The pivotal role of ZNF384: driving the malignant behavior of serous ovarian cancer cells via the LIN28B/UBD axis

Abstract

Zinc finger protein 384 (ZNF384) is a highly conserved transcribed gene associated with the development of multiple tumors, however, its role and mechanism in serous ovarian cancer (SOC) are unknown. We first confirmed that ZNF384 was abnormally highly expressed in SOC tissues by bioinformatics analysis and immunohistochemistry. We further used lentivirus packaging and transfection techniques to construct ZNF384 overexpression or knockdown cell lines, and through a series of cell function experiments, gradually verified that ZNF384 promoted a series of malignant behaviors of SOC cell proliferation, migration, and invasion. By establishing a xenotransplantation model in nude mice, it was confirmed that ZNF384 promoted the progress of SOC in vivo. Mechanistically, Overexpression of ZNF384 enhanced the transcriptional activity of Lin-28 homolog B (LIN28B), which promoted the malignant behavior of SOC cells. In addition, LIN28B could regulate the expression of the downstream factor ubiquitin D (UBD) in SOC cells, further promoting the development of SOC. This study shows that ZNF384 aggravates the malignant behavior of SOC cells through the LIN28B/UBD axis, which may be used as a diagnostic biomarker for patients with SOC.

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Cell Biol Toxicol (2024) 40:100
https://doi.org/10.1007/s10565-024-09938-6
RESEARCH
The pivotal role ofZNF384: driving themalignant behavior
ofserous ovarian cancer cells viatheLIN28B/UBD axis
YeYang· RunzeHe· DongxiaoLi· TianliMu·
ZitengKuang· MinWang
Received: 28 February 2024 / Accepted: 16 October 2024 / Published online: 19 November 2024
© The Author(s) 2024
Abstract Zinc finger protein 384 (ZNF384) is a
highly conserved transcribed gene associated with
the development of multiple tumors, however, its
role and mechanism in serous ovarian cancer (SOC)
are unknown. We first confirmed that ZNF384 was
abnormally highly expressed in SOC tissues by bio-
informatics analysis and immunohistochemistry.
We further used lentivirus packaging and transfec-
tion techniques to construct ZNF384 overexpression
or knockdown cell lines, and through a series of cell
function experiments, gradually verified that ZNF384
promoted a series of malignant behaviors of SOC
cell proliferation, migration, and invasion. By estab-
lishing a xenotransplantation model in nude mice, it
was confirmed that ZNF384 promoted the progress
of SOC in vivo. Mechanistically, Overexpression of
ZNF384 enhanced the transcriptional activity of Lin-
28 homolog B (LIN28B), which promoted the malig-
nant behavior of SOC cells. In addition, LIN28B
could regulate the expression of the downstream fac-
tor ubiquitin D (UBD) in SOC cells, further promot-
ing the development of SOC. This study shows that
ZNF384 aggravates the malignant behavior of SOC
cells through the LIN28B/UBD axis, which may be
used as a diagnostic biomarker for patients with SOC.
Keywords Serous ovarian cancer· ZNF384·
LIN28B· UBD
Introduction
Ovarian cancer comes in a variety of histologi-
cal forms, with serous ovarian cancer (SOC) being
the most prevalent morphologic subtype (Izar et al.
2020). HGSOC accounts for approximately 90% of
SOC and is the most malignant pathologic type with
the worst prognosis (Peres etal. 2019; Kurman 2013).
Due to the deep location of the ovary in the pelvic
cavity and there are no effective markers for early
diagnosis of cancer, 75% of patients are found with
advanced tumors (Dochez et al. 2019). Despite the
continuous optimization of chemotherapy regimens,
the effect of SOC treatment has been unsatisfactory,
with 5-year survival rates still hovering at 30%-40%
(Boehm etal. 2022). Therefore, further understanding
of the pathogenesis of SOC and finding new thera-
peutic targets have become the key to prolonging the
survival period and improving clinical outcomes of
patients with SOC.
Zinc finger protein 384 (ZNF384) is a transcrip-
tion factor, which contributes to the transcriptional
regulation of extracellular matrix genes (Nakamoto
Supplementary Information The online version
contains supplementary material available at https:// doi.
org/ 10. 1007/ s10565- 024- 09938-6.
Y.Yang· R.He· D.Li· T.Mu· Z.Kuang· M.Wang(*)
Department ofObstetrics andGynecology, Shengjing
Hospital ofChina Medical University, Shenyang,
People’sRepublicofChina
e-mail: wm21st@126.com
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etal. 2000). ZNF384 can be coupled with SYNRG,
EWSR1, and other genes, thereby modulating
downstream signaling factors, such as JAK/STAT3,
and inducing the development of multiple leukemia
subtypes (Yamamoto etal. 2019; Shago etal. 2016).
Furthermore, studies suggested that the reason why
ZNF384 promotes the malignant progression of
cancer may be related to the enhancement of the
proliferation and invasion ability of tumor cells (He
etal. 2019; Yan etal. 2022). Although various evi-
dence suggests that ZNF384 might be an oncogenic
factor that promotes the occurrence of tumors, the
research of ZNF384 in SOC is still lacking.
Ubiquitin D (UBD) is a ubiquitin-like protein
involved in various biological processes including
cellular immunity, apoptosis, and signal transduc-
tion (Song etal. 2021). In recent years, a growing
number of studies have revealed an association
between increased UBD expression and various
malignant tumor progression (Yan etal. 2010; Chou
etal. 2022; Yuan etal. 2014). Lee etal. found that
UBD expression increased in ovarian cancer tissues
(Lee et al. 2003). Lin-28 homolog B (LIN28B) is
a functionally conserved RNA-binding protein that
can maintain the proliferative capacity of pluripo-
tent stem cells and control embryonic development
(Tsanov etal. 2017; Zhang etal. 2018). LIN28B is
considered to be an important oncogene in a vari-
ety of solid tumors and has been reported to predict
poor prognosis (Helsmoortel et al. 2016; Xu etal.
2022; Ren etal. 2018). The expression of LIN28B
in hypothalamic-pituitary-gonad tissues has been
intensively studied over the past few decades, and
interestingly, LIN28B is widely expressed in the
ovary (Grieco etal. 2013). In addition, high expres-
sion of LIN28B can increase the risk of SOC and
the malignancy of the disease (Yong etal. 2018).
