in various cancers through multiple
mechanisms [5,6]. LncRNAs participate in gene regulation not
only at the epigenetic and transcriptional levels [7] but also at the
post-transcriptional and translational levels via interactions with
RNAs [8–10] and proteins [11]. For example, p53-induced
noncoding transcript (Pint) directly binds to polycomb repressive
complex 2 (PRC2) and is required for H3K27 trimethylation during
colon cancer progression [7]. You et al. found that PTAR in the
cytoplasm acted as a competing endogenous RNA (ceRNA),
interacting with miR-101 and regulating ZEB1 level, thereby
promoting metastasis of OC [8]. Likewise, it has been proven that
cytoplasmic SPOCD1-AS can induce OC metastasis through
interacting with protein G3BP1 to remodel mesothelial cells [11].
The lncRNA NRSN2-AS1 (NCBI: NR_109990.1, Ensemble:
ENSG00000225377) is situated on chromosome 20, spanning a
total length of 5566-bp. Research on NRSN2-AS1 is limited. The
latest research found that NRSN2-AS1 was located in both the
cytoplasm and nucleus of cancer cells. Chen et al. have first
showed that NRSN2-AS1 could facilitate ovarian cancer progres-
sion via sponging miR-744-5p to regulate PRKX expression [12].
Subsequently, Xu et al. found that NRSN2-AS1 promotes the
progression of esophageal squamous cell carcinoma by regulating
the ubiquitin degradation of PGK1 [13]. In a recent study
conducted by Huang et al., the expression level of NRSN2-AS1
exhibited a significant positive correlation with immune cell
infiltration and participated in the peroxisome and Peroxisome
proliferator-activated receptor (PPAR) signaling pathways in
hepatocellular carcinoma [14]. However, NRSN2-AS1 still needs
to be further investigated in tumors for more in-depth functions.
In present study, we verified that NRSN2-AS1 was upregulated
in OC tissues and cells, and positively corresponds with OC
progression. Through RNA pull-down liquid chromatography-mass
spectrometry (RNA pull-down LC-MS/MS), we found that NRSN2-
AS1 could directly interact with PTK2 (protein tyrosine kinase 2). In
depth, IP assays demonstrated that NRSN2-AS1 protected PTK2
from mitsugumin 53 (MG53)-mediated ubiquitinated degradation.
Therefore, accumulated PTK2 enhances OC progression by
activating the β-catenin pathway. The identification of the
NRSN2-AS1/PTK2/β-catenin signaling pathway not only enriches
our knowledge of the specific role of NRSN2-AS1 in OC but could
also provide new targets for detecting and treating OC.
Received: 19 April 2023 Revised: 17 September 2023 Accepted: 3 October 2023
1
Human Reproductive and Genetic Center, Affiliated Hospital of Jiangnan University, Wuxi 214122, China.
2
Department of Obstetrics, Suzhou Municipal Hospital, The Affiliated
Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou 215002, China.
3
Department of Breast and Thyroid Surgery, Suzhou Municipal
Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou 215002, China.
4
Suzhou Dushu Lake Hospital (Dushu Lake
Hospital Affiliated to Soochow University), Suzhou 215124, China.
5
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, School of Basic
Medical Sciences, Nanjing Medical University, Nanjing 211166, China.
6
Department of Gynaecology, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical
University, Gusu School, Nanjing Medical University, Suzhou 215002, China.
7
State Key Laboratory of Reproductive Medicine, Center for Reproduction and Genetics, Suzhou
Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou 215002, China.
8
These authors contributed
equally: Yi-Bo Wu, Shen-Yi Li, Jin-Yan Liu, Jia-Jia Xue. ✉email: qiaolongwei1@126.com; houshunyu@sina.com; biowt@163.com; congshen@nj mu.edu.cn
Edited by Dr Giovanni Blandino
www.nature.com/cddis
Official journal of CDDpress
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MATERIALS AND METHODS
Bioinformatics analysis
The Cancer Genome Atlas (TCGA) datasets (http://cancergenome.nih.gov/)
[15] were used to obtain clinical and RNA sequencing information on 419
OC and 88 normal ovarian tissue samples. The docking of NRSN2-AS1
bound to PTK2 were predicted with HDOCK (http://
hdock.phys.hust.edu.cn/) and visualized with PyMOL (https://pymol.org/
2/) as previously described [16,17]. The cut-off employed to dichotomize
OC patients in high and low NRSN2-AS1 expression is median.
Samples collection
Fresh tumor and paracancerous tissues were derived from 23 OC
patients with surgical resection from January 2021 to January 2022 at
the Suzhou Municipal Hospital. All participants signed informed consent,
and participant information will be fully protected. Approval was
received from the Research Ethics Committee of Suzhou Municipal
Hospital. Collected tissues were stored in RNA Keeper Tissue Stabilizer
(Vazyme, Nanjing, China) at −80 °C as previously described [18].
RNA extraction and real-time quantitative reverse
transcription polymerase chain reaction (RT-qPCR) assays
Tissues fixed in RNA Keeper Tissue Stabilizer were ground to powder with
liquid nitrogen. We extracted RNA with Total RNA Extraction Reagent
(TRIzol, Vazyme) and reverse transcribed it into cDNA with HiScript III RT
SuperMix for qPCR kit (Vazyme). The relative mRNA expression was
measured using an AceQ qPCR SYBR Green Master Mix kit (Vazyme) on an
Applied Biosystems 7500 RealTime PCR System. Finally, the expression
level was analyzed with 2
–ΔΔCT
and normalized to 18sRNA. Table S1 lists
the primers used in this study.
