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Liu et al. Biology Direct (2024) 19:59
https://doi.org/10.1186/s13062-024-00506-w Biology Direct
†Xiaofang Liu, Yang Chen and Ying Li contributed equally to this
work.
*Correspondence:
Yaodong Dong
dydlxf_1218@163.com
Yingying Zhou
zyy03300821@163.com
1Department of Anus and Intestine Surgery, The First Aliated Hospital of
China Medical University, Shenyang, Liaoning, People’s Republic of China
2Department of General Surgery, The First Aliated Hospital of Liaoning
University of Traditional Chinese Medicine, Shenyang, Liaoning, People’s
Republic of China
3Department of Obstetrics and Gynecology, Shengjing Hospital of China
Medical University, No. 36, Sanhao Street, Shenyang, Liaoning
110004, People’s Republic of China
4Department of Otolaryngology Head and Neck Surgery, Shengjing
Hospital of China Medical University, No. 36, Sanhao Street, Shenyang,
Liaoning 110004, People’s Republic of China
Abstract
Background To investigate the role of lncRNA LINC00665 in modulating ovarian cancer stemness and its inuence
on treatment resistance and cancer development.
Methods We isolated ovarian cancer stem cells (OCSCs) from the COC1 cell line using a combination of
chemotherapeutic agents and growth factors, and veried their stemness through western blotting and
immunouorescence for stem cell markers. Employing bioinformatics, we identied lncRNAs associated with
ovarian cancer, with a focus on LINC00665 and its interaction with the CNBP mRNA. In situ hybridization,
immunohistochemistry, and qPCR were utilized to examine their expression and localization, alongside functional
assays to determine the eects of LINC00665 on CNBP.
Results LINC00665 employs its Alu elements to interact with the 3’-UTR of CNBP mRNA, targeting it for degradation.
This molecular crosstalk enhances stemness by promoting the STAU1-mediated decay of CNBP mRNA, thereby
modulating the Wnt and Notch signaling cascades that are pivotal for maintaining CSC characteristics and driving
tumor progression. These mechanistic insights were corroborated by a series of in vitro assays and validated in vivo
using tumor xenograft models. Furthermore, we established a positive correlation between elevated CNBP levels
and increased disease-free survival in patients with ovarian cancer, underscoring the prognostic value of CNBP in this
context.
Conclusions lncRNA LINC00665 enhances stemness in ovarian cancer by mediating the degradation of CNBP mRNA,
thereby identifying LINC00665 as a potential therapeutic target to counteract drug resistance and tumor recurrence
associated with CSCs.
Keywords Ovarian cancer, Cancer stem cells, LINC00665, CNBP, Stemness regulation, mRNA decay, Long non-coding
RNA
STAU1-mediated CNBP mRNA degradation
by LINC00665 alters stem cell characteristics
in ovarian cancer
XiaofangLiu1†, YangChen2†, YingLi3†, JinlingBai3, ZhiZeng3, MinWang3, YaodongDong4* and YingyingZhou3*
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 2 of 22
Liu et al. Biology Direct (2024) 19:59
Background
Cancer stem cells (CSCs) harbor the distinctive capacity
for self-renewal, establishing themselves as a specialized
subset that contributes to the complexity and malignancy
of various solid tumors [1, 2]. ese cells are adept at
multidrug resistance [3], evading immune surveillance,
and facilitating invasion and metastasis [4–7]. In specific
microenvironments, CSCs can differentiate into diverse
cell types, including cancerous cells, which are implicated
in cancer recurrence and metastasis [2, 8, 9]. Moreover,
cancer cells can dedifferentiate back into a CSC pheno-
type [10], thereby escaping the effects of chemotherapeu-
tic drugs that target rapidly dividing cells. Investigating
the stemness dynamics within ovarian cancer cells is
therefore of critical importance, as it sheds light on the
progression, recurrence, metastasis, and drug response of
ovarian cancer, emphasizing the significance of CSCs as
targets for therapeutic strategies.
Alu elements are short, repetitive sequences widely dis-
persed throughout the human genome [11]. ey are a
type of transposable element that can affect gene expres-
sion and genome stability [11]. Alu sequences have been
implicated in various diseases, including cancer, due to
their ability to mediate genomic rearrangements and
influence gene regulation [12]. Recent studies have high-
lighted the role of Alu elements in RNA-RNA interac-
tions, contributing to the regulation of mRNA stability
and translation [12]. In the context of long non-coding
RNAs (lncRNAs), Alu sequences within lncRNAs can
facilitate the formation of RNA duplexes with comple-
mentary Alu elements in target mRNAs, thereby modu-
lating their stability and expression [13]. However, the
specific mechanisms by which lncRNAs regulate target
gene expression through the formation of RNA duplexes
with complementary Alu elements in target mRNAs,
particularly in the context of ovarian cancer progression,
remain largely unclear.
Staufen1 (STAU1) is a double-stranded RNA-binding
protein that plays a significant role in mRNA localiza-
tion, stability, and decay [14]. It is involved in a process
known as Staufen-mediated mRNA decay (SMD), where
STAU1 binds to specific mRNA 3’-untranslated regions
(3’-UTRs), leading to mRNA degradation [14]. e inter-
action between STAU1 and Alu elements within mRNAs
has been demonstrated to regulate the stability of vari-
ous transcripts, thereby influencing gene expression [14].
is action effectively regulates the mRNA’s abundance
and its post-transcriptional control [15, 16]. e involve-
ment of lncRNA in mRNA decay represents a significant
mechanism that has been validated across various tumor
entities [14]. LncRNAs can recruit STAU1 to target
mRNAs through Alu-mediated RNA duplex formation,
promoting their degradation via SMD and influencing
cancer progression [17, 18]. Nonetheless, the precise
mechanisms by which lncRNA-mediated mRNA decay
via the SMD pathway modulates the malignant features
in ovarian cancer cells remain to be fully delineated.
Cellular Nucleic Acid Binding Protein (CNBP) is a
highly conserved nucleic acid-binding protein that pos-
sesses seven zinc finger motifs of the CCHC type and a
region abundant in arginine and glycine (RG/RGG) [19].
It is capable of binding to nucleic acids and plays a role in
the regulation of various disorders, including neuromus-
cular degeneration, inflammation, autoimmune condi-
tions, and cancers [19]. In the context of cancer, CNBP
has been shown to influence tumor growth and metas-
tasis by regulating the stability and translation of specific
mRNAs [20]. Currently, little is known about the role of
CNBP in ovarian cancer.
In previous research, lncRNA LINC00665 was iden-
tified to show increased expression in ovarian can-
cer tissues, as determined by lncRNA expression
profiling microarrays and bioinformatics analysis [21]. In
this study, we investigate the role of LINC00665 in ovar-
ian cancer progression, focusing on its interaction with
Alu sequences, CNBP, and STAU1. We hypothesize that
LINC00665 forms duplex structures with the 3’-UTRs
of target mRNAs via Alu elements, facilitating STAU1-
mediated CNBP mRNA decay and thus promoting ovar-
ian cancer stem cell-related phenotypes. Building on this
discovery, our current study reveals that LINC00665
affects the shift towards stemness in ovarian cancer
cells by controlling CNBP mRNA stability via the SMD
pathway and by altering β-catenin levels in the nucleus
through the Wnt signaling pathway. ese findings sug-
gest that targeting LINC00665 could be a new approach
to modulate stemness in ovarian cancer cells, offering a
potential avenue for therapeutic intervention in the treat-
ment of cancer progression and chemoresistance.
Methods
Collection of patient samples
We acquired 40 serous ovarian cancer specimens, fixed
in formalin and embedded in paraffin, from individuals
admitted to Shengjing Hospital of China Medical Univer-
sity within the timeframe of 2012 to 2017. ese patients
underwent conventional surgical or debulking proce-
dures tailored to the stage of their cancer, with none
having been subjected to chemotherapy or radiotherapy
prior to surgery. Consent was duly obtained from all par-
ticipants, and the protocol for collecting and handling
patient data received approval from the Ethics Commit-
tee of China Medical University [No. 2019PS286K(X1)].
Cultivation of cells and pharmacological evaluation
e human serous epithelial ovarian carcinoma cell
lines COC1 and SKOV3 were acquired from the China
Center for Type Culture Collection. ese cells were
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Page 3 of 22
Liu et al. Biology Direct (2024) 19:59
incubated at 37 °C in a 5% CO2 atmosphere. e cul-
ture was sustained in RPMI-1640 medium (31800-014,
Gibco, Bristol, RI, USA), enriched with 10% fetal bovine
serum (SH30084.03, Hyclone, Logan UT, USA), and the
medium was refreshed after 24h of incubation. is pro-
cess was repeated until the cells reached 80% confluence.
After exposure to cisplatin (40 µmol/l) and paclitaxel (10
µmol/l) [22], the cells were incubated for an additional 5
days [23]. ereafter, the cells were propagated in condi-
tions conducive to stem cell growth: RPMI-1640 medium
(31800-014, Gibco), supplemented with recombinant
human insulin (5µg/ml) (11061-68-0, Solarbio, Beijing,
China), EGF (10 ng/ml) (10,605-HNAE, Sino Biological,
Beijing, China), bFGF (10 ng/ml) (10,014-HNAE, Sino
Biological, China), and LIF (12 ng/ml) (RPA085Hu01,
Cloud-Clone Corp., Katy, TX, USA). e medium was
replaced bi-daily. After 6 days, cells were harvested for
subsequent analysis of gene and protein expression.
Assay for sphere formation
To separate the spherical cell clusters, the cultures were
treated with 0.25% trypsin–EDTA for 1–2min at 37°C.
Subsequently, 100 cells were seeded per well into 96-well
plates containing 200µl of growth medium, and an addi-
tional 25µl of the medium was supplemented to each
well every two days. e count of dissociated spherical
cells in each well was tallied following a 7-day incubation
period.
Flow cytometry analysis
Separated cells underwent centrifugation at 1000 rpm
for 5 min and were subsequently retrieved. e cells
underwent a dual wash with phosphate-buffered saline
(PBS; P10033, Doublehelix, Shanghai, China) and were
gathered post-centrifugation at 1000 rpm for 5 min.
1 × 106 cells were suspended in 100µl of PBS containing
anti-CD133 (12-1339-41, APC, eBioscience, San Diego,
CA, USA), anti-CD117 (11-1178-41, APC, eBiosci-
ence, USA), and isotype control antibodies (non-specific
mouse IgG for CD133 and CD117, 70-CMG105-10, and
70-CMG104-10, MultiSciences, Hangzhou, China). e
proportions of CD133+ and CD117+ cells were ascer-
tained through flow cytometry following a period of
incubation in darkness.