Based on the above studies, our work aims to
explore the biological role of ZNF384 in SOC and
its potential mechanisms. We found that ZNF384 is
overexpressed in SOC and is related to poor prog-
nosis in patients with SOC. Functional experiments
showed that ZNF384 can promote malignant behav-
iors of SOC cells, including migration and invasion.
Mechanically, ZNF384 can enhance the transcrip-
tional activity of LIN28B by binding to the LIN28B
promoter, and then regulate the downstream target
UBD to promote SOC progression. In conclusion,
this study reveals that ZNF384 may be a potential
therapeutic target for SOC.
Materials andmethods
Patients and samples
One hundred cases of paraffin-fixed SOC tissues
and thirty cases fresh SOC tissues and thirty cases
non-tumor-ovarian tissues were collected from The
Shengjing Hospital from March. 2023 to Septem-
ber. 2023, among which the non-tumor ovarian tis-
sues were mainly derived from the ovarian tissues
removed during the operation of patients with uterine
fibroids or adenomyosis.
Online bioinformatic analysis tools
Raw data between SOC and normal ovarian tissue
were obtained from the NCBI (http:// www. ncbi. nlm.
nih. gov/ gds/) dataset and the gene expression profil-
ing (GSE14407) was selected. DEGs were selected
by |log2FC|> 1 and adj. P < 0.05 in the GSE14407
dataset. GO enrichment analysis and KEGG enrich-
ment analysis software were R packages ggplot2. The
R package clusterProfile and enrichplot were used to
analyze GSEA results. Mutation mapping of ZNF384
was obtained from the SOC datasets using the cBio-
portal database (https:// www. cbiop ortal. org/). The
GSCA database (https:// guolab. wchscu. cn/ GSCA/)
was used to analyze the effect of ZNF384 expression
on the prognosis of ovarian cancer.
Cell lines
The SOC cell lines OVCAR-8, OVCAR-3, and
SKOV3 were obtained from Saibaikang Company
(China). OVCAR-8 and OVCAR-3 cells were cultured
in RPMI-1640 medium (Solarbio, China) containing
10% FBS. SKOV3 cells were cultured in McCoy’s5A
medium (Procell, China) containing 10% FBS.
Lentivirus infection and transfection
Two shRNA fragments targeting the ZNF384
sequence: 5’-GAA TCT CAC TCA ATT CTA AC-3’ or
5’-CAA TGT TCA TCA ACA AGA TGA-3’. The scram-
ble shRNA targeting 5’-TTC TCC GAA CGT GTC
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ACG T-3’ was used as a control. These fragments
were ligated into the lentiviral vector pLKO.1-EGFP-
puro, and the enzymatic cleavage sites were AgeI
and EcoRI sites. The coding sequence of ZNF384
(NM_001039920) was ligated into the lentiviral vec-
tor pLJM1-EGFP-puro, and the enzymatic cleavage
sites were NheI and AgeI sites. The SOC cells were
infected with lentivirus at the MOI of 20.
For lipofection transfection,cells in 6-well plates
and transfected with Lipofectamine™ 3000 (Invitro-
gen, USA). Cells were maintained in a 37°C and 5%
CO2 incubator.
Western blotting
The protein extracted from the tissue or cell was
quantitatively concentrated with a BCA pro-
tein assay kit (Beyotime, China). Proteins were
separated via SDS-PAGE (Beyotime, China). Next,
samples were transferred to PVDF membranes
(Abcam, China). Samples were blocked with the
specified sealing solution (Beyotime, China) for 1h,
followed by incubating with primary antibodies and
secondary antibodies. The bands were visualized
using ECL luminol (Beyotime, China). The anti-
bodies are shown in Table1.
qPCR assay
RNA was extracted from SOC cell lines by Trizol
reagent (BioTeke, China), and its concentration was
determined. cDNA was synthesized using the Bey-
oRT II M-MLV Reverse Transcriptase (Beyotime,
China). The PCR reactions were performed using
the qPCR SYBR Green (Solarbio, China). Finally,
quantitative fluorescence analysis was done with
the Exicycler™ 96 fluorescence quantifier (Bioneer,
Korea). The primers are shown in Table2.
CCK-8 assay
We seeded 5 × 103 SOC cells in 96-well plates. 10
μL of CCK-8 reagent (KeyGen Biotech, China) was
added at the indicated times. The optical density
value was determined at 450nm by the microplate
reader (BIOTEK, USA).
Table 1 Antibodies employed in this study
Antibody Product number Company Origin Dilu-
tion
ZNF384 cat# ab176689 Abcam UK 1:3000
LIN28B cat# 24017–1-AP Proteintech China 1:1000
UBD cat# 13003–2-AP Proteintech China 1:500
Goat anti-
rabbit
cat# A0208 Beyotime China 1:5000
Goat anti-
mouse
cat# A0216 Beyotime China 1:5000
Table 2 Primers employed
in this study Gene Forward sequence (from 5’ to 3’) Reverse sequence (from 5’ to 3’)
qPCR ZNF384 GGT AGC ATC GAC CCT AAC CG CAT CCT CAG GGG AGA GGA CA
qPCR LIN28B CAG CCA AAG AAG TGCCA AGC CTC CTG AGG AAACG
qPCR UBD ATG CTT CCT GCC TCTGT GGG TAA GGT GGA TGGTC
qPCR β-actin GGC ACC CAG CAC AAT GAA TAG AAG CAT TTG CGG TGG
ChIP LIN28B-1 ATG CCA TCA TTG AGA TTA CTT AGG GCT ACT TTC TCA TTT TATT
ChIP LIN28B-2 CCT TTT CTT TTC TCC CTA AC CAC TCC AGC ACT GTT TCA C
RIP UBAP2L GAA ACT GGG AAC AAC CTC A TTT CTT TGC CTT CCT CAT
RIP UBE2T ATG CCA GAC AGT GGA CAG AG CAG GTT TAA AAG ATT TCA AAA TAC
RIP UBD TTG TAT TGG AGG GTGAC CTG GCT TTG AAT GCTCT
RIP UBE3A TTA GTT CAA GGA CAGCA GAA GAT TTC CTC CACAA
RIP UBE3C GGA GTT GTA TCC CGC ATT T TAT CCT CGT GGG TCTGG
RIP USP1 TGT TAT GGT GGT GGACT CAA TGG TTC TGG CTTAC
RIP SOX2 GGG CAA AAG TTT TAGAC AAA TGG AAA GTT GGGAT
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Wound healing assay
SOC cells were cultured in a serum-free medium and
then treated with mitomycin C (SIGMA, USA) for
1h. Subsequently, the cell monolayer was scratched
to form a scratching wound using a sterile pipette tip.