Fluorescence in situ hybridization (FISH) analysis
A tissue microarray with 48 OC and 10 normal tissue samples was
purchased from Zhongke Huaguang Biotechnology Company (Shanxi,
China). FISH assays were carried out with FISH kit (RiboBio Biotechnology,
Guangzhou, China) following the manual. A unique probe targeting
NRSN2-AS1 was synthesized by RiboBio. 4′,6-diamidino-2-phenylindole
(DAPI, Beyotime, Haimen, China) was used for staining nuclei. All
microscopy images were captured with a confocal laser microscope
(LSM 810, Carl Zeiss, Oberkochen, Germany).
Cell culture and treatments
Human OC cell lines (OVCAR3, A2780 and SKOV3) and normal ovarian
epithelial cell lines (IOSE80) were obtained from the Chinese Academy of
Cell Collection (Shanghai, China), and cultured in a humidified conditions
at 37 °C with 5% CO
2
. IOSE80, SKOV3 and OVCAR3 cells were cultivated in
RPMI-1640 (Gibco, USA) with fetal bovine serum (FBS) (ExCell Bio, New
Zealand) and 1% penicillin/streptomycin (PS) (NCM Biotech, China).
Specifically, 10%FBS was needed to IOSE80 and SKOV3, and 20% to
OVCAR3. Dulbecco’s modified Eagle medium (Gibco, USA) with 10% FBS
and 1% PS were used to culture A2780 cells.
When the cell fusion degree reached 60–70%, small interfering RNAs
(siRNAs) (GenePharma, Suzhou, China) targeting NRSN2-AS1, PTK2, and
MG53 as well as overexpression plasmids (GenePharma) pcDNA3.1-NRSN2-
AS1, pEX-1-MG53 and an empty vector (EV) were transfected into OC cells
by Lipofectamine 2000 (Invitrogen, USA) and X-treme GENE HP DNA
transfection reagent (Mannheim, Germany). The nucleotide sequences of
all siRNAs are shown on Table S2.
XAV-939 and SKL2001 (Selleck, Shanghai, China) were solubilized in
dimethyl sulfoxide (DMSO) (Sigma Aldrich, USA) at 10 mM, and used in
experiments at 15 μM. Following treatment, cells were harvested for
further analysis 48 h later.
Cell proliferation assays
A 96-well plate (2000 cells/well) was inoculated with the cells for the
CCK-8 assay. Cell viability was measured every 24 h using a Cell Counting
Kit-8 kit (Beyotime) on a microplate reader (Bio-Rad Model 680, USA) at
450 nm.
800–1000 cells were added into six-well plates for 2 weeks to evaluate
cloning formation capabilities. The clones were fixed with methanol,
stained with 0.1% crystal violet (Beyotime) and counted for analysis.
Cell migration assays
In the transwell assay, 4.5 × 10
4
cells in 300 μl serum-free medium were
seeded into the upper chamber with 8 μm pore size (Corning, USA),
whereas 700 μl complete medium was added into the lower chamber.
After 48 h, the cells outside the chamber were fixed, stained and imaged
for counting.
Xenograft in mice
Four-week-old female athymic BALB/c nude mice (Vital River Laboratory,
China) were used to construct a subcutaneous tumor model and lung
metastasis model. Mice received 12 h light/12 h dark at 22–28 °C and
50–70% humidity under specific-pathogen-free conditions. The approval of
mice investigations obtained from the Animal Ethics and Welfare
Committee of Nanjing Medical University.
For the subcutaneous tumor model [19], 1 × 10
7
cells transfected with
sh-NRSN2-AS1 or sh-NC were subcutaneously injected into the left and
right flanks of nude mice, respectively (n=5). The tumor volumes were
calculated every 3 days (V =0.5 × D × d
2
(V, volume; D, longitudinal
diameter; d, latitudinal diameter)). We sacrificed the mice at 11 days, follow
by removing and measuring the subcutaneous tumors.
For the lung metastasis model [20], the tail veins of mice were
injected with 2 × 10
7
treated cells (n=5). An eight-week period ended
with the mice being sacrificed, their lungs were removed and fixed in
formalin.
Immunofluorescence
Immunofluorescence assays were conducted as previous descriptions
[21–24]. Briefly, the slides containing target cells and tissues were
incubated with primary and Alexa-Fluor secondary antibodies (Thermo
Scientific, Waltham, USA) orderly. Confocal laser microscopes (LSM 810)
were used to observe all samples. A list of the primary antibodies used can
be found in Table S3.
Hematoxylin and eosin (H&E) staining
Staining of lung tissue sections with H&E was followed by ethanol
dehydration, xylene hyalinization, and neutral balsam closure. A micro-
scope (Axioskop 2 Plus, Zeiss) was used to photograph.
RNA pull-down assays and LC-MS/MS analysis
T7 RNA polymerase (Ambio Life, Shanghai, China) and Biotin RNA Labeling
Mix (Ambio Life) were used to transcribe and label NRSN2-AS1 according
to the manual. Next, Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo
Scientific) was utilized to carry out RNA pull-down assays [25]. Finally, the
proteins interacting with NRSN2-AS1 were identified by LC-MS/MS
analysis.