Western blotting
Proteins were isolated from COC1, SKOV3, and spher-
oid cell populations. e protein concentrations were
quantified, and aliquots were prepared by mixing with 5×
loading buffer and PBS to achieve a final concentration
of 40µg of protein in a 20µl volume. Proteins were then
subjected to SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) at 80V for 2.5h, followed by transfer onto
a polyvinylidene fluoride membrane (PVDF; IPVH00010,
Millipore Sigma, Burlington, MA, USA). e membranes
were then blocked with non-fat dry milk and incubated
with primary antibodies at 4 °C overnight, followed by
incubation with the corresponding secondary antibod-
ies at 37°C for 45min. e protein bands were visual-
ized using enhanced chemiluminescence (ECL) Western
blot detection reagents (Pierce, ermo Fisher Scientific,
Waltham, MA, USA) and captured with a Gel-Pro-Ana-
lyzer (WD-9413B; Beijing Liuyi, Beijing, China). β-actin
(WL01845; Wanleibio, Shenyang, China) and histone H3
served as internal controls for normalization. e anti-
bodies utilized for the immunoblotting are detailed in
Table1.
Quantitative PCR (qPCR)
RNA was isolated utilizing an RNA isolation kit (RP1201;
BioTeke, Beijing, China), and its purity and concentra-
tion were assessed. 1µg of the isolated RNA was incor-
porated into a 19µl mix for reverse transcription using
the PrimeScript™ RT reagent kit with gDNA Eraser
Table 1 Antibodies used in Western blotting analysis
Rabbit anti-human OCT4 antibody 1:500 Abcam, ab18976, Cambridge, UK 4°C overnight
Rabbit anti-human SOX2 antibody 1:500 Abcam, ab97959 4°C overnight
Rabbit anti-human NANOG antibody 1:500 Abcam, ab80892 4°C overnight
Rabbit anti-human ALDH1 antibody 1:500 Abclonal, A0157, Hubei, China 4°C overnight
Rabbit anti-human LGR5 antibody 1:500 Abclonal, A12327 4°C overnight
CNBP antibody 1:400 Abclonal, A15110 4°C overnight
Ki-67 antibody 1:500 Wanleibio, WL01384a 4°C overnight
STAU1 antibody 1:500 Proteintech, 4225-1-AP, Rosemont, IL, USA 4°C overnight
E-cadherin antibody 1:500 Wanleibio, WL01482 4°C overnight
β-catenin antibody 1:500 Wanleibio, WL0962a 4°C overnight
MDR1 antibody 1:500 Wanleibio, WL02395 4°C overnight
β-actin antibody 1:1000 Wanleibio, WL01845 4°C overnight
Histone H3 antibody 1:1000 Wanleibio, WL0984a 4°C overnight
Goat anti-rabbit secondary antibody 1:5000 Abcam, ab7090 37°C 45min
Goat anti-rabbit secondary antibody 1:5000 Wanleibio, WLA023 37°C 45min
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 4 of 22
Liu et al. Biology Direct (2024) 19:59
(Perfect Real Time) (RR037Q; Takara, Beijing, China).
e expression levels of genes were quantified employing
SYBR Green I nucleic acid gel stain (SY1020; Solarbio) on
the Exicycler 96 Real-Time Quantitative ermal Block
(Bioneer, Daejeon, Korea). e sequences of the primers
utilized are listed in Table2. e thermal cycling condi-
tions were set as follows: initial denaturation at 94°C for
10min, followed by 40 cycles of denaturation at 94°C for
10s, annealing at 60°C for 20s, and extension at 72°C
for 30s, with a final extension at 72°C for 2min and 30s,
and a terminal hold at 40°C for 5min and 30s. e melt-
ing curve analysis ramped from 60 to 94°C, increasing by
1.0°C every second, and concluded with a cooling step at
25°C for 1min. β-actin served as the endogenous refer-
ence gene for data normalization.
Immunocytochemistry
Cells were seeded onto 8-well chamber slides and cul-
tured for 24–48h. e slides were then fixed, rinsed, and
blocked with 1% bovine serum albumin in a tris-buffered
saline with Tween 20 (TBST) solution, followed by an
overnight incubation at 4 °C with the designated pri-
mary antibody. Afterward, the slides were washed thrice
with TBST for 10min at 25 °C and subsequently incu-
bated with the appropriate secondary antibody for 1 h
at ambient temperature. Post-secondary antibody incu-
bation, the slides were washed, stained with 0.5mg/mL
4′,6-diamidino-2-phenylindole (DAPI) for 10 min, and
then mounted using a fluorescence quenching prevention
medium (S2100; Solarbio). Images were captured with a
BX53 microscope (OLYMPUS, Tokyo, Japan). Omission
of the primary antibody in the blocking solution served
as the negative control. e primary and secondary anti-
bodies utilized are detailed in Table3.
Immunohistochemical analysis
Sections were dewaxed, immersed in antigen unmask-
ing solution, and subjected to continuous heating for
10min; the sections were then dried and treated with 3%
hydrogen peroxide, followed by a 15-minute incubation
at ambient temperature; normal goat serum was applied
in a dropwise manner, and the tissue samples were fur-
ther incubated for 15 more minutes at ambient tempera-
ture. e samples were subsequently incubated with the
primary antibody (CNBP antibody at a 1:100 dilution in
PBS) overnight within a humidified chamber at 4°C. e
samples were then treated with HRP-conjugated goat
anti-rabbit IgG (1:500, #31,460; ermo Fisher, USA) for
1h at 37°C, visualized using DAB chromogen (ermo
Fisher, USA), and counterstained with hematoxylin. Fol-
lowing this, the sections were dehydrated using abso-
lute ethanol, cleared in xylene, set with neutral gum, and
imaged using a 400× magnification microscope (DP73;
OLYMPUS).
In situ hybridization
Tissue samples underwent staining, proteinase K treat-
ment, denaturation, and subsequent in situ hybridization
with probes for LINC00665 and CNBP (synthesized by
Wanleibio, China). Probe detection was carried out using
Table 2 Sequences of primers for qPCR assay
Name Sequence Primer length Tm Product length
Linc00665 F G G T G C A A A G T G G G A A G T G T G 20 58.4 191
Linc00665 R A G T C C G G T G G A C G G A T G A G A A 21 63.9
snRNA F C T T C A A G A C T C T C T T C G T G G 20 52.0 196
snRNA R G C C A T C T G C G T G T T T G T A A G 20 56.6
TDGF1 F A T T T G C T C G T C C A T C T C G 18 53.6 139
TDGF1 R G G T T C T G T T T A G C T C C T T A C T G 22 53.5
β-actin F G G C A C C C A G C A C A A T G A A 18 57.7 137
β-actin R C G G A C T C G T C A T A C T C C T G C T 21 59.3
CNBP F T T C C A G T T T G T T T C C T C G T C 20 55 186
CNBP R G C C A C A G T T G T A G C A G C A T 19 53.9
STAU1 F A T C C G A T T A G C C G A C T G G 18 55.5 246
STAU1 R A C T T G A G T G C G G G T T T G G 18 56.2
Table 3 Antibodies used in immunouorescence staining
Rabbit monoclonal antibody Lgr5 1:100 Novus Biologicals, MAB8078-SP, Centennial, CO, USA 4°C overnight
Rabbit polyclonal antibody ALDH1 1:300 Wanleibio, WL02762 4°C overnight
Rabbit monoclonal antibody SOX2 1:100 Abclonal, A0561 4°C overnight
Rabbit monoclonal antibody OCT4 1:100 Anity Biologicals, AF0226, Shanghai, China 4°C overnight
Rabbit monoclonal antibody Nanog 1:100 Anity, AF5388 4°C overnight
FITC-conjugated goat anti-rabbit IgG 1:100 Abcam, ab6717 Room temperature, 1h
Cy3-conjugated goat anti-mouse IgG 1:200 Invitrogen, A-21,424 Room temperature, 1h
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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Liu et al. Biology Direct (2024) 19:59
a fluorescence in situ hybridization kit (GenePharma,
Shanghai, China) as per the manufacturer’s protocol,
after which the tissue samples were counterstained with
DAPI and examined under a fluorescence microscope.
Cellular transduction
Overexpression lentiviral vectors for LINC00665
(NR_038278.1, 1749bp in length) and knockdown len-
tiviral vectors for CNBP were employed to transduce
COC1 and SKOV3 cells, respectively. In addition, knock-
down vectors for LINC00665 and overexpression vec-
tors for CNBP (NM_001127192.2, CDS = 540 bp) were
used to transduce OCSCs, and knockdown vectors for
STAU1 were used to transduce OCSCs or co-transduce
COC1 cells with LV-LINC00665. Cells were harvested
for analysis at the designated time points. STAU1 knock-
down shRNA and control sequences were synthesized,
and SKOV3 cells in the exponential growth phase were
transfected with Lipofectamine 3000 to introduce the
STAU1 knockdown fragments. e shSTAU1 with the
most effective knockdown was selected for subsequent
experiments.
Luciferase reporter assay
We identified an Alu element within LINC00665 and the
3’-UTR of CNBP mRNA [1485–1631bp (5’-3’)] through
analysis with RepeatMasker software. e Alu elements
were further analyzed using RNA_RNA_Anneal software,
revealing a 132bp complementary sequence with a free
energy of -222.2kcal/mol, suggesting a potential STAU1
binding site. We constructed the luciferase reporter vec-
tors pmirGLO-CNBP-wtUTR with the wild-type (wt)
CNBP mRNA 3’-UTR segment and pmirGLO-CNBP-
mutUTR with mutations (mut) in the binding site.
Additionally, we created an overexpression vector for
LINC00665 (NR_038278.1, length = 1749 bp) and co-
transfected SKOV3 cells with either pmirGLO-CNBP-
wtUTR or pmirGLO-CNBP-mutUTR. Luciferase activity
was measured to assess the interaction.
RNA binding protein immunoprecipitation (RIP) assay
Cell lysates were prepared and combined with RIP
immunoprecipitation buffer containing immunomag-
netic beads, followed by incubation at 4°C ranging from
3h to overnight. After a brief centrifugation, the beads
were immobilized using a magnetic separator, and the
supernatant was discarded. e beads were then washed
with RIP wash buffer. RNA was isolated from the immu-
noprecipitation complexes by protein digestion, and the
presence of target RNA was detected through reverse
transcription and quantitative PCR. To generate bar
graphs from RNA immunoprecipitation (RIP) results, we
extracted RNA post-RIP with a specific antibody, evalu-
ated CNBP mRNA and LINC00665 levels via qPCR, and
normalized them to β-actin. Using the 2−ΔΔCt method,
we calculated relative expression, followed by statistical
analysis to determine the mean and standard deviation,
typically from triplicate experiments. Finally, we used
SPSS and GraphPad Prism to create bar graphs repre-
senting relative expression levels with error bars indicat-
ing standard deviation (SD).