After 24h of culture, the wound images of the cells
were taken under the microscope at 0h and 24h.
Colonyformationassay
Lentivirus-infected SOC cells were digested by
trypsin and counted. About 400 cells were inoculated
in each petri dish. Till incubation lasted for 2weeks
visible colonies were formed. Then each petri dish
was fixed by 4% paraformaldehyde (Aladdin, China)
for 25 min and stained with Giemsa (KeyGen Bio-
tech, China) for 5min.
Transwell assay
For cell invasion assays, SOC cells were cultured in
a serum-free medium. The transwell chamber con-
taining Matrigel-coated polycarbonate membrane
filter (Corning, USA) was placed into the 24-well
plates, 800 μL culture medium with 10% FBS was
added to the lower chamber, the upper chamber was
added with 200 μL cell suspension and fixed with
4% paraformaldehyde for 20min. Finally, cells were
stained with 0.4% crystal violet and counted using an
inverted light microscope. Five visual field counting
cells were selected for each sample and the number
was averaged.
ELISA
The content of MMP-2 was quantified by Human
MMP-2 ELISA Kits (Multisciences, USA). The cul-
ture supernatants were centrifuged at 300 × g for
10 min to remove sediment. The OD values were
detected at 450nm with the microplate reader.
Immunofluorescence
Each cell sheet was fixed with paraformaldehyde,
permeabilized with 0.1% Triton X-100 (Beyotime,
China) for 30min, then incubated with BSA (Sangon,
China) for 15 min at room temperature. The SOC
cells were further incubated with primary antibodies
including Vimentin (Affinity, China) at 4°C. On the
second day, the sections were followed by incuba-
tion with fluorescent secondary antibodies anti-rabbit
IgG (CST, USA) for 1h. Finally, the DAPI (Aladdin,
China) was used to stain the nuclei and the anti-fluo-
rescence quencher (Solarbio, China) was added. The
fluorescence images were photographed by the fluo-
rescence microscope.
Animal models
6–8weeks female BALB/c nude mice were obtained
from Changzhou Cavens Laboratory Animal Co.
LTD (China) and subjected to xenograft tumor exper-
iments. Mice were randomly grouped, and SKOV3
and OVCAR-8 cells (1 × 105) in the logarithmic
growth phase were injected subcutaneously into the
flanks of mice, respectively. The tumor volume was
measured every 5days, and the tumor tissue was col-
lected after 25 days for subsequent testing. SKOV3
and OVCAR-8 cells (1 × 106) were intra-peritoneally
injected into the nude mice according to groups, and
the tumor metastasis in vivo was observed by IVS-
cope8200 Small animal live imaging system (Clinx,
China) after feeding for 4weeks. All animal experi-
ments were performed with consent from China Med-
ical University (Approval No. CMU2023049).
Immunohistochemistry
Immunohistochemistry was performed using
ZNF384 antibody (Bioss, China), Ki67 antibody
(Affinity, China). In brief, the sections were sub-
jected to dewaxing and rehydration. After the heat-
induced antigen recovery, the sections were sequen-
tially incubated with 3% hydrogen peroxide and 1%
BSA for 15 min, respectively. The primary anti-
bodies were added to the sections at 4°C. On the
second day, the secondary antibodies were added.
Finally, sections were stained with diaminobenzi-
dine (Solarbio, China) and re-stained with hema-
toxylin (Solarbio, China). The images were photo-
graphed by the microscope. The H-score method
was used to quantify the ZNF384 and Ki67 expres-
sion. The staining intensity was into 4 categories:
0, no staining; 1 + , weak staining; 2 + , moderate
staining; and 3 + , strong staining, and the percent-
age of cells at different staining intensities was
determined by visual assessment. The formula for
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calculating the score is H-score = Σ (Pi x i), where
the Pi is the proportion of positive cells with a cer-
tain intensity, and the i is the staining intensity. The
H-score is based on the 200 score standard, more
than 200 score is defined as high expression, less
than 200 score is defined as low expression.
Dual-luciferase assay
OVCAR-8 cells were inoculated in 12-well plates
to 90% confluency. Dual luciferase vectors contain-
ing different LIN28B promoter sequences were con-
structed and co-transfected with ZNF384 overex-
pression plasmid into OVCAR-8 cells. Cells were
collected after 48 h to detect the promoter activity.
The luciferase assay kit (KeyGen Biotech, China) was
used to measure the luciferase activities. The ratio
of luciferase was calculated according to the results
of the multifunctional microplate reader (TECAN,
Switzerland).
ChIP assay
For the ChIP experiment, the ChIP Assay Kit (Bey-
otime, China) was used. OVCAR-8 cells were fixed
with 37% formaldehyde to crosslink the DNA and
protein, followed by ultrasonic treatment to shear the
genomic DNA. After centrifugation of the sample at
12,000–14,000 × g for 4min, the samples were incu-
bated with the target antibody or with the negative
control anti-IgG overnight. Finally, the immunopre-
cipitated DNA was analyzed by PCR. The primers for
ChIP detection are listed in Table2.
RIP assay
For the RIP experiment, the EZ-Magna RIP Kit (Mil-
lipore, USA) was used. First, OVCAR-8 cells were
lysed in the RIP lysis buffer. The suspension was cen-
trifugated at 14,000 rpm for 10 min and cell super-
natant was incubatedwith the RIP immunoprecipita-
tion buffer which contained anti-LIN28B-conjugated
beads. The above samples were further incubated
with protease K for 30min. Finally, the immunopre-
cipitated RNA was analyzed by PCR. The primers for
RIP detection are listed in Table2.
RNA pull-down assay
For the RNA pull-down experiment, the BersinBio™
RNA pulldown Kit (Bersinbio, China) was used. In
brief, according to the length/mass ratio of 1 ug/1000
nt, biotin-labeled target RNA probes and NC probes
with corresponding mass were respectively taken to
form the RNA secondary structure. The RNA probes
forming the RNA secondary structure were added to
the beads and incubated with the cell lysate. Finally,
the RNA complex bound to the beads was eluted and
extracted for immunoblot analysis.
