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Cellular Oncology
https://doi.org/10.1007/s13402-023-00891-w
RESEARCH
Cancer/testis‑45A1 promotes cervical cancer cell tumorigenesis
anddrug resistance byactivating oncogenic SRC anddownstream
signaling pathways
MeiMeng1,2,3,4,5· YanGuo6 · YuChen1· XuLi1· BinZhang7· ZhijiaXie8· JuntaoLiu1· ZheZhao9· YuxiLiu1·
TongZhang1· YingnanQiao1· BingxueShang10,11 · QuanshengZhou1,2,3,4,5
Accepted: 8 October 2023
© The Author(s) 2023
Abstract
Background Cancer/testis antigen-45A1 (CT45A1) is overexpressed in various types of cancer but is not expressed in healthy
women. The role of CT45A1 in cervical cancer has not yet been described in the literature.
Purpose The aim of this research was to study the role of CT45A1 in cervical cancer progression and drug resistance,
elucidate the mechanisms underlying CT45A1-mediated tumorigenesis and investigate CT45A1 as a biomarker for cervical
cancer diagnosis, prognostic prediction, and targeted therapy.
Methods The CT45A1 levels in the tumors from cervical cancer patients were measured using immunohistochemical stain-
ing. The role and mechanisms underlying CT45A1-mediated cervical cancer cell tumor growth, invasion, and drug resistance
were studied using xenograft mice, cervical cancer cells, immunohistochemistry, RNA-seq,real-time qPCR, Chromatin
immunoprecipitationand Western blotting.
Results CT45A1 levels were notably high in the tumor tissues of human cervical cancer patients compared to the paracancer-
ous tissues (p < 0.001). Overexpression of CT45A1 was closely associated with poor prognosis in cervical cancer patients.
CT45A1 promoted cervical cancer cell tumor growth, invasion, neovascularization, and drug resistance. Mechanistically,
CT45A1 promoted the expression of 128 pro-tumorigenic genes and concurrently activated key signaling pathways, includ-
ing the oncogenic SRC, ERK, CREB, and YAP/TAZ signaling pathways. Furthermore, CT45A1-mediated tumorigenesis
and drug resistance were markedly inhibited by the small molecule lycorine.
Conclusion CT45A1 promotes cervical cancer cell tumorigenesis, neovascularization, and drug resistance by activating
oncogenic SRC and downstream tumorigenic signaling pathways. These findings provide new insight into the pathogenesis
of cervical cancer and offer a new platform for the development of novel therapeutics against cervical cancer.
Keywords Cervical cancer· CT45A1· Tumorigenesis· Biomarker· Lycorine· Cancer therapy
1 Introduction
Although the human papillomavirus (HPV) vaccine has
effectively reduced the incidence of cervical cancer in
developed countries, the annual global incidence of cervical
cancer remains high. Globally, there are 604,127 new cases
and 341,831 deaths from cervical cancer annually, and the
five-year survival rate of metastatic and advanced cervical
cancer patients is a mere 10% [1, 2]. The main reasons for
this catastrophe are that the mechanisms of cervical cancer
metastasis and progression are enigmatic [2, 3] and there are
no effective drugs against metastatic and advanced cervical
Mei Meng, Yu Chen and Xu Li contributed equally to this work.
* Yan Guo
guoyansdfyy@163.com
* Bingxue Shang
bingchengliren@163.com
* Quansheng Zhou
zhouqs@suda.edu.cn
Extended author information available on the last page of the article
M.Meng et al.
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Cancer/testis‑45A1 promotes cervical cancer cell tumorigenesis anddrug resistance by…
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cancer available in the clinical setting [4]. Therefore, there is
a critical need for elucidation of the mechanisms underlying
cervical cancer metastatic and progression and development
of effective drugs against cervical cancer.
HPV is a trigger in the initiation of cervical cancer [1, 2].
The Pap test has long been used to identify HPV-induced
cellular disorder, and HPV mRNA and DNA have recently
been utilized for the screening and diagnosis of cervical
cancer [5–7]. However, most HPV-infected women do not
suffer from cervical cancer in their lifetime; while 291 mil-
lion women have been infected by HPV worldwide, only
a small portion of HPV-infected women go on to develop
cervical cancer [2, 3, 8]. Emerging evidence indicates that
some cervical cancers are independent of HPV [9, 10]. Of
note, it has been reported that the risk of cancer metastasis
in HPV-negative cervical cancer patients is higher than that
in HPV-positive cases [10–12], implying that many patho-
logical factors, other than HPV, also play a critical role in
cervical cancer metastasis and progression.