MS2-RIP analysis
We constructed a LINC00665 expression and mutant
construct that included an Alu sequence and an MS2
hairpin loop with an MS2 binding site (Supplymen-
tary Table2). ese constructs were co-transfected into
OCSCs along with GFP expression vectors (pMS2-GFP).
Forty-eight hours post-transfection, we conducted a
RIP assay on the cells using an anti-GFP antibody with
the Magna RIP RNA binding protein immunoprecipita-
tion kit (Millipore, Bedford, MA, USA) according to the
manufacturer’s protocol. We assessed CNBP mRNA lev-
els in the immunoprecipitants by qPCR and STAU1 pro-
tein levels by western blot analysis. After altering STAU1
levels, we performed the RIP assay again and measured
CNBP mRNA levels in the immunoprecipitants using
qPCR.
Proliferation assay
Each group’s cells were plated in 96-well plates at 3.5 × 103
cells per well, with quintuplicate wells per group. After
overnight incubation, cells underwent viral transduc-
tion or co-transduction to alter the expression of spe-
cific genes. Post 48-h incubation at 37°C and 5% CO2,
cell proliferation was evaluated using the CCK-8 assay as
per the kit’s instructions. In chemosensitivity assays, cells
were treated with varying cisplatin concentrations (0, 10,
30, 50 µM) 48h post-viral transduction. After an addi-
tional 48-h incubation, cell viability was determined by
adding 10µl of CCK-8 solution to each well and incubat-
ing for 2h at 37°C in 5% CO2. Absorbance at 450nm was
recorded using a microplate reader.
Apoptosis and cell cycle analysis
Apoptosis assay: Cells from each group were cultured in
6-well plates at 5 × 105 cells per well and harvested at the
specified time point. Cells were centrifuged and resus-
pended in 500µl of binding buffer. Following the apop-
tosis detection kit’s protocol, 5 µl of Annexin V-Light 650
was added and mixed thoroughly. en, 10µl of prop-
idium iodide was added, mixed, and incubated at room
temperature in the dark for 15min before flowcytometry.
Cell cycle analysis: Cells were collected, fixed with 70%
ethanol at 4°C for 2h, centrifuged, and washed. e cells
were then treated with 100µl of RNase A at 37°C for
30min. Following this, 500 µl of propidium iodide was
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Page 6 of 22
Liu et al. Biology Direct (2024) 19:59
added, and the cells were incubated at 4°C in the dark for
30min before being analyzed by flow cytometry.
Migration and invasion assay
Transwell inserts with a polycarbonate membrane were
placed in 24-well plates and coated with a Matrigel matrix
(ermo Fisher, USA) to solidify. e lower chamber was
filled with 800 µl of medium containing 10% FBS, and
the upper chamber was filled with 200µl of cell suspen-
sion (3 × 105 cells/well for COC1 and OCSC, and 2 × 104
cells/well for SKOV3 cells). For the migration assay, the
same medium setup was used, and 200µl of cell suspen-
sion (1 × 105 cells/well for COC1 and OCSC, and 5 × 103
cells/well for SKOV3 cells) was added to the upper cham-
ber. After incubation, Transwell inserts with invaded or
migrated cells were washed, fixed, stained, and rinsed at
room temperature. OCSCs, COC1, and SKOV3 cells on
the underside of the membrane were counted using an
inverted microscope. e average number of cells from
three fields of view was calculated for each sample.
Assessment of colony formation
OCSC and COC1 cells were subjected to a colony for-
mation evaluation by seeding them in a medium supple-
mented with 0.8% methylcellulose at a concentration of
500 cells per plate. ese plates were incubated at 37°C
in an atmosphere containing 5% CO2 for a duration of
14 days before image capture. SKOV3 cells were simi-
larly seeded and incubated under the same conditions.
Post-incubation, the cells were treated with R2 reagent
for staining and subsequently scanned to identify colo-
nies. e rate of colony formation was calculated using
the formula: (total colonies formed/initial cells seeded) ×
100%.
Assessment of RNA stability
Two days after transfection, the cells were exposed to
actinomycin D (10 µg/ml). e culture was continued,
and at predetermined intervals (0, 0.5, 1, 2, 4, 8, and
16 h), mRNA was isolated. e abundance of CNBP
mRNA at these time points was quantified via qPCR, and
the mRNA half-life was deduced.
Ovarian cancer xenograft model
Female BALB/c athymic nude mice, aged six weeks, were
acclimatized for one week in a controlled environment
with a 12-h light/dark cycle, at 22 ± 1 °C and 45–55%
humidity, with free access to food and water. e xeno-
graft model was established by subcutaneous injection
of 1 × 105 OCSC (spheroids) into the mice. After a four-
week period, the mice were sacrificed, and the tumors
were harvested for further examination. e onset and
growth of tumors were closely monitored, and tumor vol-
umes were calculated using the formula: Tumor volume
(mm3) = (longest diameter × shortest diameter2) × 0.5.
Chromatin immunoprecipitation (ChIP)
Primers were designed based on the predicted NFYA
binding sites within the LINC00665 promoter region,
as suggested by Jaspar. e effectiveness of these prim-
ers was confirmed by PCR using genomic DNA as a
template. Cells were treated with formaldehyde for DNA-
protein cross-linking, and the chromatin was subse-
quently fragmented ultrasonically. Protein Agarose, and
Anti-NFYA antibody (100,575, Sino Biological, Wayne,
PA, USA) was introduced to the supernatant for incuba-
tion. Following this, the immunoprecipitated complexes
were isolated after further incubation with Protein Aga-
rose beads. e complexes were then washed, and the
cross-links between DNA and protein were reversed
using NaCl. Finally, the DNA of interest was purified and
subjected to PCR analysis to confirm the presence of the
target sequences. e sequences of all primers utilized in
the experiments are detailed in Table4.
Bioinformatics analysis
To ascertain the long non-coding RNAs (lncRNAs) and
protein-encoding genes, we utilized the RNA V5 platform
(4*180K, Design ID: 076500; Agilent Technologies, Inc.,
Santa Clara, CA, USA) to scrutinize the lncRNAs and
genes that were differentially expressed between OCSCs
and COC1 cells. e procedures were executed in align-
ment with the guidelines provided by the manufacturer.
e differentially expressed lncRNAs (DELs) and genes
(DEGs) were pinpointed by evaluating the fold change
(FC), with the cut-off for upregulated and downregulated
genes set at an absolute FC value of 2.0 or greater. In the
end, we selected a cohort of 325 lncRNAs that exhibited
an absolute FC value of 2 or higher and arranged them
in an ascending sequence based on the absolute value
of the log fold change (Refer to Supplementary Table 1
for the leading 50 lncRNAs). Given that Staufen-medi-
ated mRNA decay (SMD) is contingent upon the base
pairing between the Alu sequence in the lncRNA and
Table 4 Primer sequences for PCR
Name Sequence Product length
LINC00665 F A G G A A A C A G C A C C A A G G G 192
LINC00665 R C G C T C A G T C A G C C T C A A A
GAPDH F T A C T A G C G G T T T T A C G G G C G 166
GAPDH R T C G A A C A G G A G G A G C A G A G A G C G A
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Liu et al. Biology Direct (2024) 19:59
the Alu sequence in the 3’-UTR of the mRNA, our ini-
tial focus was to identify lncRNAs harboring an Alu
sequence. Employing the RepeatMasker tool, we discov-
ered an Alu sequence within LINC00665 (Supplementary
Fig.1). We retrieved the harmonized and standardized
pan-cancer dataset from the UCSC database (PANCAN,
N = 19,131, G = 60,499; https://xenabrowser.net/), which
allowed us to extract the expression data for LINC00665
(ENSG00000232677) across various sample types. We
then conducted a more refined screening of the sam-
ples, including solid tissue normal, primary solid tumor,
primary tumor, normal tissue, primary blood-derived
cancer from bone marrow, and primary blood-derived
cancer from peripheral blood. Each expression value was
transformed using a log2(x + 1) conversion.
Collection of clinical information
We compiled data from 80 individuals diagnosed with
epithelial ovarian cancer who underwent standard sur-
gical intervention or tumor debulking tailored to the
stage of cancer, with comprehensive clinical records and
pathological paraffin-embedded specimens. is cohort
consists of 40 cases collected during the initial pre-exper-
imental stage from 2012 to 2017, and an additional 40
cases collected from January 2019 to December 2023, fol-
lowing the establishment of a new clinical sample bank in
our department at Shengjing Hospital of China Medical
University. ese patients had not received any preopera-
tive treatments. e cut-off for the follow-up period was
set for December 2023. Pathological confirmation of epi-
thelial ovarian cancer was obtained for all tissue samples,
which were preserved in paraffin blocks. Clinical data of
these patients have been shown in Supplementary Table
3. Additionally, a control group consisting of 15 patients
with ovarian serous cystadenoma, who underwent either
cyst nucleotomy or resection of the affected adnexa, was
established. Informed consent was duly obtained from
all participants, and the study received approval from
the Ethics Committee of China Medical University [No.
2019PS286K(X1)].
Statistical evaluation
e statistical analysis was conducted using software
packages SPSS 27.0 (IBM, SPSS, Chicago, IL, USA) and
GraphPad Prism 9.0 (GraphPad Software Inc., Bos-
ton, MA, USA). Quantitative data were expressed as
mean ± standard deviation, while categorical data were
presented in percentages. e comparison of mean val-
ues between two groups with similar variances was per-
formed using either the Student’s t-test (for two samples)
or one-way analysis of variance (ANOVA). To assess
significant differences, non-parametric tests such as the
Unpaired Wilcoxon Rank Sum and Signed Rank Tests
were employed. Survival rates were determined using
the Kaplan–Meier plotter [24] and the log-rank test,
while Pearson correlation analysis was utilized to exam-
ine the relationships between different groups. A P-value
of less than 0.05 was considered to indicate statistical
significance.