Western blot assays
We performed Western blotting according to our previous protocol
[20,26]. In short, radioimmunoprecipitation assay (RIPA, Beyotime) buffer
containing 1% protease inhibitor phenylmethylsulfonyl fluoride (PMSF,
Beyotime) was used to lysis protein samples. Subsequently, the proteins
were denaturalized at 100 °C for 10 min, electrophoresed with 10% SDS-
polyacrylamide gel (GenScript, China) and transferred to polyvinylidene
difluoride membranes (Millipore, Billerica, USA). Primary antibodies and
horseradish peroxidase-conjugated secondary antibodies were incubated
on membranes. In the end, an Image-Pro Plus (Media Cybernetics, USA)
was employed to measure the bands signal detected with the BeyoECL
Plus kit (Beyotime). Tubulin antibody was used as a control. There is
detailed information about the antibodies in Table S3. The original
western blots were shown on the Original Data File of Supplemental
Material.
RNA immunoprecipitation (RIP) assays
RIP assays were conducted with Magna RIPTM RNA-Binding Protein
immunoprecipitation kit (Millipore) as previously described [25]. Briefly,
cell lysate, which lysed by RIP lysis buffer, was incubated with anti-PTK2
and anti-IgG antibodies at 4 °C. Subsequently, the protein–RNA
complexes were obtained using 0.5 mg/ml proteinase K with 0.1%
SDS.RT-qPCRweredonetodeterminetheinteractionbetweenPTK2
and NRSN2-AS1.
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Cell Death and Disease (2023) 14:696
TOP/FOP Flash luciferase assays
TOP/FOP Flash luciferase assays were performed to prove the effect of
the NRSN2-AS1/PTK2 axis on β-catenin activity [12,27,28]. In brief,
OVCANR3 and A2780 cells treated with si-NC, si-NRSN2-AS1, or si-PTK2
separately were co-transfected with TOP/Flash, FOP/Flash and Renilla
luciferase plasmids (Beyotime) using Lipofectamine 2000. After
48 hours, the cells were lysed with the lysis buffer and the luciferase
activities were detected with Luciferase Reporter Gene Assay Kit
(Beyotime). The TOP/FOP-Flash values were normalized to Renilla
luciferase activity.
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Cell Death and Disease (2023) 14:696
Protein half-life assays
Cycloheximide (CHX, 100 μg/ml) was added into OC cells to interrupt
protein synthesis. Western blotting was conducted to detect the
expression of PTK2 at 0, 1, 2, 4 h.
Immunoprecipitation (IP) assays
IP assays were carried out according to our previously described procedure
[16,29]. Cell lysates were incubated with anti-PTK2 or anti-IgG antibodies
overnight, followed by washed beads (Santa Cruz) for 2 h at 4 °C. Finally,
the IP product, was detected by specific antibodies via western blotting.
Antibody information is provided in Table S3.
Ubiquitination assays
RIPA buffer containing 1% PMSF was used to lysed OC cells transfected
with siRNAs or overexpression plasmids. Anti-PTK2 or anti-IgG antibodies
were incubated overnight with cell lysates, followed by protein A/G beads
for 2 h at 4 °C. Finally, the products were analyzed using immunoblotting
with anti-Ub and anti-Ub-K48 antibodies.
Statistical analysis
The data were provided as mean ± standard deviation and analyzed using
Student’s t-tests for two groups, and one-way analyses of variance for three
or more groups on SPSS 17.0 (SPSS, Inc., USA) and GraphPad Prism 7.0
(GraphPad Software, USA). Survival curves were profiled with Kaplan–Meier
survival plots. P< 0.05 was represented as statistical significance.
RESULTS
NRSN2-AS1 expression is positively correlated with OC
progression
Based on TCGA data, NRSN2-AS1 was remarkably overregulated in
OC tissues (Fig. 1A), and high NRSN2-AS1 level was associated with
poor prognosis, including lower disease-specific survival and
overall survival rates (Fig. 1B, C). Additionally, NRSN2-AS1, age
and stage were found significantly associated with OC prognosis
by multivariate Cox regression analysis (Fig. S1). High NRSN2-AS1
levels were also found in OC patient tissues, as verified by RT-qPCR
and FISH assays (Fig. 1D–F). Relative to normal ovarian epithelial
cells (IOSE80), the OVCAR3 and A2780 cell lines displayed higher
levels of NRSN2-AS1 expression (Fig. 1G). Therefore, we used the
OVCAR3 and A2780 cell lines for further analysis. Silencing NRSN2-
AS1 using si-RNA strikingly decreased the cell viability, colony
formation, and migration of both OVCAR3 and A2780 cells in vitro
(Fig. 1H–M). Besides, smaller volumes and lower numbers of Ki67
+
cells were observed in xenograft tumors in the sh-NRSN2-AS1
group. There are decreased metastatic nodules, increased
E-cadherin positive cells, and reduced N-cadherin and vimentin-
positive cells in the lung of the sh-NRSN2-AS1 group (Fig. 1N–W).
NRSN2-AS1 interacts with PTK2
An RNA pull-down LC-MS/MS assay was performed to isolate
potential proteins interacting with NRSN2-AS1 (Fig. 2A). From
Venn diagrams of three independent RNA pull-down assays, 30
proteins overlapped (Fig. 2B and Table S4). These included PTK2,
also known as focal adhesion kinase (FAK), which is reported to
engaged in the progression of various tumor types. Based on
TCGA data, we observed that PTK2 was expressed excessively in
OC tissues than normal tissues (Fig. 2C).