Increasing evidence indicates that multiple factors, includ-
ing hypoxia [13], microbiome-induced chronic inflammation
[14], overexpression of various oncogenes due to aberrant
genetic and epigenetic alterations, activation of multiple
tumorigenic signaling pathways [15–17], and the generation
of cancer stem cells [18, 19], trigger robust tumor growth,
neovascularization, cancer metastasis, and drug resistance [2,
3, 15–19]. However, the mechanisms underlying HPV-inde-
pendent cervical cancer initiation and progression are unclear.
Cancer/testis antigens (CTAs) are proteins that are restric-
tively expressed in the male testes and are not expressed
in healthy females. However, various CTAs are aberrantly
overexpressed in several types of cancer [20–24]. To date,
more than 700 CTAs have been identified; however, the
effects of most CTAs on tumorigenesis and cancer progres-
sion remain unclear [21].
Cancer/testis antigen-45A1 (CT45A1) is a proto-onco-
gene overexpressed in various types of cancer; it is not
expressed in normal tissues and cells in healthy women
[25–28]. Overexpression of CT45A1 enhances tumor cell
motility [25] and promotes cancer metastasis to the lungs
[26] and bones [27]. Aberrant CT45A1 overexpression is
also closely associated with the poor prognosis of malignant
tumors [25–27], such as ovarian cancer [28]. However, the
role of CT45A1 and other CT45 family members (CT45) in
cervical cancer has not yet been reported in the literature.
There are nine CT45 family members in the human
genome with 97% identity in amino acid sequences and high
tumor specificity and antigenicity [25–28]. CT45 has been
targeted for cancer immunotherapy, the addition of CT45-
mediated immunotherapy to chemotherapeutics raises the
efficacy of ovarian cancer therapy [29]. Additionally, CT45
has been targeted using SiRNA and nano micelles for can-
cer therapy [30, 31]. However, the small molecules against
CT45-mediated carcinogenesis remain to be explored.
In the current study, we found that CT45A1 levels were
notably high in the tumor tissues of human cervical cancer
patients. Overexpression of CT45A1 was closely associated
with poor prognosis in these cancer patients. CT45A1 pro-
moted tumorigenesis, neovascularization, cancer metastasis,
and drug resistance. Interestingly, the small molecule lyco-
rine effectively inhibited CT45A1-mediated tumorigenesis,
neovascularization, and drug resistance. This study is the first
to unravel the role of CT45A1 in cervical cancer progression
and demonstrate that CT45A1 is a new biomarker for cervical
cancer diagnosis, prognostic prediction, and therapy.
2 Results
2.1 CT45A1 promotes tumorigenesis andisanew
biomarker forcervical cancer diagnosis
andprognostic prediction
CT45A1 expression was investigated in cervical cancer
patients. Immunofluorescence (IF) staining revealed that
CT45A1 was overexpressed in the tumor tissues of cervical
cancer patients, but not in the paired paracancerous tissues
(Supplementary Fig.S1A). Immunohistochemical (IHC)
staining revealed that the CT45A1 level in the tumor tissues
obtained from 119 cervical cancer patients (Supplementary
TableS1) was markedly higher than that in the paired para-
cancerous tissues (Fig.1A, B, p < 0.001), with 88% specific-
ity and 62% sensitivity without classification of cancer stage.
Fig. 1 CT45A1 is overexpressed in tumor tissues from cervical can-
cer patients and promotes tumor growth and metastasis in xenograft
mice. Immunohistochemical staining showed that CT45A1 was over-
expressed in the tumor tissues of cervical cancer patients (A-1 and
A-2), but there was very little expression in the para-cancerous tis-
sues (A-3 and A-4) and in the benign uterine myoma tissues (C).
The results were scored and statistically analyzed (B). The CT45A1
expression levels in uterine myoma, cervical cancerstages I/II, and
cervical cancerstages III/IV were also statistically analyzed (D). The
correlation between CT45A1 levels and the overall survival of cer-
vical cancer patients was assessed by gene expression profiling in
the TCGA cohort and Kaplan–Meier analysis (E). The expression
levels of CT45A1 in the organs and tissues of healthy individuals
were measured by RT-PCR (F). Cervical cancer Caski cells trans-
fected with either CT45A1-vector (CT45A1) or vector as a control
(Vector) were subcutaneously injected into nude mice (n = 5/group).