Results
Isolation and identication of ovarian cancer stem cells
e isolation of cancer stem cells can be achieved by
sorting side population (SP) cells from ovarian cancer
cell populations through the efflux of Hoechst 33,342
dye, a process that can be inhibited by verapamil treat-
ment [25]. Alternatively, the presence of cancer stem
cells can be confirmed by identifying cells expressing sur-
face markers indicative of pluripotency, such as CD44+,
CD133+, and CD117+, using flow cytometric analysis
[24–26]. In our quest to understand the factors influ-
encing the stemness characteristics of ovarian cancer
cells, we adopted a low-density cell culture technique.
e COC1 ovarian cancer cell line was cultivated in a
serum-deprived medium supplemented with various
growth factors to promote the emergence of cells with
stem-like properties. e surface markers CD117+ and
CD133+ were utilized to identify ovarian cancer stem
cell-like cells. Following treatment with chemothera-
peutic agents (cisplatin and paclitaxel), COC1 cells were
maintained in a medium conditioned for stem cells,
which included recombinant human insulin, EGF, bFGF,
and LIF. Analysis via flow cytometry of the resultant cell
aggregates indicated a significantly greater frequency of
CD117+/CD133+ cells compared to untreated COC1
cells (*P < 0.05). e presence of stem cell-associated pro-
teins was assessed through western blotting and immu-
nofluorescence techniques, with a comparative analysis
against COC1 cells. is analysis revealed elevated levels
of stemness-related proteins SOX2, OCT4, and NANOG
in the treated cells. Additionally, the levels of ALDH1 and
LGR5, two specific markers for ovarian cancer stem cells,
were found to be enhanced (Supplementary Fig.2). ese
CD117+/CD133+ cells were thus designated as ovarian
cancer stem cells (OCSCs).
Bioinformatics-based prediction of protein-coding and
long non-coding RNAs involved in stemness regulation in
ovarian cancer cells
Dysregulated lncRNAs play a pivotal role in the stem-
ness transition of neoplastic cells [27–30]. LncRNAs can
modulate the expression of protein-coding genes either
directly or indirectly [29]. Our findings indicated that
in epithelial ovarian cancer, LINC00665 levels were sig-
nificantly elevated in tumor samples compared to normal
ovarian tissues (tumor: 5.75 ± 0.92; normal: 4.12 ± 0.51,
P = 0.013) (Fig. 1A, a). Kaplan–Meier survival analysis of
ovarian cancer cases from the GEO, EGA, and TCGA
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Page 8 of 22
Liu et al. Biology Direct (2024) 19:59
Fig. 1 Prediction and validation of RNAs associated with the regulation of stemness of ovarian cancer cells (A) a. Dierential expression of LINC00665
in epithelial ovarian cancer versus normal tissues analyzed by TCGA TARGET GTEx. b. Relationship between LINC00665 and ovarian cancer prognosis
analyzed by Kaplan–Meier plotter. c. Expression and cellular localization of LINC00665 in benign and malignant ovarian tumors detected by in situ hy-
bridization. d. Dierential expression of LINC00665 in OCSC and epithelial ovarian cancer cells detected by qPCR. (B) a. Dierential expression of CNBP
in epithelial ovarian cancer versus normal tissues analyzed by GENT2. b. The relationship between CNBP expression and ovarian cancer prognosis was
analyzed by the Kaplan–Meier plotter. c. Dierences in CNBP expression in samples from ovarian cancer of dierent clinical stages. d. Dierences in CNBP
expression between responders and non-responders. e. Expression and cellular localization of CNBP in benign and malignant ovarian tumors detected
by immunohistochemical assays. f. The diagnostic value of ROC curve analysis of CNBP in epithelial ovarian cancer. g. Dierential expression of CNBP
protein in drug-resistant and sensitive ovarian cancer tissues (left) and the diagnostic value of ROC curve analysis of CNBP in chemotherapy resistance
of ovarian cancer (right). h. Dierences in the expression of CNBP mRNA and CNBP in OCSC and epithelial ovarian cancer cells detected by qPCR and
western blotting, *P < 0.05
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Page 9 of 22
Liu et al. Biology Direct (2024) 19:59
databases revealed that patients with high LINC00665
expression had reduced overall survival (OS, 36.4 months
vs. 45 months, HR = 1.33 [1.05–1.68], P = 0.019) and dis-
ease-free survival (DFS, 12.83 months vs. 18.27 months,
HR = 1.63 [1.31–2.03], P = 9.2e-06) compared to those
with low LINC00665 expression (Fig.1A, b).
e subcellular distribution of LINC00665 was pre-
dicted using an online tool (http://www.csbio.sjtu.
edu.cn/bioinf/lncLocator/ [33]), which suggested that
LINC00665 transcripts are predominantly located in the
cytosol and cytoplasm (Supplementary Fig. 3). In situ
hybridization and qPCR confirmed that LINC00665 was
mainly found in the cytoplasm of ovarian cancer tissues,
whereas it was present in both the nucleus and cytoplasm
of serous cystadenoma tissues (Fig. 1A, c; Supplemen-
tary Fig. 3). LINC00665 levels were higher in OCSCs
compared to COC1 and SKOV3 ovarian cancer cell lines
(Fig.1A, d).
Utilizing datasets GSE80373 and GSE145374 from the
GEO repository, a cohort of 1016 DEGs were identified
as the differentially expressed mRNAs between OCSC
and the respective control. ese genes were predomi-
nantly associated with critical cellular pathways such as
p53 signaling, Notch signaling, and metabolic processes,
hinting at their potential role in the modulation of stem-
ness within ovarian cancer cells (Supplementary Fig.4).
e StarBase platform [34] was employed to pinpoint
mRNAs of protein-coding genes that might interact
with LINC00665, and these were cross-referenced with
the previously identified 1016 DEGs. is comparative
approach yielded nine genes of interest: HKR1, SUN1,
TMTC4, IDH1, CNBP, RBM19, EIF4A2, EEF1A1, and
PSMD9. An Alu element within the 3’-UTR of CNBP
mRNA was discovered through RepeatMasker analy-
sis. Subsequent scrutiny using RNA_RNA_Anneal soft-
ware [11] disclosed a 132 bp sequence within this Alu
element that is complementary to the Alu sequence
in LINC00665, exhibiting a significant free energy of
− 222.2kcal/mol (Supplementary Fig.5).
Using the GENT2 database, we extracted expression
data for the ENSG00000169714 (CNBP) gene to assess
its mRNA levels in ovarian cancer. e analysis showed
that CNBP expression was markedly increased in ovar-
ian cancer tissues compared to normal ovarian tissues
(P < 0.001) (Fig.1B, a). To determine the impact of CNBP
levels on patient outcomes, survival analysis was con-
ducted using the Kaplan–Meier plotter, incorporating
data from the GEO, EGA, and TCGA databases. Patients
with elevated CNBP expression exhibited a decreased
overall survival (OS) (35 months vs. 45 months, HR = 1.32
[1.06–1.66], P = 0.014), a reduced progression-free sur-
vival (PFS) (11.53 months vs. 18 months, HR = 1.7 [1.37–
2.12], P = 1.6e-06), but an extended post-progression
survival (PPS) (42.63 months vs. 37 months, HR = 0.77
[0.65–0.92], P = 0.0045) (Fig.1B, b). e CNBP expression
was more pronounced in stage II and III ovarian cancers
compared to stage IV (P < 0.01), with no significant dif-
ference between stages II and III (P = 0.16) (Fig. 1B, c).
In grade 3 ovarian cancer patients treated with platinum
and paclitaxel chemotherapy (https://www.rocplot.org/
ovarian/index) [35], those who responded to chemother-
apy within 6 months had higher CNBP expression than
those who did not respond (Fig.1B, d). e gene-based
classification of treatment response showed potential of
CNBP expression in distinguishing between responder
and non-responder patients with an AUC of 0.625;
P = 0.0039 (Fig.1B, d). ese findings underscore the sig-
nificant association of CNBP with the progression and
prognosis of ovarian cancer.
Immunohistochemical analysis revealed that in serous
ovarian carcinoma, CNBP predominantly resided in
the cytoplasm, irrespective of its expression levels, with
occasional nuclear presence observed in certain cells. In
contrast, in serous cystadenoma, high CNBP expression
was noted within the nuclei of cells. Comparative immu-
nohistochemical studies between epithelial ovarian can-
cer tissues and ovarian serous cystadenoma indicated a
significantly higher expression of CNBP protein in the
former (Fig. 1B, e). ROC curve analysis suggested that
elevated CNBP levels could serve as a potential marker
for distinguishing between epithelial ovarian tumors
(AUC = 0.91, P < 0.05) (Fig. 1B, f). e patient cohort
under study received 6–8 cycles of paclitaxel-carbopla-
tin (TC) chemotherapy. Sixteen cases were categorized
as the drug-resistant group due to recurrence within
six months post-treatment (evidenced by increased
CA125 and imaging) or lack of response to therapy, while
another 16 cases, showing no recurrence or recurrence
after six months, were deemed the sensitive group. e
analysis indicated that CNBP protein expression was sig-
nificantly higher in the sensitive group compared to the
drug-resistant group (P < 0.05). Additional ROC curve
analysis demonstrated that CNBP expression could effec-
tively predict the response of epithelial ovarian tumors to
chemotherapy (AUC = 0.71, P < 0.05) (Fig. 1B, g). In both
serous ovarian cancer and serous cystadenoma, high
LINC00665 expression correlated with reduced CNBP
expression. Furthermore, CNBP mRNA levels and pro-
tein expression in OCSC were found to be lower than
those in COC1 and SKOV3 cells (Fig.1B, h). e com-
prehensive flow diagram of the study is depicted in Fig.2.
Interaction between LINC00665 and CNBP mRNA 3’-UTR
through alu elements facilitates mRNA degradation
Subsequently, we confirmed the direct interaction
between the Alu sequence of LINC00665 and the Alu
sequence within the 3’-UTR of CNBP mRNA. By employ-
ing a luciferase reporter assay in 293T cells, we observed
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Page 10 of 22
Liu et al. Biology Direct (2024) 19:59
that LINC00665 markedly decreased the luciferase activ-
ity of constructs containing the wild-type CNBP mRNA
3’-UTR Alu sequence (P < 0.05). is suppressive effect
of LINC00665 was not observed with mutant constructs
(Fig.3A).
To validate the direct association of STAU1 with both
LINC00665 and the CNBP mRNA 3’-UTR, we used anti-
STAU1 antibodies to precipitate the complex from OCSC
lysates. e qPCR analysis confirmed the enrichment of
both CNBP 3’-UTR and LINC00665 in the precipitated
samples (Fig.3B).
To demonstrate the direct interaction of LINC00665
with the CNBP mRNA 3’-UTR mediated by STAU1, we
constructed an overexpression vector for LINC00665
with an MS2 stem-loop and co-transfected it with a GFP
overexpression vector pMS2-GFP into OCSCs. Immuno-
precipitation with anti-GFP antibodies and subsequent
western blotting analysis showed STAU1 presence in
the MS2-LINC00665-wt immune complex containing
the intact LINC00665 Alu sequence. In contrast, STAU1
was absent in the complex with the mutated MS2-
LINC00665-mut (Fig. 3C, a). qPCR analysis indicated
a significantly higher CNBP mRNA level in the MS2-
LINC00665-wt immune complex compared to the MS2-
LINC00665-mut and the negative control (Fig. 3C, b).