Statistical analysis
The GraphPad Prism 9.5 software was used to ana-
lyze data. All data were expressed as mean ± SD. For
two-group comparisons, the statistical differences
were assessed by Studen’s t-test. For multiple com-
parisons, the statistical differences wereevaluated by
ANOVA followed by Tukey’s test. The criterion for
significance was p < 0.05 for all comparisons.
Results
Identification of DEGs and their functional
annotation in serous ovarian cancer
We performed DEGs screening on the SOC data-
set GSE14407 with |log2FC|> 1, adj. P < 0.05, and
then performed GO and KEGG enrichment analysis
of the up-regulated DEGs (Fig.1a). We found that
many up-regulated DEGs were enriched in molecu-
lar functions related to DNA transcriptional activity
as well as in pathways associated with cell cycle and
cell adhesion. Furthermore, to reduce the impact of
DEGs screening on enrichment results, we further
performed GSEA analysis on the GSE14407 data-
set and annotated GO and KEGG (Fig.1b-c). The
results of the GSEA analysis indicated that genes
under this gene set were significantly enriched
in molecular functions related to DNA transcrip-
tional activity and epithelial cell proliferation, and
pathways related to the cell cycle. After excluding
transcription factors known to have a role in ovar-
ian cancer, we noted that Zinc finger protein 384
(ZNF384) is a common C2H2 type zinc finger pro-
tein transcription factor, which can bind to DNA,
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Fig. 1 Identification of DEGs and their functional annotation
in serous ovarian cancer. a GO and KEGG enrichment analy-
sis of up-regulated DEGs in GSE14407 serous ovarian cancer
database. Raw data between serous ovarian cancer and normal
ovarian tissue were obtained from the Gene Expression Omni-
bus (GEO; http:// www. ncbi. nlm. nih. gov/ gds/) dataset and the
gene expression profiling (GSE14407) was selected. Differ-
entially expressed genes (DEGs) were selected by |log2FC|> 1
and adj. P < 0.05 in the GSE14407 dataset. Gene Ontology
(GO) enrichment analysis and Kyoto Encyclopedia of Genes
and Genomes (KEGG) enrichment analysis software were
R packages ggplot2. b Enrichment fraction diagram of GO-
GSEA in the GSE14407 dataset. c Enrichment fraction dia-
gram of KEGG-GSEA in the GSE14407 dataset. Gene Set
Enrichment Analysis (GSEA) analysis software was R pack-
age clusterProfiler + enrichplot. GO, Gene Ontology; KEGG,
Kyoto Encyclopedia of Gene and Genome; DEG, differentially
expressed genes; GSEA, Gene Set Enrichment Analysis
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activate RNA polymerase, and promote the down-
stream gene expression (extracellular matrix genes,
cell cycle regulatory proteins) (He etal. 2019; Zhu
et al. 2023). In addition, ZNF384 has been exten-
sively studied in gynecological tumors, which can
promote the malignant progression of cervical can-
cer and breast cancer (Meng etal. 2022; Mori etal.
2015). Therefore, we chose ZNF384 for follow-up
study.
ZNF384 is highly expressed in serous ovarian cancer
The ZNF384 expression level in normal ovarian
epithelial tissues and SOC tissues of the GSE14407
database was further analyzed, and it can be seen
that ZNF384 was overexpressed in SOC tissues. In
addition, we demonstrated the location of the up-
regulated DEGs in the dataset on the chromosome by
circos plot, and the location of ZNF384 on the chro-
mosome is shown in Fig. 2a. We further collected
30 cases fresh SOC tissues and 30 cases non-tumor-
ovarian tissues to detect the expression of ZNF384
by qPCR. The results demonstrated that the ZNF384
expression was higher in SOC tissues compared to the
non-tumor-ovarian tissues (Fig. 2b). In addition, in
silico data analysis showed that ZNF384 was altered
in 7% of SOC and the mutation type was predomi-
nantly amplification (Fig. 2c). We further analyzed
whether ZNF384 expression affects the prognosis of
SOC by Gene Set Cancer Analysis dataset, and the
results demonstrated that patients with high ZNF384
expression had significantly lower OS and DSS than
patients with low expression. In addition, there was a
trend of decreased PFS and DFI in patients with high
ZNF384 expression. The above analysis suggested
that high expression of ZNF384 may indicate a poor
disease prognosis (Fig.2d). We further collected data
from 100 clinical samples of SOC patients to analyze
whether its expression affects clinicopathological fea-
tures. The data indicated that ZNF384 expression was
closely associated with the FIGO stage and Lymph
node metastasis (Table 3). Figure 2e showed repre-
sentative immunohistochemical staining maps of low
and high expression of ZNF384 and the immunohis-
tochemical score of the samples was shown in Sup-
plementary Table1. The above analysis indicated that
ZNF384 was abnormally highly expressed in SOC
and was related to poor tumor prognosis.
ZNF384 promotes the growth of serous ovarian
cancer cells invitro
Next, we specifically reduced or increased endog-
enous expression of ZNF384 in SOC cell lines by
lentiviral interference or overexpression techniques.
We first detected the expression of ZNF384 in nor-
mal ovarian epithelial cells (IOSE-80) and 5 lines
of serous ovarian cancer cells (OVCAR-8, HEY
A8, OVCAR-3, HEY and SKOV3) by western blot,
and found that ZNF384 expression was higher in
OVCAR-8 and OVCAR-3 cells and lower in SKOV3,
so we selected OVCAR-8 and OVCAR-3 cells to
ZNF384 knockdown treatment and selected SKOV3
cells to overexpression treatment (Fig.3a). As shown
in Fig. 3b, protein and mRNA levels of ZNF384
were decreased in ZNF384-shRNA lentivirus-
infected SOC cells. The above analyses confirmed
that ZNF384 was specifically depleted by ZNF384
shRNA. Similarly, the ZNF384 expression was sig-
nificantly increased in SKOV3 cells transfected with
ZNF384 overexpression lentivirus (Fig.3c). We next
assessed the effects of ZNF384 knockdown or overex-
pression on SOC cell proliferation, including cell via-
bility and colony-forming ability. The results demon-
strated that compared with the shCon group, ZNF3B4
knockdown dramatically inhibited cell viability, while
forced expression of ZNF384 in SOC cells enhanced
cell viability (Fig. 3d). In clone formation assay,
ZNF384 knockdown groups were observed to restrain
SOC cells proliferation whereas ZNF384 overexpres-
sion groups showed the opposite phenotype (Fig.3e).