Tumor infiltration in each group was shown in G; the blue and pink
circle points to the junction between the subcutaneous tumor and
the peritoneum. The tumor volume was calculated (H). The tumors
were weighted and analyzed (I). Data represent the mean (± SE) of
the tumor vascular diameter in 17 tumor tissue fields of five tumor-
bearing mice (J). The blood vessel number in the tumors with
CT45A1 expression (K, tumor) was greater than in the tumors with-
out CT45A1 expression; the red arrows refer to the blood vessels in
the tumor. P values calculated by the log-rank test. Data are shown as
the mean ± SE. *p < 0.05, **p < 0.01 in an unpairedt-test
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Among the 20 benign uterinemyoma patients, IHC staining
showed that 16 patients did not express CT45A1, 3 patients
barely expressed CT45A1, only 1 patient expressed CT45A1
at a moderate level (Fig.1C, D), indicating that there was
virtually no expression of CT45A1 in most of the benign
uterinemyoma patients. The CT45A1 level of patients with
early-stage cervical cancer (I–II) was significantly higher
than that of benign uterinemyoma patients (p < 0.001)
(Fig.1D). The CT45A1 level of patients with advanced-
stage cervical cancer (III-IV) was much higher than that
of benign uterinemyoma patients (Fig.1D, p < 0.001).
Strikingly, the specificity and sensitivity of CT45A1 in the
advanced-stage (III-IV) cervical cancer patients reached
98% and 91%, respectively. These data indicate that CT45A1
is a new biomarker for the diagnosis of cervical cancer.
Additionally, the t-test showed a significant positive corre-
lation between CT45A1 levels and pathological cancer grade
(Fig.1D, p < 0.001, supplementary TablesS2 and S3). More
importantly, Kaplan–Meier plots indicated that high expres-
sion of CT45A1 was associated with a poor prognosis in
cervical cancer patients (kmplot.com) (Fig.1E). The average
survival time was shortened by 2.5years in the cervical can-
cer patients with high CT45A1 levels as compared to those
with low CT45A1 levels (p = 0.035). Further more, CT45A1
expression was investigated in 12 organs or tissues from
healthy people by RT-PCR (Fig.1F) and Real-time PCR
(Supplementary Fig.S1B), respectively. The results showed
that CT45A1 was overexpressed in the male testis but was not
expressed or had extremely low expression in other normal
tissues. Together, these data suggest that CT45A1 has high
tumor specificity and sensitivity and is a new biomarker for
the diagnosis and prognostic prediction of cervical cancer.
Next, the effect of CT45A1 on cervical tumor growth was
examined in xenograft mice. Nude mice (n = 5/group) were
subcutaneously injected with Caski cells with or without
CT45A1 expression (Fig.1G, Supplementary Fig.S2A, S2B).
Forty-five days later, the tumor volume and weight were signif-
icantly increased in the CT45A1 expression group compared
to the control group (Fig.1H, I). Strikingly, the tumors with
CT45A1 expression exhibited irregularly shaped edges and
were invaded into the deep skin layer (Fig.1G); the tumors in
three out of five mice had disseminated to the peritoneum. By
contrast, the tumors without CT45A1 expression had smooth
surfaces or soft textures and were easily stripped from the skin
layer. H&E staining indicated that the number of blood vessels
was increased 3.5-fold in the tumors with CT45A1 expression
compared to the control tumors without CT45A1 expression
(Fig.1J, K), suggesting that CT45A1 enhances tumor growth.
Additionally, we performed invitro tube formation assay to
assess the effect of CT45A1 on tumor cell-mediated neovas-
cularization. The result showed that the numbers of tube-like
structures in CT45A1-overexpressing cervical cancer Caski
cells were more than that of the control Caski cells without
CT45A1 expression (Supplementary Fig.S1C and S1D), sug-
gesting that CT45A1 promotes tumor cells-mediated neovas-
cularization. In short, these data imply that CT45A1 enhances
tumor malignant progression and neovascularization.
Moreover, CT45A1 significantly increased the migration
and invasion of both cervical cancer Caski and Siha cells
(Fig.2A–H); convincingly, silencing of CT45A1 resulted in
a significant decrease in HeLa cell migration and invasion
(Fig.2I–L), implying that CT45A1 increases cervical cancer
cell motility. Additionally, a colony formation assay showed
that CT45A1 increased the cervical cancer cell colony num-
ber 2.6-fold (Fig.2M–P), suggesting that CT45A1 increases
cervical cancer cell tumorigenesis. Collectively, these data
indicate that CT45A1 enhances tumor growth, neovasculari-
zation, and metastasis invivo and promotes cervical cancer
cell tumorigenesis, tube-like structure formation, migration,
and invasion invitro.