Based on the three-dimensional (3D) structure, the interaction
interface between NRSN2-AS1and PTK2 was predicted using
HawkDock and visualized with PyMOL (Fig. 2D). The crossing
surface was composed of residues 35–82 of NRSN2-AS1 and
414–670 of PTK2 (Fig. 2E and Table S5). Subsequently, the
colocalization of NRSN2-AS1 and PTK2 in OC cells was confirmed
by co-immunostaining (Fig. 2F, G). Furthermore, RNA-pulldown
western blotting and RIP-qPCR experiments verified that NRSN2-
AS1 interacted with PTK2 (Fig. 2H, I).
PTK2 facilitates cell proliferation and migration and is
associated with β-catenin signaling activation in OC cells
OVCAR3 and A2780 cells were transfected with si-PTK2 or si-NC,
respectively to explore the effect of PTK2 on OC cells. The
downregulation of PTK2 reduced cell proliferation, including cell
viability and colony formation (Fig. 3A–D). The migratory capacity
was also dramatically reduced after PTK2 silencing (Fig. 3E, F). In
short, the knockdown of PTK2 suppressed OC cell proliferation
and migration in vitro.
β-catenin signaling is already being implicated in multiple
aspects of human cancers, including tumorigenesis, progression,
and malignant invasion [30]. In various cancers [31–33], β-catenin
is abnormally activated, including OC [34,35]. Most β-catenin is
located in the cytoplasm, where it maintains cell-cell adhesion
through interactions with E-cadherin [36]. In the nucleus,
β-catenin binds T-cell factor/lymphoid enhancer to activate
downstream genes, causing uncontrolled proliferation and
migration of neoplastic cells [37]. As illustrated (Fig. 3G), studies
have demonstrated that PTK2 participates in activating β-catenin
signaling. On the one hand, PTK2 can directly phosphorylate
β-catenin
Y142
to promote its translocation into the nucleus [38,39].
On the other hand, it phosphorylates GSK3
Y279/Y216
to promote its
ubiquitinated degradation mediated by E3 ligase β-TrCP
(β-transducin repeats-containing proteins), thereby stabilizing
the expression of total β-catenin [40] (Fig. 3G). Through western
blotting, we found that PTK2 silencing resulted in decreased
expression of β-catenin
Y142
, GSK3
Y279/Y216
, and total-β-catenin in
OVCAR3 and A2780 cells (Fig. 3H, I). Overall, the above results
indicate that PTK2 might also trigger the β-catenin pathway in
OC cells.
Knockdown of NRSN2-AS1 reduces protein expression of PTK2
and blocks β-catenin activation
Western blot was applied to clarify the influences of NRSN2-AS1
on the PTK2/β-catenin pathway. Reduced PTK2, β-catenin
Y142
,
Fig. 1 NRSN2-AS1 is significantly upregulated in OC and its levels are tightly correlated with OC progression both in vitro and in vivo.
ATCGA data indicated differences in expression of NRSN2-AS1 between 419 tumor tissues and 88 normal tissues. B, C Kaplan–Meier curves
showed a negative correlation between NRSN2-AS1 expression and disease-specific survival and overall survival in OC patients. DRT-qPCR
was used to detect the relative expression of NRSN2-AS1 in 23 paired OC samples. EFISH assays showed the expression of NRSN2-AS1 (red) in
OC tumor tissues (n=48) and normal tissues (n=10), with nuclei stained by DAPI (blue). Scale bar: 50 μm. FQuantification of fluorescence
intensity from (E). GRT-qPCR was used to test NRSN2-AS1 expression in normal human ovarian epithelium cell (IOSE80) and OC cells (OVCAR3,
A2780, and SKOV3), n=3. HRelative expression of NRSN2-AS1 in OVCAR3 and A2780 cells transfected with si-NRSN2-AS1 and si-NC tested by
RT-qPCR, n=3. IThe viability of OC cells was detected by CCK8 assays, n=6. J, K The proliferation ability of NRSN2-AS1-transfected OC cells
were determined with colony-formation assay, n=3. L, M Transwell assays were done to investigate the migratory ability of OC cells, n =3.
Scale bar: 100 μm. N-R A total of 5 nude mice were injected with OVCAR3 cells transfected with sh-NC or sh-NRSN2-AS1. NTumor volumes
were calculated every 3 days. O-P Tumors were collected and weighted. (Q, R) Ki-67 expression in tumors were evaluated with
immunofluorescence. Scale bar: 50 μm.(S-W OVCAR3 cells transfected with sh-NC or sh-NRSN2-AS1 were injected into the tails of nude mice
(n=5 each). SIn both sh-NC and sh-NRSN2-AS1 mice, entire lungs were obtained, and Tnodules on the lung surfaces were counted. ULung
sections were stained with H&E. VRelative expression of E-cadherin, N-cadherin, and vimentin in lung nodules was detected by
immunofluorescence; n=5. Scale bar: 50 μm. WQuantification of fluorescence intensity of (V). *P< 0.05, **P< 0.01, ***P< 0.001.