These data suggested that ZNF384 promoted the pro-
liferation of SOC cells.
ZNF384 promotes the invasion and migration of
serous ovarian cancer cells
Meanwhile, we also evaluated the effect of ZNF384
on SOC metastasis. The cells of ZNF384 knock-
down groups had a greater migrating distance and
slower wound healing rate compared to the cells
of shCon group after 24 h, while up-regulation of
ZNF384 significantly increased the migratory capac-
ity of SOC cells (Fig.4a). In addition, the transwell
assay results showed similar trends in the invasion of
SOC cells (Fig. 4b). The overexpression of MMP-2
in ovarian cancer is related to tumor invasion and
metastasis (Vos et al. 2016). Therefore, we verified
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whether ZNF384 affects the production of MMP-2.
As shown in Fig.4c, the MMP-2 concentration in the
shZNF384 groups decreased extremely compared to
the shCon groups, whereas ZNF384 overexpression
resulted in an increase in MMP-2 concentration. Past
studies have suggested that vimentin overexpression
Fig. 2 ZNF384 is highly expressed in serous ovarian cancer.
a Circos plot showed the location of up-regulated genes in
chromosomes of the GSE14407 dataset. The outermost track
showed the chromosome information of these genes. The box
plot in the middle of the Circos plot showed the expression of
ZNF384 in normal and cancer tissue in the GSE14407 data-
base. b Relative expression of ZNF384 in 30 serous ovar-
ian cancer tissues and 30 normal ovarian tissues by qPCR. c
The genomic profile of ZNF384 was obtained from the serous
ovarian cancer data set (TCGA) using cBioportal database
(https:// www. cbiop ortal. org/). d The association between
ZNF384 expression and OS, PFS, DFI, and DSS in serous
ovarian cancer from the GSCA database (https:// guolab. wch-
scu. cn/ GSCA/). e Immunohistochemistry staining images of
high and low expression of ZNF384. The scale bar indicates
50 µm. TCGA, The Cancer Genome Atlas; GSCA, Gene Set
Cancer Analysis; OS, overall survival; PFS, progression-free
survival; DFI, disease-free interval; DSS, disease-specific sur-
vival. ****p < 0.0001 as compared to the Normal group. Data
are presented as mean ± SD. qPCR, quantitative real-time PCR
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may be associated with increased metastatic capacity
of SOC cells through the epithelial to mesenchymal
transformation (Psilopatis etal. 2023). On this basis,
we further evaluated the regulatory effect of ZNF384
on vimentin protein expression. Immunofluorescence
assay showed that ZNF384 knockdown inhibited
vimentin expression compared with control cells,
while ZNF384 overexpression showed the opposite
results (Fig. 4d). These data strongly indicated that
ZNF384 promotes SOC cell invasion, possibly in part
through the influence of MMP-2.
ZNF384 promotes tumor growth and metastasis
invivo
We further explored the effect of ZNF384 on the
SOC cells by establishing a vivo xenograft animal
model. The results indicated that the tumor volume
and weight in the ZNF384 knockdown group were
significantly reduced, indicating that the tumor
growth in the shZNF384 group was slower. In con-
trast, overexpression of ZNF384 promoted the growth
rate of tumors (Fig. 5a-c). In addition, we demon-
strated that the expression levels of Ki67, a marker
for tumor growth, were downregulated after ZNF384
knockdown using immunohistochemical staining,
while the overexpression of ZNF384 was the oppo-
site (Fig. 5d). Next, we further verified whether it
affects the metastasis of SOC cells invivo. ZNF384
knockdown or overexpressed SOC cells were intra-
peritoneally injected into the nude mice, respectively,
and the bioluminescence of the mice was observed
in intravital imaging after 4 weeks. As shown in
Fig.5e, compared with the shCon group, the biolumi-
nescence level of the shZNF384 group was reduced,
while ZNF384 overexpression significantly enhanced
the bioluminescence level in mice. In addition, the
number of tumor nodules was detected and counted,
and the results were consistent with the expectation
that ZNF384 overexpression significantly increased
the number of metastatic nodules (Fig. 5f). Taken
together, our results demonstrated that ZNF384 may
have a promoting effect on the development of SOC
invivo.