2.2 CT45A1 triggers theoverexpression
ofoncogenic genes andactivates tumorigenic
signaling pathways
DNA microarray showed that CT45A1 up-regulated the
expression of 128 genes in cervical cancer Caski cells, includ-
ing 68 pro-tumorigenic genes, such as fibronectin-1 (FN1),
OXTR, PLAC8, LCP1, DACT1, KIAA1462, COL4A1,
ABCA1, CNN1, GRB10, TNC, LMCD1, CPE, PLAC8,
GNB4, TGFBI, LTBP1, CHML, KRT8, and COL4A2 (> two-
fold, p < 0.05, Fig.3A, Supplementary TableS4). In particu-
lar, the expression level of tumorigenic FN1 was increased
14-fold in cervical cancer Caski cells with CT45A1 expres-
sion compared to control cells without CT45A1 expression
(Fig.3B). Many other oncogenic genes, including PLAC8,
DACT1, KISS1, and GRB10, were also markedly increased
(Supplementary Fig.S2C). In contrast, CT45A1 down-
regulated 126 genes (> twofold, p < 0.05, Supplement ary
TableS4). Additionally, CT45A1 overexpression changed
multiple signaling pathways in cervical cancer cells, includ-
ing the ECM-receptor interaction, focal adhesion, Hippo, and
PI3K-AKT signaling pathways (Supplementary Fig.S2D).
Further investigation revealed that CT45A1 markedly
elevated both FN1 mRNA and protein levels in cervical can-
cer cells (Fig.3B–E, Supplementary Fig.S3A and S3B).
Fig. 2 CT45A1 enhances cervical cancer cell migration, invasion and
colony formation. CT45A1 promoted the migration of cervical cancer
Caski (A, C) and Siha cells (E, G) and also enhanced the invasion of
cervical cancer Caski (B, D) and Siha cells (F, H). By contrast, silenc-
ing of CT45A1 in HeLa cells inhibited cell migration (I, K) and invasion
(J, L). The colony numbers in CT45A1-overexpressed Caski cells (M,
CT45A1) and CT45A1-silenced HeLa cells (N, shRNA) were counted
and statistically analyzed (O and P). Data are shown as the mean ± SE
of three independent replicates. *p < 0.05, **p < 0.01 in an unpairedt-test
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Mechanistically, CT45A1 promoted FN1 gene transcription
by enhancing FN1 gene promoter activity (Fig.3F and G) and
interacting with the transcription factor CREB (Supplemen-
tary Fig.S3C-S3I). By contrast, the Caski cells that overex-
pressed CT45A1 was silenced by shRNA significantly reduced
FN1 levels (Fig.3H and I) and inhibited Caski cell migration
(Fig.3J–K). Together, these data suggest that CT45A1 regu-
lates FN1 gene transcription in cervical cancer cells.
CT45A1 is a nuclear protein. We recently identified the
CT45A1 protein-binding consensus sequence CGC CCC
(Fig.3L). In the current study, we first explored whether
the CT45A1 protein-binding CGC CCC exists in the FN1
gene promoter region (Fig.3L). The result showed that
there are two CGC CCC sequences in the FN1 gene pro-
moter region (Fig.3L), named as the site 1 and site 2,
respectively. Next, the direct binding between purified
CT45A1 recombinant protein and these two sites GAT
CCG AAAG CGC CCC GCG GAA TCT (site 1) and TCT
CTC CCCCC CGC CCC G GGC CTC CAG (site 2) was
accessed by CHIP. The results showed that CT45A1
directly bound to the site 1 in cervical cancer cells but
did not bind to the site 2 (Fig.3M–N, Supplementary
Fig.S2E). Convincingly, Protein-DNA binding Dot blot
confirmed the results (Fig.3O), suggesting that although
the core nucleic acid sequence CGC CCC is important for
CT45A1 binding, the front and downstream nucleic acids
of the CGC CCC is also critical for the binding of CT45A1
protein to the FN1 gene promoter. In brief, these data indi-
cate that CT45A1 binds to the FN1 gene promoter and
drives transcription of the gene.
We next examined whether CT45A1 affects the FN1 down-
stream oncogene SRC. Western blotting showed that CT45A1
overexpression significantly increased SRC phosphorylation,
whereas silencing of CT45A1 in Caski-CT45A1 cells reduced
SRC phosphorylation (Fig.4A, B). Notably, silencing of FN1
by shRNA completely abolished CT45A1-induced SRC acti-
vation (Fig.4C and D), implying that FN1 is at the down-
stream of CT45A1.