Y. Wu et al.
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Fig. 2 NRSN2-AS1 interacts with PTK2. A Flow chart of the RNA pull-down assays. BVenn diagram of proteins identified by MS from three
independent RNA pull-down assays. CTCGA data indicated the expression of PTK2 in 419 tumor tissues and 88 normal tissues. DVisualization
of the docking of NRSN2-AS1 (yellow) and PTK2 (blue) from front and back perspectives. Interacting residues are colored red for NRSN2-AS1
and purple for PTK2. The cartoon mode displays the backbone as well as the secondary structures of the corresponding proteins. The surface
mode represents the solvent-accessible surface area. The merged mode combines cartoon and transparent surface views. EVisualization of
the binding interface of the NRSN2-AS1/PTK2 complex. The interface view zooms and labels the residues (in stick mode) involved in the
binding between NRSN2-AS1 and PTK2. F, G Co-immunostaining of NRSN2-AS1 and PTK2 in OC cells. Scale bar: 50 μm. HWestern blot assays
showed the expression levels of PTK2 after the NRSN2-AS1 pull-down assay. IRIP assays for NRSN2-AS1 binding to PTK2 in OC cells lysates,
with rabbit anti-IgG as the negative control (n =3). *P< 0.05, **P < 0.01, ***P< 0.001.
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Cell Death and Disease (2023) 14:696
GSK3
Y279/Y216
, and total β-catenin expression were observed in OC
cells with NRSN2-AS1 silencing (Fig. 4A, B). Furthermore,
immunofluorescence confirmed that silencing either NRSN2-AS1
or PTK2 resulted in a dramatic loss of β-catenin expression. The
reduction was more pronounced in the nucleus (Fig. 4C, D). Aside
from that, the knockdown of NRSN2-AS1 or PTK2 produced a
remarkable reduction of TOP/FOP luciferase activity (Fig. S2).
Therefore, the knockdown of both NRSN2-AS1 and PTK2 blocks
β-catenin activation.
NRSN2-AS1 promotes OC proliferation and migration in a
manner dependent on β-catenin signaling
To further verify the key role of β-catenin in OC progression
associated with NRSN2-AS1, SKL2001 [41], a small-molecule
β-catenin agonist, was applied to OVCAR3 and A2780 cells with
silencing NRSN2-AS1. The expression of both β-catenin
Y142
and
total β-catenin was significantly enhanced after treatment with
SKL2001 (Fig. 5A, B). In addition, loss-of-function assays revealed
that activation of β-catenin in the NRSN2-AS1 knockdown group
could reverse cell proliferation and migration in vitro (Fig. 5C–H).
NRSN2-AS1 promotes OC progression through targeting
PTK2/β-catenin axis
To further prove the engagement of the PTK2/β-catenin axis in
NRSN2-AS1-mediated OC progression, si-PTK2 and XAV-939 [42], a
small-molecule β-catenin inhibitor, were applied to OVCAR3 and
A2780 cells overexpressing NRSN2-AS1. As a result, the expression
of PTK2, GSK3
Y279/Y216
,β-catenin
Y142
, and total β-catenin were
significantly increased after overexpression of NRSN2-AS1,
whereas silencing of PTK2 reduced the expression of PTK2,
GSK3
Y279/Y216
,β-catenin
Y142
, and total β-catenin. Moreover,
depression of β-catenin via XAV-939 also diminished the protein
Fig. 3 Effects and underlying mechanisms of PTK2 in OC cells. A, B CCK8 assays detected the OC cells viability with PTK2 silencing, n=6. C,
DThe proliferation ability of cells transfected with si-PTK2 was assessed by colony formation assays, n=3. E, F The migration abilities of cells
transfected with si-PTK2 were examined by transwell assays, n=3. Scale bar: 100 μm. GSchematic diagram of the pathway from PTK2 to
β-catenin. H, I Western blots to determine the expression of PTK2, GSK3
Y279/Y216
,β-catenin, and β-catenin
Y142
in PTK2-downregulated OC cells,
n=3. *P< 0.05, **P< 0.01, ***P< 0.001.
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Cell Death and Disease (2023) 14:696
Fig. 4 Potential molecular mechanisms involving NRSN2-AS1 and PTK2/β-catenin pathway in OC cells. A, B An internal control was
tubulin, western blotting was used to determine the protein level of PTK2, GSK3
Y279/Y216
,β-catenin, and β-catenin
Y142
in OC cells with silencing
of NRSN2-AS1, n=3. CImmunofluorescence experiments were performed to detect β-catenin expression levels (green) in the si-NRSN2-AS1
and si-PTK2 groups; DAPI was used to stain nuclei. Scale bar: 50 μm. DQuantification of fluorescence intensity from (C), n=100 cells for each
group. *P < 0.05, **P < 0.01, ***P < 0.001.
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Cell Death and Disease (2023) 14:696
level of β-catenin
Y142
and total β-catenin but not PTK2 or
GSK3
Y279/Y216
(Fig. 6A, B). Furthermore, cell function assays
demonstrated that inhibition of both PTK2 and β-catenin
noticeably abolished the proliferation and migration of OC cells
overexpressing NRSN2-AS1 (Fig. 6C–G).
According to OC patients cohort downloaded from the TCGA
database, there were significant positive correlation between
NRSN2-AS1 and PTK2, PTK2 and β-catenin, and NRSN2-AS1 and
β-catenin (Fig. S3A). Moreover, K-M survival analyses with the
integrated NRSN2-AS1/PTK2/β-catenin gene signature demon-
strated that the higher the expression, the worse the overall
survival in OC (Fig. S3B). Additionally, time-dependent receiver
operating characteristic (ROC) curve analyses further elucidated
the potential prognostic value of the integrated NRSN2-AS1/PTK2/
β-catenin gene signature. At 3, 5 and 8 years the Area Under
Curves (AUCs) were 0.520, 0.580 and 0.683 (Fig. S3C).