ZNF384 promotes transcriptional activity of LIN28B
in serous ovarian cancer
In our previous bioinformatics analysis, Lin-28
homolog B (LIN28B) was screened as a poten-
tial target of ZNF384. We hypothesized that the
oncogenic effect of ZNF384 might be exerted by
promoting the function of the RNA-binding pro-
tein LIN28B. Both western blot and qPCR assays
showed a positive correlation between ZNF384
treatment and LIN28B expression in SOC cells,
suggesting that LIN28B is indeed regulated by
ZNF384 (Fig. 6a-b). Next, we examined whether
ZNF384 promoted LIN28B expression via increas-
ing its promoter activity. We downloaded the
-2000/ + 100 bp DNA sequence of LIN28B from
the UCSC website, and then uploaded this sequence
to the JASPER database to predict the sequence
that may be recognized by ZNF384. The binding
sites were shown in Fig.6c. We constructed lucif-
erase reporters containing different lengths of the
LIN28B promoter and co-transfected them with
Table 3 Correlation of ZNF384 expression and clinicopatho-
logical parameters in serious ovarian cancer tissues
Differences between groups were done by the Chi-square test
p < 0.05 was considered significantly different
Characteristics No ZNF384
expression
level
P value
Low High
Overall
Patient age
 ≤ 55 58 23 35
 > 55 42 12 30 0.25141
Pathological grade
Well differentiated 13 4 9
Moderately differentiated 3 1 2 0.093951
Poorly differentiated 84 30 54
FIGO stage
I-II 43 22 21
III-IV 57 13 44 0.0325
Lymph node metastasis
Yes 42 8 34
No 58 27 31 0.00443
Intestinal metastasis
Yes 39 12 27
No 61 23 38 0.47817
Diaphragmatic metastasis
Yes 9 2 7
No 91 33 58 0.39951
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ZNF384 overexpression plasmid into OVCAR-8
cells. As shown in Fig. 6c, the luciferase activ-
ity was enhanced in the ZNF384 overexpression
group compared with the vector group. Further-
more, the luciferase activity of the LIN28B pro-
moter sequence (-1364 ~ + 31) was significantly
lower than that of the sequence (-1969 ~ + 31)
under ZNF384 overexpression conditions, there-
fore, we speculated that the main regulatory
region of ZNF384 on LIN28B promoter was the
-1969 ~ -1364 region, which contained three binding
sites: site7, site8, and site9. Next, we proposed to
design primers for each of these three sites to fur-
ther verify whether ZNF384 directly binds to these
sites using ChIP assay, but due to the close dis-
tance between site 7 and site 8, only a pair of prim-
ers can be used for detection. As demonstrated in
Fig.6d, the left panel corresponds to sites 7 and 8,
and the right panel corresponds to sites 9. The posi-
tive products were detected in both the Input group
and the anti-ZNF384 group, indicating that ZNF384
could directly bind to these sites, while negative
products were detected in the anti-IgG group, sug-
gesting that the binding of ZNF384 to the above
Fig. 3 ZNF384 promotes the growth of serous ovarian cancer
cells invitro. a Western blot analysis of ZNF384 expression in
normal ovarian epithelial cells (IOSE-80) and 5 lines of serous
ovarian cancer cells (OVCAR-8, HEY A8, OVCAR-3, HEY
and SKOV3). b Western blot and qPCR analysis of ZNF384
expression in OVCAR-8 and OVCAR-3 cells with ZNF384
knockdown. c Analysis of ZNF384 expression in SKOV3 cells
with ZNF384 overexpression. ****p < 0.0001 as compared to
the shCon or Vector group. d Cell proliferation of OVCAR-
8, OVCAR-3, and SKOV3 cells after lentivirus infection was
detected by CCK8 assay. *p < 0.05, **p < 0.01 as compared
to the shCon or Vector group. e Colony formation assay of
OVCAR-8, OVCAR-3, and SKOV3 cells was performed.
The experiments were repeated three times. **p < 0.01,
***p < 0.001, ****p < 0.0001 as compared to the shCon or
Vector group. Data are presented as mean ± SD. qPCR, quan-
titative real-time PCR
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Fig. 4 ZNF384 promotes the invasion and migration of serous
ovarian cancer cells. a The effects of ZNF384 on the migration
of serous ovarian cancer cells by wound healing assay. Photo-
graphs showed cell migration before and after injury under the
microscope at 100 × magnification field. Quantification of cell
migration by measuring wound closure areas before and after
injury. b Effect of ZNF384 on the invasion of serous ovarian
cancer cells by transwell assay (crystal violet staining × 200).
c MMP-2 levels in the supernatants of OVCAR-8, OVCAR-3,
and SKOV3 cells were detected by ELISA assay. *p < 0.05,
**p < 0.01, ****p < 0.0001 as compared to the shCon or Vec-
tor group. d Representative immunofluorescence images
of vimentin expression (red) in OVCAR-8, OVCAR-3, and
SHOV3 cells. Cell nuclei were stained by DAPI (blue). The
scale bar indicates 50 µm. Data are presented as mean ± SD.
DAPI, 4′6’-diamidino-2-phenylindole dihydrochloride
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sites is specific. Next, we further assessed whether
the regulation of LIN28B by ZNF384 affects the
malignant phenotype of SOC cells by constructing
the LIN28B overexpression plasmid and transfect-
ing it into ZNF384-knockdown stably transfected
cells. The transfection efficiency was verified by
western blot and qPCR (Fig. 6e). The viability of
cells transfected with LIN28B was enhanced com-
pared to the vector group, indicating that LIN28B
partially restored the knockdown effect of ZNF384
(Fig. 6f). Similarly, both wound healing and tran-
swell experiments demonstrated that LIN28B medi-
ated the regulation of ZNF384 on the migration and
invasion capacity of SOC cells (Fig.6g-h). Taken
together, ZNF384 mediated the malignant behavior
of SOC cells through transcriptional activation of
LIN28B expression.
LIN28B promotes the malignant behavior of serous
ovarian cancer cells by regulating UBD
Based on previous experiments, we have clarified the
role of LIN28B in the development of SOC, but its
molecular mechanism is still unclear. In this part, we
mainly carried out relevant studies on the downstream
molecular targets of LIN28B as well as the regula-
tory mechanism. Firstly, we predicted that UBAP2L,
UBE2T, ubiquitin D (UBD), UBE3A, UBE3C, and
USP1 might be the downstream factors of LIN28B
through literature research and ENCORI website,
Fig. 5 ZNF384 promotes tumor growth and metastasis
in vivo. a-c Xenograft tumors were formed by subcutaneous
injection of OVCAR-8 or SKOV3 cells into BALB/c nude
mice. The tumors were stripped after 25 days, photographed,
measured, and weighed. *p < 0.05, **p < 0.01, ****p < 0.0001
as compared to the shCon or Vector group. d Immunohis-
tochemical staining images of ZNF384, Ki67 expression in
tumor tissues. The scale bar indicates 50 µm. e Biolumines-
cence images of BALB/c nude mice after intraperitoneal injec-
tion of OVCAR-8 or SKOV3 cells. f Representative images
of abdominal cavity metastasis of tumors in different groups
of nude mice (left panel) and quantification of the num-
ber of tumor metastatic nodules (right panel). ***p < 0.001,
****p < 0.0001 as compared to the shCon or Vector group.
Data are presented as mean ± SD
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and the binding relationship between the above fac-
tors and LIN28B has not been reported. Next, we
used RIP and qPCR assays to detect the enrichment
of the above predictors in anti-IgG and anti-LIN28B
antibodies, using SOX2 as a negative control. As
shown in Fig.7a, UBAP2L, UBE2T, UBD, UBE3A,
UBE3C, and USP1 were notably enriched in the anti-
LIN28B RIP but not anti-IgG RIP. Notably, the role
Fig. 6 ZNF384 promotes transcriptional activity of LIN28B
in serous ovarian cancer. a-b Western blot and qPCR analy-
sis of LIN28B expression in OVCAR-8, OVCAR-3, and
SKOV3 cells with ZNF384 knockdown or overexpression.