Upon STAU1 knockdown in the MS2-LINC00665-wt
complex, qPCR revealed a reduction in CNBP mRNA
levels, although they remained elevated compared
to the MS2-LINC00665-mut and negative control
(Fig.3D). ese findings suggest that the Alu element of
LINC00665 is associated with the CNBP mRNA 3’-UTR
in an STAU1-dependent manner.
To further substantiate the influence of STAU1 on
CNBP, we assessed the levels of STAU1 and CNBP in
OCSCs and COC1 cells through western blotting. e
findings indicated that STAU1 levels were elevated in
OCSCs compared to COC1 cells. In contrast, CNBP lev-
els were found to be lower in OCSC than in COC1 cells,
displaying an inverse relationship with STAU1. To delve
deeper into the effects of STAU1 on CNBP regulation,
we suppressed STAU1 expression in OCSCs. Subsequent
evaluations revealed an increase in both mRNA and
protein levels of CNBP in OCSCs with reduced STAU1
expression (Fig. 3E, a, b). However, LINC00665 levels
Fig. 2 Flowchart of bioinformatics analysis of LINC00665 and CNBP
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Page 11 of 22
Liu et al. Biology Direct (2024) 19:59
remained unchanged, implying that STAU1 alterations
do not influence LINC00665 expression (Fig.3E, c).
Additionally, we examined the impact of STAU1 on
CNBP mRNA stability by employing actinomycin D to
inhibit transcription. e data indicated that the down-
regulation of STAU1 extended the half-life of CNBP
mRNA in OCSCs (2.56h) compared to the control group
(1.44h) (P < 0.05, Fig.3F).
Altering CNBP expression did not result in changes
in LINC00665 levels across all cell lines tested (Fig.3G).
Experiments modulating LINC00665 expression demon-
strated that in OCSCs with reduced LINC00665, there
was an increase in CNBP mRNA and protein levels, and
the half-life of CNBP mRNA was extended, suggest-
ing enhanced mRNA stability. Conversely, in COC1 and
SKOV3 cells with increased LINC00665 expression, there
was a decrease in CNBP mRNA and protein levels, and
the half-life of CNBP mRNA was reduced, suggesting
reduced mRNA stability. All observed differences were
statistically significant (P < 0.05, Fig.3H and I).
Fig. 3 LINC00665 promotes CNBP mRNA decay by forming duplexes with 3’-UTRs via Alu elements (A) Eect of LINC00665 on the uorescent expression
of reporter gene vectors containing the CNBP mRNA 3’-UTR Alu element in 293T cells 48h after transfection in the luciferase reporter assay. (B) RNA im-
munoprecipitation (RIP) was performed with a STAU1-specic antibody. The RNA was extracted, and CNBP mRNA and LINC00665 levels were evaluated
by qPCR. a. The relative expression of CNBP mRNA, b. The relative expression of LINC00665. C. MS2-RIP followed by western blotting and qPCR to detect
STAU1 and CNBP mRNA separately associated with LINC00665. a. Western blot for STAU1, b. qPCR for CNBP mRNA. D. MS2-RIP followed by qPCR to detect
CNBP mRNA associated with LINC00665 after inhibiting the expression of STAU1. E. Expression of STAU1 and CNBP in OCSC and COC1 cells, and the eect
of STAU1 inhibition on the expression of CNBP and LINC00665. a. Western blotting results after inhibition of STAU1. b. CNBP mRNA detected by qPCR. c.
LINC00665 detected by qPCR. F. The stability of CNBP mRNA in OCSCs treated with shSTAU1. G. Expression of LINC00665 in each group of cells detected
by qPCR after modulating CNBP expression: a. CNBP overexpression in OCSCs; b. Suppression of CNBP expression in COC1 and SKOV3 cells. H. After
modulating LINC00665 expression, the expression of CNBP mRNA and CNBP protein in each group of cells was detected by qPCR and western blotting,
respectively: a. qPCR assay after inhibiting LINC00665 expression in OCSC; b. qPCR assay after overexpression of LINC00665 in COC1 and SKOV3; and c.
Western blot results. I. The stability of CNBP mRNA in cells transfected with the indicated vectors: a. OCSCs treated with shLINC00665, b. COC1 and c.
SKOV3 ovarian cancer cells treated with LV-LINC00665, *P < 0.05
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Liu et al. Biology Direct (2024) 19:59
CNBP is involved in the modulation of stemness dynamics
in ovarian cancer cells
A lentiviral vector for the overexpression of CNBP was
engineered and used to transduce OCSCs. At 48h post-
transduction, a comparative analysis with the control
group revealed a notable reduction in the sphere for-
mation of CNBP-overexpressing OCSCs, a significant
decline in the proportion of CD133+/CD117+ cells rela-
tive to the total cell population (Fig.4A, Supplementary
Fig.6), and a marked decrease in the proliferative poten-
tial of CNBP-overexpressing OCSCs (Fig.4B, a).
We developed a vector to suppress CNBP expres-
sion and introduced it into COC1 and SKOV3 cells.
Post-transfection at 48 h, the proliferative capacity of
Fig. 4 CNBP regulates the stemness transition of ovarian cancer cells (A) Sphere formation of OCSC overexpressing CNBP, and percentage of CD133+/
CD117+ cells overexpressing CNBP to total cells detected by ow cytometry. (B) Eects of CNBP on cell proliferation detected by using CCK-8 assay: a.
Overexpression of CNBP in OCSCs; b. Inhibition of CNBP expression in COC1 and SKOV3 cells. C. Dierences in the inhibition rate of each group of cells
treated with dierent concentrations of cisplatin after modulating the expression of CNBP: The cells of each group were added with dierent concentra-
tions of cisplatin (0, 10, 30, 50 µM) and then continued to be cultured for 48h. The eect of dierent concentrations of cisplatin on the inhibition rate of
each group of cells after regulating the expression of CNBP was detected by using CCK-8 assay: a. OCSC overexpressing CNBP; b and c. Suppression of
CNBP expression in COC1 and SKOV3 cells. Detection of cell biological behaviors after modulating CNBP expression in each group of cells: overexpression
of CNBP in OCSC and inhibition of CNBP expression in COC1 and SKOV3 cells. D. Cell migration and invasion were measured by transwell assays. E. Colony
formation assays for each group of cells; F. Cell cycle percentage assays for each group of cells; G. Apoptotic percentage assays for each group of cells; H.
Detection of relevant protein expression in each group of cells by western blotting. I. The eect of CNBP on the activity of the Wnt pathway in each group
of cells was detected. The relative uorescence activity of each group of cells was assayed after transfection with TOPFlash/FOPFlash vectors: a. OCSC
overexpressing CNBP; b. COC1 and SKOV3 cells with suppressed CNBP expression, *P < 0.05
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Liu et al. Biology Direct (2024) 19:59
these cells was significantly augmented (Fig.4B, b). Sub-
sequently, we treated the cells with varying cisplatin
concentrations (0, 10, 30, 50 µM) and continued the incu-
bation for an additional 48h. e findings demonstrated
that the suppressive impact of cisplatin on OCSC with
elevated CNBP expression was significantly intensified in
a dose-dependent manner, whereas the suppressive effect
was notably reduced in COC1 and SKOV3 cells with
diminished CNBP expression (Fig.4C).
Additionally, OCSCs with increased CNBP expression
exhibited reduced capabilities for invasion, metastasis,
and colony formation (Fig.4D, E). Conversely, COC1 and
SKOV3 cells with downregulated CNBP expression dis-
played increased invasive, metastatic, and colony-form-
ing activities (Fig.4D, E). When compared to the control
group, the overexpression of CNBP led to a reduced
entry of OCSCs into the S-phase of the cell cycle and a
substantial elevation in apoptotic events (Fig.4F, G). On
the other hand, the suppression of CNBP expression in
COC1 and SKOV3 cells resulted in an increased transi-
tion into the S-phase, a significant reduction in apopto-
sis, and associated alterations in Cyclin-D1 expression
(Fig. 4F, G). ese observations imply that CNBP has
the capacity to suppress cellular proliferation and trig-
ger apoptosis by inducing arrest in the S-phase of the cell
cycle.
In our analysis of GSE106918, the CNBP-binding
mRNAs identified were subjected to KEGG pathway
analysis and network construction. is analysis high-
lighted pathways pertinent to CSC, including those
involved in Wnt, p53, Hippo, and VEGF signaling path-
ways, as well as those implicated in cancer, resistance
to platinum-based drugs, PD-L1 expression and PD-1
checkpoint pathways, and Notch, MAPK, and HIF-1 sig-
naling pathways, including those that regulate the plu-
ripotency of stem cells (Supplementary Fig. 7). ese
pathways are linked to the preservation and transition of
stemness, leading us to select proteins within these path-
ways for further validation in OCSCs.
Our investigation demonstrated that in OCSCs, the
enhancement of CNBP levels led to a suppression of stem
cell markers such as OCT4, SOX2, NANOG, LGR5, and
ALDH1, as well as a reduction in the surface markers
CD117 and CD133. is was accompanied by a decrease
in Ki-67 expression, indicative of reduced cellular pro-
liferation. Furthermore, the upregulation of CNBP was
associated with an increase in E-cadherin levels and a
decrease in VE-cadherin, N-cadherin, and Vimentin
expression, aligning with the observed reduction in inva-
sive and metastatic capabilities of OCSCs. e expression
of NOTCH1 was also diminished, implying a potential
inhibition of the Notch signaling pathway by CNBP over-
expression. Additionally, there was a decrease in MDR1
expression, which is linked to multidrug resistance, and a
reduction in nuclear β-catenin levels, although cytoplas-
mic β-catenin levels remained unchanged (Fig. 4H, left
panel). e introduction of TOPFlash/FOPFlash vectors
and subsequent dual-luciferase assays indicated a lower
TOPFlash/FOPFlash fluorescence ratio in the group with
CNBP overexpression, suggesting a dampening effect on
Wnt pathway activity (Fig.4I, a). Conversely, the down-
regulation of CNBP in COC1 and SKOV3 cells resulted
in an upsurge of the aforementioned stem cell mark-
ers and proteins associated with invasiveness and drug
resistance, a decrease in E-cadherin expression, and an
increase in nuclear β-catenin, without impacting cyto-
plasmic β-catenin (Fig.4H, middle and right panels). e
fluorescence ratio was also found to be higher in these
cells, indicating an activation of the Wnt pathway, likely
due to the suppression of CNBP (Fig.4I, b). Our findings
suggest that enhanced CNBP levels in OCSCs suppressed
stem cell markers, reduced proliferation, and inhibited
invasion, potentially via modulation of the Notch and
Wnt pathways.