Metastatic progression heavily relies on the EMT procedure. To
investigate the involvement of the NRSN2-AS1/PTK2/β-catenin
network in modulating the expression of typical mesenchymal/
epithelial markers in OC cells, the RT-qPCR analyses of the NRSN2-
AS1/PTK2/β-catenin network on EMT markers were carried out. As
a result, silencing NRSN2-AS1 could downregulate the expression
of some mesenchymal markers (TWIST, NCAD, SNAIL, SLUG, ZEB1)
and upregulate epithelial markers (ECAD), and SKL2001 could
reverse it partially in OVCAR3 and A2780 (Fig. S2A) (Fig. S4A).
Contrarily, overexpressed NRSN2-AS1 could promote EMT,
whereas si-PTK2 and XAV939 could partially reverse it (Fig. S4B).
A trend to reduction/increase on EMT were detected with the
change of NRSN2-AS1/PTK2/β-catenin network, although some
differences were not statistically significant.
Finally, we analyzed the expression of PTK2 and β-catenin in
xenograft tumors derived from OVCAR3 cells. As showed by
immunofluorescence, there were reduced PTK2 and β-catenin
signals in the sh-NRSN2-AS1 group, when compared with these in
control group (Fig. S4C, D).
Based on these results, it appears that NRSN2-AS1 promotes OC
progression by targeting the PTK2/β-catenin axis.
NRSN2-AS1 protects PTK2 from polyubiquitinated
degradation by E3 ligase MG-53 in OC
In this study, we observed that NRSN2-AS1 affected the protein
level of PTK2 (Fig. 4A, B). We therefore assessed the stability of
PTK2 in the absence of NRSN2-AS1 via CHX assays in OC cells. The
protein stability of PTK2 significantly diminished after down-
regulation of NRSN2-AS1 (Fig. 7A, B). Furthermore, IP assays
showed that OC cells transfected with si-NRSN2-AS1 displayed
higher levels of polyubiquitination of PTK2. (Fig. 7C). The
proteasome could specifically recognize substrate proteins labeled
Fig. 5 Effects of NRSN2-AS1/β-catenin pathway in OC cells. OC cells were transfected with si-NC, si-NRSN2-AS1, si-NRSN2-AS1 +SKL2001
(β-catenin activator, 15 μM), or si-NRSN2-AS1 +DMSO. A, B Western blot was used to identify the expression of β-catenin and β-catenin
Y142
,
n=3. C, D CCK8 assays to detect cell viability, n=6. E, F The cells proliferation ability was determined by colony formation assays, n=3. G, H
Investigating migratory abilities with transwell assays, n=3. Scale bar: 100 μm. *P< 0.05, **P< 0.01, ***P< 0.001.
Y. Wu et al.
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Cell Death and Disease (2023) 14:696
Fig. 6 Effects of NRSN2-AS1/PTK2/β-catenin pathway in OC cells. OC cells were transfected with EV, OE-NESN2-AS1, OE-NRSN2-AS1+si-
PTK2, OE-NRSN2-AS1+si-NC, OE-NRSN2-AS1 +XAV-939 (β-catenin inhibitor, 15 μM), or OE-NRSN2-AS1 +DMSO. A, B Western blot analysis of
the expression of PTK2, GSK3
Y279/Y216
,β-catenin and β-catenin
Y142
,n=3 for each group. CCCK8 assays were performed to assess cell viability,
n=6. D, E Colony formation assays were done to detect proliferation ability, n=3. F, G Transwell assays were used to investigate changes in
migratory abilities, n=3. Scale bar: 100 μm. *P< 0.05, **P< 0.01, ***P< 0.001.
Y. Wu et al.
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Cell Death and Disease (2023) 14:696
with the K48 ubiquitin chain, which in turn degrades them. In this
study, it was obvious that Lys-48 was the critical lysine for PTK2
ubiquitination (Fig. 7C). The striated muscle-specific MG53, also
known as tripartite motif-containing 72 (TRIM72), has been
reported to induce PTK2 ubiquitination and degradation during
skeletal myogenesis [43]. In our study, IP assays also verified that
the interaction between MG53 and PTK2 was enhanced in NRSN2-
AS1-knockdown OC cells (Fig. 7C), which raised the possibility that
NRSN2-AS1 might stabilize PTK2 through blocking the ubiquitina-
tion and degradation mediated by MG53 in OC cells.
Y. Wu et al.
10
Cell Death and Disease (2023) 14:696
Based on TCGA data (Fig. 7D) and 23 paired clinical samples
(Fig. 7E), OC tissues had significantly lower MG53 levels than
normal tissues. Furthermore, gain-of-function assays demon-
strated that MG53 inhibited OC cell proliferation and migration
in vitro (Fig. 7F–J). These results suggest that MG53 appears to
play a tumor-suppressive role in OC based on all of these results.