***p < 0.001, ****p < 0.0001 as compared to the shCon or
Vector group. c Predicted binding site and sequence names of
LIN28B promoter (left panel). Dual-luciferase vectors contain-
ing LIN28B promoter sequences with different binding sites
were co-transfected with ZNF384 overexpression plasmid
into OVCAR-8 cells, and luciferase reporter gene assay was
performed after 48h (right panel). d The binding of ZNF384
to the LIN28B promoter was verified by ChIP experiments. e
OVCAR-8 cells were transfected with LIN28B overexpres-
sion plasmid, and the transfection efficiency was verified by
western blot and qPCR. f Cell proliferation of OVCAR-8 cells
with LIN28B overexpression was detected by CCK8 assay. g-h
The migration (g) and invasion (h) capacity of the OVCAR-8
cells transfected with LIN28B by wound healing and transwell
assays. *p < 0.05, **p < 0.01. Data are presented as mean ± SD.
qPCR, quantitative real-time PCR; ChIP, chromatin immuno-
precipitation
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of UBE2T and USP1 in ovarian cancer has been pre-
viously reported, while the GEPIA database showed
no significant changes in the expression of UBAP2L,
UBE3A, and UBE3C in ovarian cancer (Cui et al.
2022; Song et al. 2022). In addition, we found that
UBD expression was higher in ovarian cancer sam-
ples than in normal tissues through GEPIA database
analysis, so UBD was selected as the downstream fac-
tor of LIN28B for follow-up studies. The information
on the predicted binding motif of LIN28B was shown
in Fig. 7b. To demonstrate the physical interaction
between LIN28B and UBD mRNA, we performed
RNA pull-down experiments, which showed that
LIN28B could bind to the wild-type UBD mRNA
(lane 3). We further generated a UBD mutant Probe
(UBDdel probe), which the predicted LIN28B binding
site (AUG AGA A) was deleted. The results showed
that LIN28B could bind to wild-type UBD mRNA
(UBDWT probe, lane 3). In addition, the binding of
UBD to LIN28B was attenuated after the deletion of
the predicted UBD binding site (UBDdel probe; lane
4), suggesting that LIN28B may bind UBD by spe-
cifically recognizing this site. We further explored
the relationship between UBD mRNA and LIN28B.
Western blot and qPCR assays confirmed that knock-
down of LIN28B resulted in decreased UBDL mRNA
and protein levels (Fig.7c). On this basis, we used
actinomycin D for mRNA degradation experiments to
inhibit mRNA transcription. As shown in Fig.7d, the
results indicated that LIN28B knockdown enhanced
the degradation of UBD mRNA. In summary, the
above observations suggested that LIN28B can bind
to UBD and enhance the stability of UBD mRNA.
Next, we further verified whether LIN28B could
promote SOC by targeting UBD. UBD overexpres-
sion plasmid was co-transfected with shLIN28B into
Fig. 7 LIN28B promotes the malignant behavior of serous
ovarian cancer cells by regulating UBD. a Downstream fac-
tors that LIN28B may regulate were verified by RIP assay.
SOX2 was used as a negative control. b Schematic diagram
of predicted LIN28B motif and UBD binding sites. The bind-
ing of LIN28B and UBD was verified by RNA pull-down
assay, and the expression of LIN28B in OVCAR-8 cells was
detected by western blot. c Western blot and qPCR analysis of
UBD expression in OVCAR-8 cells with LIN28B knockdown.
d The half-life of UBD mRNA was detected in cells depicted
in (c). e OVCAR-8 cells were transfected with UBD overex-
pression plasmid, and the transfection efficiency was verified
by western blot and qPCR. Cell proliferation (f), migration (g),
and invasion (h) ability of OVCAR-8 cells after transfection
with UBD were detected by CCK-8, wound healing, and tran-
swell assays, respectively. *p < 0.05, **p < 0.01, ***p < 0.001,
****p < 0.0001. Data are presented as mean ± SD. RIP, RNA-
binding protein immunoprecipitation; qPCR, quantitative real-
time PCR
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Cell Biol Toxicol (2024) 40:100 Page 15 of 18 100
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OVCAR-8 cells to further assess the cell prolifera-
tion, migration, and invasion abilities. The UBD pro-
tein expression in OVCAR-8 cells transfected with
shLIN28B or co-transfected with shLIN28B + UBD
was detected by western blot. the UBD expression
decreased with the knockdown of LIN28B, and qPCR
also showed similar experimental results, suggesting
that LIN28B may regulate the expression of UBD in
SOC cells (Fig. 7e). The proliferation of OVCAR-8
cells transfected with shLIN28B was slower than that
of the shNC group, while UBD partially reversed the
inhibitory effect of shLIN28B (Fig. 7f). In addition,
transfection with shLIN28B inhibited the migration
and invasion of SOC cells compared to the shNC
group, whereas UBD significantly restored this trend
(Fig.7g-h). Collectively, these data demonstrated that
LIN28B promoted SOC progression by regulating
UBD expression in SOC cells.
Discussion
SOC is the most lethal malignancy of the female
reproductive system (Prat 2015). Research con-
ducted over the past 20years has led to a profound
understanding of its cellular and molecular basis. For
example, the origin of HGSOC has been elucidated,
and serous tubal epithelial carcinoma is the precur-
sor lesion of HGSOC which helps guide the develop-
ment of prevention programs for patients with genetic
susceptibility (Yamamoto et al. 2016). Although the
diagnosis and treatment technology of SOC has made
great progress, its clinical outcome is still not optimis-
tic due to the late clinical stage of most patients at the
time of diagnosis and the high recurrence rate after
treatment. Therefore, the discovery of new potentially
effective targeted genes and biomarkers is the key to
early tumor prevention and effective treatment.
ZNF384 belongs to a family of zinc finger pro-
teins and acts as transcription factors that can shut-
tle between nucleoplasm and respond to changes in
cell shape and cytoskeletal structure by altering DNA
structure (Janssen and Marynen 2006). In addition,
ZNF384 is consistently highly expressed in a variety
of malignant tumors, such as lung cancer, hepatocel-
lular cancer, and has a significant clinical correlation
with worsening prognosis (He etal. 2019; Yan etal.