LINC00665 fosters stemness characteristics in ovarian
cancer cells through the downregulation of CNBP
Simultaneous manipulation of LINC00665 and CNBP
levels was performed in our study. Initially, we observed
that the suppression of LINC00665 led to a reduction in
both the sphere formation capacity of OCSCs and the
proportion of CD133+/CD117+ cells relative to the total
cell population when compared to the control group. On
the other hand, the downregulation of CNBP resulted in
a resurgence of sphere-forming OCSCs and an increased
ratio of CD133+/CD117+ cells (Supplementary Fig. 8).
e growth rate of OCSCs with diminished LINC00665
levels was notably lower than that of the control group.
is reduction in cell proliferation was reversed upon
the subsequent downregulation of CNBP. Noting that
LINC00665 levels in COC1 and SKOV3 cells were infe-
rior to those in OCSCs, we proceeded to induce over-
expression of LINC00665 in these cell lines. e data
indicated that LINC00665 augmentation markedly pro-
moted cell proliferation across the groups in comparison
to the control. Yet, when both LINC00665 and CNBP
were overexpressed in COC1 and SKOV3 cells, a signifi-
cant decline in cell proliferation was recorded (Fig.5A).
Consistent with previous steps, we treated the various
cell groups with escalating doses of cisplatin and moni-
tored the outcomes. e findings demonstrated that the
rate of cisplatin-induced inhibition was substantially
elevated in OCSCs with LINC00665 downregulation at
higher concentrations, reduced in OCSCs with concur-
rent suppression of LINC00665 and CNBP, diminished
in COC1 and SKOV3 cells with LINC00665 upregula-
tion, and escalated in OCSCs with upregulation of both
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Liu et al. Biology Direct (2024) 19:59
Fig. 5 LINC00665 promotes stemness transition of ovarian cancer cells by mediating CNBP expression A. The eects of CNBP and LINC00665 expression
regulation on cell proliferation were detected using CCK-8 assays: a. LINC00665 and CNBP expression was modulated in OCSC; b. LINC00665 and CNBP
expression was modulated in COC1 and SKOV3 cells. B. Dierences in the inhibition rate of cells in each group with a modulated expression of LINC00665
and CNBP by dierent concentrations of cisplatin were detected: cells in each group were added with dierent concentrations of cisplatin (0, 10, 30, and
50 µM) and then continued to be cultured for 48h. The inhibition rate of (a) OCSCs, (b) COC1 cells, and (c) SKOV3 cells with modulated expression of
LINC00665 and CNBP by dierent concentrations of cisplatin was detected by using CCK-8 assays. The cell biological behaviors of each group of cells after
modulating the expression of CNBP and LINC00665 were detected: the expression of LINC00665 and CNBP was co-repressed in OCSC and co-overex-
pressed in COC1 and SKOV3 cells. C. Cell migration and invasion were measured by transwell assays. D. Colony formation assays. E. Cell cycle percentage
assays. F. Detection of apoptotic percentages. G. Detection of relevant protein expression by western blotting. H. Detection of the eect of LINC00665
and CNBP on the activity of the Wnt pathway. The relative uorescence activity of each group of cells was assayed after transfection with TOPFlash/
FOPFlash vectors: a. OCSCs with suppressed expression of LINC00665 and CNBP; b. COC1 and SKOV3 cells overexpressing LINC00665 and CNBP, *P < 0.05
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Liu et al. Biology Direct (2024) 19:59
LINC00665 and CNBP (with all observed differences
being statistically significant; Fig.5B).
Patterns in the invasive, metastatic, and clonogenic
potential of these cells were consistently observed. When
LINC00665 was downregulated in OCSCs, there was a
marked decrease in their ability to invade, metastasize,
and form colonies compared to the control group. How-
ever, when both LINC00665 and CNBP expressions were
inhibited, these cells demonstrated a significant increase
in these capabilities compared to OCSCs with only
LINC00665 downregulation. Furthermore, the forced
expression of LINC00665 in COC1 and SKOV3 cells led
to a notable enhancement in their invasive, metastatic,
and colony-forming activities relative to the control. Yet,
when LINC00665 and CNBP were both overexpressed in
COC1 and SKOV3 cells, there was a reduction in these
activities (Fig.5C, D).
e suppression of LINC00665 alone in OCSCs
resulted in a lower proportion of cells progressing to the
S-phase and a higher rate of apoptosis when compared
to the control. In contrast, OCSCs with both LINC00665
and CNBP suppression showed an increased entry into
the S-phase and a reduced apoptotic rate compared
to OCSCs with only LINC00665 downregulated. Con-
versely, the overexpression of LINC00665 alone signifi-
cantly raised the percentage of COC1 cells entering the
S-phase and lowered apoptosis rates. However, with the
concurrent overexpression of LINC00665 and CNBP,
there was a decline in the S-phase entry and an elevation
in apoptosis among COC1 cells (Fig.5E, F). ese find-
ings highlight the antagonistic roles of LINC00665 and
CNBP in the modulation of stemness transition within
ovarian cancer cells. LINC00665 appears to drive cell
proliferation and reduce apoptosis by facilitating S-phase
entry, and it also enhances invasion, metastasis, and col-
ony formation, which can be counteracted by CNBP.
Subsequent investigations were directed at under-
standing the impact of CNBP and LINC00665 on the
expression of stem cell markers, markers of epithe-
lial-mesenchymal transition, and proteins involved in
the Wnt signaling pathway, as well as their interplay
in ovarian cancer cells. In OCSCs, the suppression of
LINC00665 led to a cascade of molecular alterations:
a reduction in the levels of stem cell markers (such as
OCT4, SOX2, NANOG, ALDH1, and LGR5), as well as
the cell surface markers (CD117 and CD133). Addition-
ally, there was a decline in Ki-67 expression, indicative
of a diminished proliferative ability of the cells. Con-
currently, there was a downregulation of VE-cadherin,
N-cadherin, and Vimentin, along with an upregulation
of E-cadherin expression, pointing to a reduced poten-
tial for cell invasion and metastasis. e expression of
MDR1, linked to multidrug resistance, and NOTCH1
also saw a decrease. e levels of nuclear β-catenin were
lowered, while cytoplasmic β-catenin levels did not show
significant alteration (Fig.5G).
Concomitant inhibition of both LINC00665 and CNBP
expression resulted in elevated levels of OCT4, SOX2,
NANOG, ALDH1, LGR5, CD117, CD133, Ki-67, VE-
cadherin, N-cadherin, Vimentin, NOTCH1, MDR1, and
nuclear β-catenin proteins compared to cells with only
LINC00665 suppression. Nevertheless, these expres-
sion levels were still below those observed in the control
OCSCs. E-cadherin expression was found to be induced
in the OCSCs group with only LINC00665 suppression
but lower than in the OCSCs group with LINC00665 and
CNBP inhibition. No significant changes were noted in
the cytoplasmic β-catenin levels (Fig.5G, left panel).
Elevating LINC00665 levels in COC1 and SKOV3 cells
led to an increase in the expression of stem cell mark-
ers OCT4, SOX2, NANOG, ALDH1, LGR5, CD117,
and CD133, as well as Ki-67, VE-cadherin, N-cadherin,
Vimentin, NOTCH1, MDR1, and nuclear β-catenin. Con-
versely, E-cadherin expression was reduced, and cyto-
plasmic β-catenin levels remained unchanged (Fig.5G).
e augmentation of CNBP expression mitigated the
upregulation effects of LINC00665 on these markers,
with the exception of E-cadherin expression, which was
marginally lower than the control group, and cytoplasmic
β-catenin levels, which remained stable (Fig.5G).
e transfection of TOPFlash/FOPFlash vectors into
each cell group, followed by a dual-luciferase assay, indi-
cated a reduction in fluorescence activity in OCSCs with
LINC00665 knockdown, which was restored upon CNBP
knockdown (Fig. 5H). In COC1 and SKOV3 cells with
LINC00665 overexpression, fluorescence activity was
increased, but this was attenuated when both LINC00665
and CNBP were overexpressed (Fig.5H), suggesting that
LINC00665 and CNBP exert reciprocal regulatory influ-
ences on the Notch and Wnt signaling pathways. In sum-
mary, we established that CNBP and LINC00665 assume
antagonistic functions in the governance of stemness
dynamics in ovarian cancer cells, with LINC00665 acting
as a facilitator and CNBP serving as a repressor of this
critical cellular transition. ese results corroborate that
LINC00665 participates in the regulation of the stem-
ness of ovarian cancer cells by degrading CNBP mRNA
through SMD.
In vivo modulation of CNBP and LINC00665 expression
yields divergent eects on tumor development
In our investigation of the impact of CNBP and
LINC00665 on tumorigenesis in vivo, we utilized a xeno-
graft model with ovarian cancer stem cells in nude mice.
e findings indicated that mice injected with OCSCs
exhibiting increased CNBP and reduced LINC00665
(CNBP+/LINC00665-OCSC) developed smaller tumors
compared to the control group. Notably, the group
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 16 of 22
Liu et al. Biology Direct (2024) 19:59
receiving CNBP overexpression presented with the least
tumor volume across all tested groups. In contrast, mice
with dual suppression of both LINC00665 and CNBP
(LINC00665-/CNBP-) exhibited a significant increase in
tumor size relative to the group with only LINC00665
suppression (Fig.6A, B, C).
Tumor tissue analysis showed an upsurge in CNBP
mRNA and protein levels in mice injected with OCSC
with suppressed LINC00665 expression (Supplemen-
tary Fig.9). In these tumor samples, there was a notable
decline in the levels of stem cell markers (OCT4, SOX2,
NANOG, ALDH1, and LGR5), as well as cell surface
markers (CD117 and CD133). is was accompanied by
a reduction in Ki-67 expression, indicative of a decrease
in cellular proliferation. Additionally, a decrease in VE-
cadherin and an increase in E-cadherin expression were
observed, suggesting a potential reduction in the invasive
and metastatic capabilities of the cells. e expression
of proteins associated with multidrug resistance, such
as MDR1, CyclinD1, NOTCH1, and nuclear β-catenin,
was also diminished, while cytoplasmic β-catenin levels
remained unchanged. In neoplasms from the group with
Fig. 6 Eect of CNBP and LINC00665 on ovarian cancer tumor growth in vivoA. Tumor-bearing nude mice and tumor samples from each group. B.