To clarify the function of MG53 in the interaction between
NRSN2-AS1 and PTK2, IP assays were carried out. We found that
MG53 markedly promoted both total and K48-linked polyubiqui-
tination of PTK2 in OC cells (Fig. 7K). Furthermore, the knockdown
of MG53 reduced the increase in ubiquitination level of PTK2
caused by NRSN2-AS1 silencing in OC cells (Fig. 7L). Similarly,
NRSN2-AS1 lost the ability to inhibit PTK2 ubiquitination when
MG53 was simultaneously overexpressed (Fig. 7M).
Finally, western blot assays showed that silencing MG53 could
recover the expression of PTK2/β-catenin induced by NRSN2-AS1
silencing (Fig. 7N, O), whereas overexpression of MG53 diminished
the PTK2/β-catenin signaling activated by NRSN2-AS1 upregula-
tion (Fig. 7P, Q).
As a consequence, we conclude that NRSN2-AS1 regulates the
PTK2/β-catenin pathway in a manner that inhibits PTK2 poly-
ubiquitination and degradation induced by MG-53 in OC cells.
DISCUSSION
Recent evidence suggests that the novel oncogene NRSN2-AS1 is
hyperexpressed in esophageal squamous cell carcinoma [13] and
OC tissues [12], and promotes OC cells proliferation and migration
via interaction with miR-744-5p to modulate PRKX. In this study,
we further demonstrated that NRSN2-AS1 is highly expressed in
OC tissues based on analysis of patient samples and OC tissue
microarrays. Through RNA pull-down LC-MS/MS assays, we found
that NRSN2-AS1 could interact with many proteins in addition to
its previously reported role as a ceRNA. Among this, PTK2 was first
identified as a tyrosine-phosphorylated protein located at focal
adhesions [44,45]. Over the decades, evidence suggests that PTK2
is implicated in cell adhesion, migration, invasion, survival, and
proliferation via kinases or without them [46,47]. Most studies
have found that PTK2 is often upregulated in solid epithelial
cancers, where it contributes to tumor malignant behavior
[40,48–50]. As a susceptibility gene for OC [51], PTK2 is frequently
overexpressed in OC and is associated with immunosuppression
[52] and chemotherapy resistance [53,54]. Downregulation of
PTK2 inhibits OC growth in vivo [55,56]. Thus, in this study, PTK2
was selected as a candidate protein regarding its interactions with
NRSN2-AS1 in OC. Previously, studies have shown that lncRNAs
could influence the activation of the PTK2 pathway, thereby
facilitating tumor advancement [57–62]. For example, lncRNA
KCNQ1OT1 regulates proliferation and cisplatin resistance in
tongue cancer via miR-211-5p mediated Ezrin/Fak/Src signaling
[63]. Nevertheless, there was no evidence for a direct binding
between lncRNAs and PTK2 protein. In this study, we propose this
interaction for the first time. 3D structures, co-localization analysis,
RNA-pulldown western blotting, and RIP-qPCR experiments were
further verified the direct interaction between NRSN2-AS1 and
PTK2. Furthermore, the proliferation and migration of OC cells
were suppressed by PTK2 silencing. A key role of NRSN2-AS1/PTK2
axis in OC cells seems to be revealed by these results.
Numerous investigations have demonstrated that the β-catenin
pathway governs many aspects of OC development, including stem
cell self-renewal, metastasis, chemoresistance, angiogenesis, and
immune evasion [64]. It has reported that PTK2 could participate in
the β-catenin pathway in two ways. First, activated PTK2 could
localize at cell junctions and phosphorylate β-catenin
Y142
directly
[38,39]. Second, PTK2 phosphorylates GSK3
Y279/Y216
to stabilize
β-catenin, thereby promoting tumorigenesis [40]. Nevertheless, the
relevance of PTK2 and β-catenin in OC has remained obscure. In this
study, we found that PTK2 activated β-catenin both directly and
indirectly in OC cells. More importantly, NRSN2-AS1 exerted a pro-
cancer effect in OC cells through stabilizing PTK2 and subsequently
promoted PTK2/β-catenin pathway activation.
We then considered how NRSN2-AS1 regulated the stability of
PTK2. Previous studies have reported that PTK2 could been
degraded in a polyubiquitylation-related manner via the E3 ligase
MG53 during myogenesis [43,65]. Our results also indicated that
silencing of NRSN2-AS1 could enhance the degradation of PTK2
depending on MG53-mediated ubiquitination in OC cells. Several
reports have shown that MG53 inhibits tumor progression in non-
small-cell lung [66], tongue [67], colorectal [68], and hepatocellular
[69] cancers. Here, we clarified the tumor suppressor role of MG53
in OC. Furthermore, rescue experiments showed that NRSN2-AS1
regulates the PTK2/β-catenin pathway in a manner that blocks
ubiquitinated degradation of PTK2 mediated by MG-53 in OC.
There has been less research on the potential applications of
PTK2 and theβ-catenin pathway in cancer therapies. A few
preclinical and clinical trials (I, Ib, or II) have shown that PTK2
inhibitors could enhance activity in combination with cytotoxic
drugs or agents targeting angiogenesis [65,70–72]. Zhai and
colleagues reported that APG-2449, a novel ALK/ROS1/FAK
inhibitor, is effective against human ovarian tumor either alone
or in combination with other therapies [73]. Moreover, researchers
have studied many potential compounds targeting the β-catenin
signaling pathway in pre-clinical and phase I/II trials [74]. For
instance, WNT974 has been shown to produce cytostatic effects in
ascites cells from patients with primary OC [75]. Despite various
pre-clinical evaluations of potential therapies targeting PTK2 and
β-catenin, clinical applications still face significant challenges.