2022; Young et al. 2016). Recent studies have iden-
tified ZNF384 as a potential biomarker for psoriasis
and linked it to excessive inflammation and metabolic
disorders, further demonstrating the critical role of
ZNF384 in the disease process (Liu et al. 2022). In
our research, we found that ZNF384 has a gene vari-
ant rate of 7% in SOC, was overexpressed in SOC, and
ZNF384 expression was strongly related to the prog-
nosis of patients. It is suggested that ZNF384 may be
involved in the progression of SOC as an oncogene.
On this basis, we revealed that ZNF384 knockdown
inhibited the proliferation, migration, and invasion
of SOC cells, while overexpression of ZNF384 pro-
moted the malignant behavior of SOC cells. Mean-
while, by constructing a xenograft mice model, we
found that ZNF384 knockdown invivo significantly
inhibited tumor growth as well as the invasive ability
of tumor cells. Combined with the above findings, we
reasonably hypothesized that SOC cells express high
levels of ZNF84 to enhance their malignant behaviors
through some unknown mechanisms.
The pathogenic mechanism of ZNF384 has been
extensively studied in a variety of cancers. In hepa-
tocellular carcinoma, ZNF384 can proliferate tumor
cells by increasing cell cycle protein expression (He
et al. 2019; Xiao et al. 2022). In addition, ZNF384
has been shown to bind to the APOBEC3B promoter
and regulate A3B expression in cervical cancer,
thereby accelerating cancer progression (Mori et al.
2015). To further explore the molecular mechanisms
by which ZNF384 regulates SOC cell progression,
we validated the target genes of ZNF384. The analy-
sis results of the JASPER website suggested that the
LIN28B promoter could be recognized by ZNF384,
indicating that ZNF384 might transcriptionally acti-
vate LIN28B.
LIN28B is a highly conserved RNA-binding pro-
tein discovered in 1997, and in recent years the
expression and active participation of LIN28B has
been found in embryonic stem cell differentiation
(Moss et al. 1997; Zhou et al. 2020). LIN28B has
previously been reported to be important in the trans-
formation of cancer stem-like cells, promotes tumor
invasiveness and metastasis, and is overexpressed in
multiple tumor types associated with advanced dis-
ease (Viswanathan etal. 2009; Xu etal. 2023; Yuan
and Tian 2018). LIN28B is associated with poor
prognosis of ovarian cancer by a molecular mecha-
nism related to the negative regulation of let-7 fam-
ily microRNAs, altering the expression of down-
stream genes and tumor development (Busch et al.
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2016). Our study showed that ZNF384 and LIN28B
expression are positively correlated in SOC cells,
and ZNF384 could recognize specific sequences on
the LIN28B promoter, thereby promoting the tran-
scriptional activity of LIN28B. Moreover, LIN28B
could partially restore the tumor suppressor effect
of ZNF384 knockdown. Therefore, we suggested
that the ZNF384/LIN28B regulatory axis has a very
important role in SOC. As an RNA-binding protein,
LIN28B may have multiple target RNAs. We found
that LIN28B could bind UBD mRNA by RBPsuite
and ENCORI website analysis, and the analysis of
the GEPIA database showed that UBD expression
was elevated in ovarian cancer tissues. UBD is also
a marker factor for precancerous lesions, and past
studies revealed that UBD imparts malignant char-
acteristics to non-tumorigenic cells and enhances
malignancy-associated features in cancer cells (Gao
etal. 2014; Zhang etal. 2020). In addition, we noted
an interesting phenomenon that UBD expression in
tumors was somewhat tissue-specific, and transcrip-
tional upregulation was observed in the ovary (Lee
et al. 2003). Our study verified that LIN28B could
bind to UBD and thus enhance the stability of UBD
mRNA. In tumor cells, the expression of UBD was
positively correlated with that of LIN28B. Further-
more, the rescue experiment results showed that UBD
partially reversed the inhibitory effect of LIN28B
knockdown on the malignant behaviors of SOC
cells. Overall, these results suggested that ZNF384
is deeply involved in SOC carcinogenesis and pro-
gression by upregulating the expression of UBD via
promoting the transcriptional activity of LIN28B and
promoting the malignant behavior of tumor cells.
In summary, we systematically demonstrated
the biological role of ZNF384 in SOC occurrence
and development, confirmed the oncogene role of
ZNF384 in SOC, and then revealed the molecular
mechanisms by which ZNF384 regulates cell prolif-
eration (Fig.8). Our results suggested that ZNF384
regulates the malignant development of SOC and is
a potential prognostic marker for SOC.
Author contributions Min Wang conceives and designs
experiments. Ye Yang, Runze He, Dongxiao Li, Tianli Mu
and Ziteng Kuang conduct the experiments. Runze He collects
clinical samples. Ye Yang and Runze He perform statistical
analysis. Ye Yang writes initial draft of manuscript. Min Wang
revises manuscript. All authors have critically revised manu-
script and approved its final version.
Funding This study was supported by the Outstanding Sci-
entific Fund of Shengjing Hospital (under Grant 201705).
Data availability No datasets were generated or analysed
during the current study.
Declarations
Ethics approval All procedures involving human subjects
were approved by Shengjing Hospital of China Medical Univer-
sity (Approval No. 2023PS426K), and animal experiments were
performed with consent from the ethics committee of China
Medical University (Approval No. CMU2023049).
Fig. 8 Mechanism of ZNF384 promoting serous ovarian can-
cer progression. ZNF384 is involved in the malignant progres-
sion of serous ovarian cancer by promoting the proliferation,
migration, and invasion of serous ovarian cancer cells. Its
mechanism of action is related to the binding of the LIN28B
promoter to promote its transcriptional activity, which further
binds UBD mRNA, affects mRNA stability, and enhances its
expression
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Cell Biol Toxicol (2024) 40:100 Page 17 of 18 100
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Consent for publication All authors have read the paper and
agree that it can be published.
Competing interests The authors declare no competing inter-
ests.
Open Access This article is licensed under a Creative Com-
mons Attribution-NonCommercial-NoDerivatives 4.0 Interna-
tional License, which permits any non-commercial use, shar-
ing, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence,
and indicate if you modified the licensed material. You do
not have permission under this licence to share adapted mate-
rial derived from this article or parts of it. The images or other
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you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://creativecom-
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