Tumor volume was calculated every three days after injection. All tumors were excised four weeks after injection, and the tumor growth curve of CNBP-
overexpressing OCSCs was compared with that of the control group. C. Comparison of tumor growth curves of OCSCs with LINC00665 inhibition alone
or OCSCs with co-suppression of LINC00665 and CNBP expression with that of the control group. D. Expression of stemness marker proteins, proteins
related to cell biological behaviors, and pathway proteins detected by western blotting. E. Disease-free survival analysis of ovarian cancer samples. The
survival time of patients after surgery was compared between high-CNBP and low-CNBP expression groups. F. ChIP assay showing endogenous NFYA
bound to the LINC00665 promoter in COC1 cells. G. LINC00665 increases CNBP degradation in OCSC via the SMD pathway to participate in OCSC stem-
ness regulation, *P < 0.05
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 17 of 22
Liu et al. Biology Direct (2024) 19:59
diminished LINC00665 levels, there was a notable reduc-
tion in the expression of stem cell markers (OCT4, SOX2,
NANOG, ALDH1, and LGR5), as well as cell surface
markers (CD117 and CD133), coupled with a decrease in
Ki-67 levels, indicating a suppression of cellular prolifera-
tion. Additional alterations in the tumor tissues included
a decline in VE-cadherin levels, an increase in E-cadherin
levels, and a reduction in MDR1, Cyclin D1, NOTCH1,
and nuclear β-catenin levels. However, the levels of cyto-
plasmic β-catenin remained largely unchanged (Fig.6D).
When comparing tumor samples from OCSC with
only LINC00665 suppression to those from OCSCs
with concurrent suppression of both LINC00665 and
CNBP, the latter demonstrated elevated levels of OCT4,
SOX2, NANOG, ALDH1, LGR5, CD117, CD133, Ki-67,
VE-cadherin, NOTCH1, MDR1, CyclinD1, and nuclear
β-catenin, along with a reduction in E-cadherin lev-
els. e expression of cytoplasmic β-catenin, however,
did not show significant variation (Fig. 6D). In line
with these findings, an analysis of clinical data on ovar-
ian cancer revealed that patients with higher CNBP
expression exhibited longer periods of disease-free sur-
vival compared to those with lower CNBP expression
(P<0.05, Fig. 6E). To further investigate the regulatory
role of LINC00665 in ovarian cancer, the NCBI data-
base was utilized to identify the sequence upstream of
LINC00665’s transcription start site, and the transcrip-
tion factor ‘NFYA’ was predicted to bind to LINC00665
using the Jaspar online system. Primers were designed
based on binding sites with high scores, and genomic
DNA served as the template for PCR to confirm primer
efficacy. A ChIP assay was conducted to validate the
binding of NFYA to this site in COC1 cells (Fig.6F). e
comprehensive regulatory mechanism uncovered in this
study is depicted in Fig.6G.
Discussion
e ineffectiveness of ovarian cancer therapies is fre-
quently linked to the emergence of chemoresistance,
which encompasses both inherent resistance and
acquired resistance, the latter being defined by relapse
within six months following an initially successful treat-
ment [36]. is phenomenon is notably prevalent among
individuals receiving chemotherapy for ovarian cancer.
A deeper investigation into the preservation of stemness
features in OCSCs is imperative to elucidate the mecha-
nisms behind the development and potential reversal
of chemoresistance in ovarian cancer. In this study, we
found that CNBP plays a suppressive role in the transi-
tion of stemness within ovarian cancer cells, while its
expression is modulated by the SMD pathway driven by
LINC00665. In the presence of STAU1, the 3’-UTR of
CNBP mRNA and LINC00665 interact through match-
ing Alu elements, forming a complex that leads to the
SMD-mediated degradation of CNBP mRNA. is pro-
cess influences the transition of stemness by impacting
the Wnt and Notch signaling pathways (Fig.6G).
e SMD pathway necessitates the presence of Alu
elements within the lncRNA and the 3’-UTR of the
corresponding mRNA [37]. Alu elements are unique
sequences of nucleic acids that are widespread through-
out the human genome, typically extending over roughly
300 base pairs [37]. ese elements are predominantly
located within intronic regions, the 3’-UTR, and inter-
genic spaces. Named for their inclusion of a specific rec-
ognition site (AGCT) at the 170bp mark, which can be
cleaved by the Alu I restriction enzyme, Alu elements
can modulate the expression of protein-coding genes
via cis-activation mechanisms during both transcription
and translation [37]. e current research has uncovered
a novel mechanism in ovarian cancer where the lncRNA
LINC00665 exploits the SMD pathway to interact with
and degrade CNBP mRNA. is discovery adds a new
layer of understanding to the complex regulatory mecha-
nisms that govern gene expression in ovarian cancer and
may provide a basis for developing targeted therapies
that disrupt this pathway.
LINC00665 has been identified as a key factor in a vari-
ety of tumors, with its abnormal expression being closely
associated with clinical features and poor prognosis in
several cancer types [38]. It can also significantly influ-
ence the response to chemotherapeutic agents such as
gemcitabine, cisplatin, and paclitaxel [38]. Functioning
as a competitive endogenous RNA (ceRNA), LINC00665
regulates cellular functions in cancer by acting as a
molecular sponge for tumor-suppressive miRNAs, thus
upregulating various oncogenes implicated in cancer
progression. LINC00665 also influences the modula-
tion of several key signaling cascades, such as Wnt/β-
catenin, TGF-β, MAPK1, NF-κB, ERK, and PI3K/AKT,
and it can enhance tumor metastasis by facilitating the
epithelial-mesenchymal transition (EMT) process [39].
For instance, in ovarian cancer, LINC00665 upregulates
the expression of genes like KLF5, E2F3, and FHDC
by competitively binding with miRNA-148b-3p [40],
miRNA-34a-5p [41], and miRNA-181a-5p [42], respec-
tively, contributing to the progression of the disease. On
the other hand, LINC00665 can inhibit the progression
of triple-negative breast cancer [43] or encourage the
progression of hepatocellular carcinoma by producing
micropeptides [44]. Our study provides novel insights
into the role of LINC00665 in ovarian cancer by iden-
tifying it as a crucial regulator of the multiple signaling
pathways, which are related to cancer stemness. Unlike
previous studies that primarily focused on in vitro exper-
iments and specific miRNA axes, our research includes
evidence demonstrating that LINC00665 can form
duplex structures with the 3’-UTRs of target mRNAs
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Page 18 of 22
Liu et al. Biology Direct (2024) 19:59
through the base pairing of Alu elements, regulat-
ing ovarian cancer development via STAU1-mediated
mechanisms. is dual regulatory role, coupled with the
modulation of multiple downstream molecular signaling
pathways, highlights the complex molecular mechanisms
by which LINC00665 promotes ovarian cancer progres-
sion. e inclusion of in vivo experiments provides a
more comprehensive understanding of LINC00665’s
function, further proposing a promising and comprehen-
sive approach to target LINC00665 for innovative thera-
peutic strategies in ovarian cancer.
Moreover, lncRNAs not only functions as a ceRNA, but
they also can form duplex structures with the 3’-UTRs
of target mRNAs through base pairing of Alu elements,
playing a crucial role in the regulation of cancer devel-
opment, progression, and spread via STAU1-mediated
mechanisms [43–46] For instance, LINC00665 can form
a physical complex with double-stranded RNA-activated
protein kinase (PKR), enhancing its activation and pre-
venting its degradation via the ubiquitin/proteasome
pathway. is interaction stabilizes PKR, which in turn
positively influences the NF-κB signaling cascade in
hepatocellular carcinoma cells, ultimately fostering can-
cer development [49]. Additionally, evidence from glioma
studies indicates that LINC00665 plays a role in control-
ling the malignant behaviors of glioma cells through the
SMD pathway [50], further demonstrating LINC00665’s
ability to bind mRNA. In our research, we observed
that LINC00665 levels were elevated in OCSCs com-
pared to regular ovarian cancer cells. Utilizing Repeat-
Masker software, we pinpointed an Alu element within
LINC00665. In situ hybridization experiments confirmed
LINC00665’s localization in the cytoplasm of ovarian
cancer cells. rough the integration of GEO database
and microarray analysis, we identified 1147 differentially
expressed genes (DEGs) between OCSCs and ovarian
cancer cells. Additionally, StarBase analysis helped us
recognize 9 genes (HKR1, SUN1, TMTC4, IDH1, CNBP,
RBM19, EIF4A2, EEF1A1, and PSMD9) with potential
binding affinity to LINC00665. Notably, another Repeat-
Masker search uncovered an Alu element on the 3’-UTR
of CNBP mRNA, which could engage in partial base pair-
ing with the Alu element on LINC00665. Using MS2-RIP
and RIP assays in the presence of STAU1, we confirmed
the interaction between the Alu element of LINC00665
and the Alu element on the 3’-UTR of CNBP mRNA. We
further established that LINC00665 facilitates the deg-
radation of CNBP mRNA via this interaction, thereby
exerting post-transcriptional control over CNBP expres-
sion. us, out study adds to this body of knowledge
by demonstrating that LINC00665 promotes the deg-
radation of CNBP mRNA in ovarian cancer stem cells,
thereby regulating cancer stemness and contributing to
the malignancy of ovarian cancer.
CNBP, a highly conserved nucleic acid-binding protein,
possesses seven zinc finger motifs of the CCHC type and
a region abundant in arginine and glycine (RG/RGG) [19,
51]. is protein is capable of binding to single-stranded
nucleic acids, playing a role in the regulation of disor-
ders such as neuromuscular degeneration, inflammation,
autoimmune conditions, and various cancers [19, 51].
Significantly, CNBP has an affinity for guanine (G)-rich
sequences in DNA and RNA that can form G-quadru-
plexes (G4), secondary structures that serve as regulatory
elements influencing gene transcription near transcrip-
tion start sites or modulating translation on mRNAs
[19, 51]. Proteins that can modulate G4 structures are
thus promising targets for cancer therapy. CNBP spe-
cifically binds to sequences containing GGAG [52],
functioning as a nucleic acid chaperone that rearranges
secondary structures, impacting biological processes by
altering chromatin or RNA configurations. Within the
cell nucleus, CNBP plays a role in transcriptional regu-
lation, unwinding G4 structures at gene promoters to
control the expression of downstream genes [53]. For
instance, CNBP is known to upregulate the transcription
of KRAS and c-MYC, while downregulating NOG/nog3
by resolving G4 structures at their respective promoter
regions, influencing the process of tumorigenesis [54].