This study describes a novel mechanism by which the NRSN2-
AS1/PTK2/β-catenin axis underlies OC progression (Fig. 8). NRSN2-
AS1 interacts with PTK2 and protects it from MG53-mediated
polyubiquitination and subsequent degradation, thereby regulat-
ing the activity of β-catenin. In the absence of NRSN2-AS1, MG53
mediates the degradation of PTK2 via ubiquitination signaling.
Despite its significant results, this study had some shortcomings.
Fig. 7 Effects of MG53 on NRSN2-AS1/PTK2/β-catenin pathway in OC cells. A OC cells treated with CHX for 0 h, 1 h, 2 h, and 4 h were
analyzed by western blotting for PTK2 expression. BQuantification of results from (A), n=3. CAfter MG132 (20 μM) treatment for 6 h, IP assays
were performed with anti-IgG or anti-PTK2 in OVCAR3 and A2780 cells transfected with si-NRSN2-AS1. Immunoblot (IB) analysis of total-
ubiquitin (Ub), Lys-48 ubiquitin (Ub-K48), MG53 and PTK2 in IgG- and PTK2-immunoprecipitated products and whole-cell lysate (WCL).
DExpression of MG53 in 419 tumor and 88 normal tissues based on TCGA. ERelative mRNA level of MG53 was detected in 23 paired OC tissues
by RT-qPCR. FCCK8 assays were used to verified OC cell viability after overexpressing MG53 (n=6). G, H Colony formation assays were used to
determine the proliferation ability (n =3). I, J Transwell assays were used to investigate OC cell migration (n=3). Scale bar: 100 μm. KIB analysis
of Ub and Ub-K48 levels of PTK2 in IgG- and PTK2-immunoprecipitated products and WCL-derived cells transfected with EV and OE-MG53. LIB
analysis of Ub level of PTK2 in IgG- and PTK2-immunoprecipitated products and WCL-derived cells transfected with si-NC, si-NRSN2-AS1, or si-
NRSN2-AS1+si-MG53. MIB analysis of Ub level of PTK2 in IgG- and PTK2-immunoprecipitated products and WCL-derived cells transfected with
EV, OE-NRSN2-AS1, or OE-NRSN2-AS1 +OE-MG53. N, O With tubulin as an internal control, western blot analysis was used to determine the
expression of PTK2, GSK3
Y279/Y216
,β-catenin, and β-catenin
Y142
in OC cells transfected with si-NC, si-NRSN2-AS1, or si-NRSN2-AS1+si-MG53,
n=3 for each group. P, Q The protein level of PTK2, GSK3
Y279/Y216
,β-catenin, and β-catenin
Y142
were determined by western blot in OC cells
transfected with EV, OE-NRSN2-AS1, or OE-NRSN2-AS1 +OE-MG53, n=3 for each group. *P< 0.05, **P< 0.01, ***P< 0.001.
Y. Wu et al.
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Cell Death and Disease (2023) 14:696
All clinical specimens came from the same source and were
relatively few. Therefore, the findings regarding clinical features
such as TNM or grade cannot be generalized. Multi-center studies
with a larger sample size are needed. However, this study provides
a better understanding to the role of NRSN2-AS1 in the PTK/
β-catenin pathway in OC. Further study of the clinical implications
of the NRSN2-AS1/PTK2/β-catenin pathway may contribute to
early OC diagnosis and treatment.
DATA AVAILABILITY
All data are available from the corresponding author upon reasonable request.
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ACKNOWLEDGEMENTS
The authors wish to thank all study participants, research staff, and students who
assisted with this work.This work was supported by the National Key Research and
Development Program of China (2021YFC2700602), the Suzhou Municipal Hospital
Gynecological Clinical Trial and Improvement Project (SLT201955); the Maternal and
Child Health Research Project of Jiangsu Province (F201904); the Top Talent Support
Program for Young and Middle-aged People of Wuxi Health Committee (BJ2020047),
the Introduce Project of Clinical Medicine Experts of Suzhou Industrial Park
(SZYQTD202104), and the Non-profit Central Research Institute Fund of Chinese
Academy of Medical Sciences (2019PT310002).
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AUTHOR CONTRIBUTIONS
YW: Conceptualization, Funding acquisition. SL: Investigation, Data curation, Writing –
original draft, Formal analysis. JL: Investigation, Data curation, Writing –original draft.
Jiajia Xue: Investigation, Data curation, Methodology. Jinfu Xu: Investigation,
Validation, Visualization. Ting Chen: Investigation, Data curation. Tianyue Cao:
Investigation. HZ: Investigation. TW: Validation, Visualization. Chunlin Dong:
Investigation. W-FQ: Writing revised draft. LQ: Project administration. SH: Supervision,
Project administration, Funding acquisition. TW: Investigation, Project administration.
CS: Investigation, Methodology.
COMPETING INTERESTS
The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict
of interest.
ETHICS
The approval of patients was received from the Research Ethics Committee of Suzhou
Municipal Hospital. All animal experimental were approved by the Animal Ethics and
Welfare Committee of Nanjing Medical University.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41419-023-06214-z.
Correspondence and requests for materials should be addressed to Long-Wei Qiao,
Shun-Yu Hou, Ting Wang or Cong Shen.
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