CNBP can also induce the formation of G4 structures, as
seen when its overexpression leads to the suppression of
hnRNP K transcription through G4 structure induction
at its promoter, reducing the malignancy and invasive-
ness of fibrosarcoma cells [55]. In the context of hepato-
cellular carcinoma, CNBP’s interaction with the PGM1
promoter encourages G4 structure formation, which
decreases PGM1 expression, affects glucose metabolism,
and thus hampers cancer progression [56]. Cytoplas-
mically, CNBP is involved in RNA stability regulation,
capable of dismantling stable G4 structures in mRNAs to
enhance translation without altering mRNA levels [52]. It
also aids in the resolution of G4 structures in viral RNA,
such as the SARS-CoV-2 genome, facilitating the synthe-
sis of viral proteins [57]. Recent discoveries have unveiled
novel mechanisms by which CNBP modulates RNA sta-
bility. Research in pancreatic ductal adenocarcinoma
revealed that CNBP can recognize m6A-modified RNA
and enhance its stability [58]. By binding to the 5’-UTR
of mRNA, CNBP collaborates with a lncRNA to enhance
mRNA stability, thereby promoting cell division and pro-
liferation in tumor cells [59]. is multifaceted role of
CNBP in gene regulation, both at the level of transcrip-
tion and mRNA stability, underscores its potential as a
therapeutic target in various diseases, including cancer.
Despite the lack of extensive research on the role and
regulatory mechanisms of CNBP in ovarian cancer, par-
ticularly its involvement in the regulation of stemness
transition, this study has revealed that elevated CNBP
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Page 19 of 22
Liu et al. Biology Direct (2024) 19:59
levels have diagnostic significance in epithelial ovarian
cancer. e expression of CNBP may serve as an indica-
tor of the response to chemotherapy, offering potential
clinical utility not only in treatment but also in diagno-
sis and predicting the effectiveness of chemotherapy.
e resistance of tumors to chemotherapeutic agents,
such as drug resistance, is associated with the stem cell-
like characteristics of tumor cells. Consequently, our
research shifted focus to the interplay between CNBP
and the stem cell attributes of ovarian cancer cells. Our
findings indicate that CNBP acts as a negative regulator
of the stemness transition in ovarian cancer cells, and
higher CNBP expression diminishes the expression of
stem cell markers and disrupts the maintenance of stem-
ness in these cells. Additionally, elevated CNBP levels
were found to inhibit key genes and their activity within
the Wnt, Notch, and other pathways, suggesting that its
inhibitory effect on stemness transition may be mediated
through these pathways. We also observed that CNBP
could influence the concentration of β-catenin within the
nucleus without notably altering its cytoplasmic levels.
us, we hypothesize that CNBP may either prevent the
nuclear import of β-catenin or enhance its degradation
within the nucleus, consequently inhibiting the interac-
tion of β-catenin with TCF/LEF and the subsequent tran-
scription of Wnt pathway target genes. is hypothesis
warrants further investigation for validation.
Recent research has shed light on a spectrum of
post-transcriptional regulatory mechanisms that influ-
ence CNBP’s function in various cancers. For instance,
the ceRNA mechanism is instrumental in the post-
transcriptional control of CNBP expression, as seen in
colorectal cancer cells where CircPACRGL captures
miRNA-330-3p, thereby modulating tumor proliferation
through the miRNA-330-3p/CNBP pathway [60]. Meth-
ylation also plays a critical role, with PRMT1 in HeLa
cells methylating CNBP’s arginine- and glycine-rich seg-
ment, a modification that, despite not altering CNBP’s
nuclear localization, restricts its ability to bind RNA
and consequently reduces its activity [61]. In the realm
of drug resistance, CircFMN2 contributes to height-
ened sorafenib resistance in multidrug-resistant hepa-
tocellular carcinoma by blocking CNBP ubiquitination,
leading to increased CNBP expression [62]. Phosphory-
lation is another regulatory factor, and phosphorylated
CNBP enhances the annealing of oligonucleotides to
the CT element of the c-MYC promoter, thereby acting
as a transcriptional enhancer for c-MYC [63]. Further-
more, the phosphorylation of CNBP by AMP-activated
protein kinase, triggered by Hedgehog pathway activa-
tion in adult neural tube cell tumors, strengthens the
CNBP-Sufu interaction, stabilizes CNBP, and decreases
its proteasomal degradation, which is linked to the
proliferation of medulloblastoma cells. Lastly, redox
proteomics have detected increased CNBP oxidation in
various tumors [64], suggesting that CNBP-Cys oxida-
tion is a crucial aspect of redox homeostasis in tumori-
genesis [51]. Although it has been demonstrated that the
pre-mRNA of CNBP is regulated by alternative splicing
and has multiple isoforms, no structural or functional
differences between these isoforms have been reported
[51]. Our study identified a novel mechanism in which
LINC00665 and STAU1 degrade CNBP mRNA through
the SMD pathway and thereby regulate CNBP expres-
sion. is mechanism can release the inhibition of Wnt,
Notch, and other pathways resulting from the increased
CNBP expression, promoting ovarian cancer cell stem-
ness transition.
Our findings indicated that CNBP expression is ele-
vated in malignant tumors relative to benign ones, yet
it is reduced in OCSCs compared to non-stem ovar-
ian cancer cells, suggesting a link to tumor heterogene-
ity. is heterogeneity could be shaped by the cellular
microenvironment, genetic and epigenetic factors, with
CSCs contributing to the diversity within tumor cell
subpopulations and initiating tumorigenesis [65]. In
colorectal cancer (CRC), E-cadherin exhibits strong
positivity across primary lesions, metastatic peritoneal
tissues, and malignant ascites cells. However, Tamura,
S. et al. identified CRC pathological subtypes with vary-
ing E-cadherin levels, where E-cadherin positive (EC+)
CSCs displayed a higher in vivo proliferation potential
than E-cadherin negative (EC-) CSCs, potentially due
to elevated NANOG-driven cyclin D1 and B1 expres-
sion. Conversely, EC- CSCs, characterized by low cyclin
D1 expression, are considered quiescent and capable of
reverting to EC + status depending on their microenvi-
ronment [66]. Genetic reprogramming during tumor
progression may influence SCs/CSCs behavior, preserv-
ing SC pluripotency and driving differentiation [67, 68].
KRT19’s role in cancer progression is paradoxical; it
is upregulated in both colon and breast cancers, yet its
suppression leads to divergent outcomes. In colon can-
cer, KRT19 downregulation hampers tumor growth
by attenuating Wnt/Notch signaling without affecting
NUMB transcription. In contrast, breast cancer experi-
ences increased malignancy characteristics upon KRT19
knockdown due to diminished Wnt signaling and aug-
mented Notch signaling [69]. Further research has
linked high KRT19 expression in breast cancer to inva-
siveness, while breast cancer-derived CSCs with high
CD133/CXCR4/ALDH1 expression show low KRT19
and high NOTCH1 levels, implicating KRT19 in CSC
reprogramming and drug sensitivity regulation [70, 71].
Moreover, KRT19’s impact varies between HER2 + and
HER2- breast cancer subtypes [72, 73]. Collectively, these
findings underscore that not all cells within a line express
uniform stemness levels but rather exhibit a plasticity
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Page 20 of 22
Liu et al. Biology Direct (2024) 19:59
influenced by their microenvironment [74]. Our data
suggest CNBP’s role in inhibiting OCSC stemness, yet
its regulatory function may differ from that of OCSCs
compared to other ovarian cancer cells at different stages
of tumorigenesis and development or within different
microenvironments. While CNBP expression in ovarian
cancer tissues was found to be higher than in serous cyst-
adenoma in our study, analysis of TCGA and GTEx data
suggested that CNBP expression in epithelial ovarian
cancer is lower than in normal ovarian tissues. is dis-
crepancy indicates that CNBP may have distinct roles in
normal ovarian tissue, ovarian serous cystadenoma, and
ovarian cancer, necessitating further investigation.
Conclusion
Our investigation has uncovered a previously unrecog-
nized pathway in ovarian cancer, wherein LINC00665
engages CNBP mRNA via a STAU1-dependent mecha-
nism, culminating in mRNA degradation. is axis is
crucial for the control of stemness in ovarian cancer
cells, with CNBP playing a significant role in this process.
ese findings pave the way for novel therapeutic strat-
egies, proposing that disrupting the LINC00665-STAU1
interaction with targeted inhibitors, including small mol-
ecules, might provide an effective means to combat drug
resistance and avert ovarian cancer relapse by altering
the stem-like qualities of the cancer cells.
Abbreviations
CSCs Cancer stem cells
CNBP CCHC-type zinc nger nucleic acid binding protein
DEGs Dierentially expressed genes
DELs Dierentially expressed lncRNAs
lncRNA Long non-coding RNA
OCSCs Ovarian cancer stem cells
OS Overall survival
PKR RNA-activated protein kinase
SMD Staufen-mediated mRNA decay
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s13062-024-00506-w.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Acknowledgements
Not applicable.
Author contributions
Conceptualization, methodology, funding acquisition were performed by YZ
and YD; sample collection was performed by MW; experiment was performed
by XL, YC and YL; data analyze was performed by XL, JB and ZZ; writing-
original draft was performed by XL, YC and YL; gures and/or tables prepare
was performed by MW and JB; writing-reviewing and editing was performed
by YZ. All authors read and approved the nal manuscript.
Funding
This study was supported by grants from the National Natural Science
Foundation of China for Young Scientists of China (Grant No. 81902658,
YZ; Grant No.82303040, XL); the National Natural Science Foundation of
China (Grant No. 92168115); the Science Foundation for Outstanding Young
Scholars of Liaoning Province (Grant No. 2022-YQ-16, YD); 345 Talent Project of
Shengjing Hospital (Grant No. M1400, YD); and the Project of City-University
Cooperation (Grant No. 2400022047, YD);Liaoning Province Science and
Technology Plan Joint Program Project (Grant No.2023JH2/101700105, XL).
Data availability
The datasets generated during and/or analyzed during the current study are
available in the UCSC database repository, (TCGA TARGET GTEx (PANCAN,
N=19131, G=60499), https://xenabrowser.net/).
Declarations
Ethics approval and consent to participate
This study was performed in line with the principles of the Declaration of
Helsinki. Approval was granted by the Ethics Committee of China Medical
University [No. 2019PS286K(X1) and No. 2019PS285K(X1)]. Informed consent
was obtained from all individual participants included in the study.
Consent for publication
The authors arm that human research participants provided informed
consent for publication of the images in Fig.1B.
Competing interests
The authors declare no competing interests.
Received: 1 February 2024 / Accepted: 22 July 2024
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