Additionally, co-immunoprecipitation (Co-IP) revealed
that CT45A1 was able to directly bind to the SRC protein in
cervical cancer cells (Fig.4E, F). Immunofluorescence imag-
ing indicated that CT45A1 changed the SRC protein localiza-
tion from the sub-cellular membrane to the cytoplasm and
nucleus in cervical cancer cells (Fig.4G, H). Furthermore,
the direct binding between CT45A1 and SRC proteins was
further confirmed by a pulldown assay (Fig.4I). Moreover,
an invitro protein kinase activity assay showed that CT45A1
markedly increased SRC protein kinase activity in an aden-
osine triphosphate (ATP)-dependent manner (Fig.4J, K).
Convincingly, the SRC-specific inhibitor 6-dimethylamino-
2-phenyl-3(2H)-pyridazinone (PP2) markedly suppressed
CT45A1-mediated SRC-ERK-CREB activation (Fig.5A–D)
and inhibited cervical cancer cell migration (Fig.5E and F).
Additionally, silencing of CT45A1 in HeLa cells significantly
reduced the levels of oncogenic FN1, p-SRC, p-ERK, and
p-CREB (Supplementary Fig.S4A-4E). Together, there data
suggest that CT45A1 is a new activator of oncogenic SRC,
and there is a novel pro-tumorigenic CT45A1-FN1-SRC-
ERK-CREB signaling pathway in cervical cancer (Fig.5G),
importantly, CT45A1 is at the front of the signaling pathway;
and silencing of CT45A1 inhibits multiple oncogenic signal-
ing pathways.
Next, the effects of CT45A1 on the expression of the onco-
genic proteins Yes-associatedprotein (YAP)/ tafazzin (TAZ)
downstream of the Hippo signaling pathway were investi-
gated. Western blotting revealed that the levels of the YAP/
TAZ proteins were notably increased in cervical cancer Siha
cells with CT45A1 expression (Fig.6A–C), but did not sig-
nificantly affect several other signaling pathways (Supplemen-
tary Fig.S4F and S4G). By contrast, silencing of CT45A1
in HeLa cells by shRNA markedly reduced the levels of the
YAP/TAZ proteins (Fig.6D–F), whereas the YAP and TAZ
mRNA levels were not significantly changed (Supplementary
Fig.S4H). Additionally, CT45A1 interacted with the YAP
and TAZ proteins (Fig.6I–K) and co-localized with YAP and
TAZ in the nucleus (Fig.6L, Supplementary Fig.S4I-S4K).
Convincingly, theSRC inhibitor PP2 abolished CT45A1-
induced YAP expression (Fig.6G and H). These data suggest
that CT45A1 is a new inducer of tumorigenic YAP/TAZ, and
there is a new oncogenic CT45A1-SRC-YAP/TAZ signaling
pathway in cervical cancer cells. Collectively, CT45A1 plays
an important role in triggering tumorigenesis and is a target
for anti-cervical cancer therapy.
Fig. 3 CT45A1 upregulates fibronectin-1 (FN1) in cervical cancer
cells. DNA microarray analysis showed CT45A1 induced differential
expression of genes between Caski cells with and without expres-
sion of CT45A1; asterisk indicates that FN1 is the most up-regulated
gene among the CT45A1-regulated genes (A). QT-PCR confirmed
that CT45A1 markedly increased FN1 mRNA levels (B). Western
blot showed that the overexpression of CT45A1 notably increased
FN1 protein levels in Caski cells and in the supernatant of the cell
culture (SPN FN1) (C), and the data were statistically analyzed (D
and E). The FN1 gene promoter region (-1354 bp to + 247 bp) and
the location of the transcription factor CREB are shown (F). The
luciferase assay indicated that CT45A1 markedly increased FN1 pro-
moter activity (G). Silencing of CT45A1 reduced FN1 protein levels
(H, I) and decreased Caski-CT45A1 cell migration (J, K). Computer
analysis predicates potential CT45A1 protein-binding site 1 and site
2 in the FN1 gene promoter region (L). ChIP showed that CT45A1
bound to the site 1, but did not bind to the site 2 in FN1 promoter
region (M). QT-PCR also indicated that CT45A1 bound to the site
1 (N). The site 1 and site 2 nucleic acids, and control nucleic acids
were spotted on nitrocellulose membranes, and blocked with 5%
Nonfat-Dried Milk buffer. After incubation with CT45A1 protein,
the binding of CT45A1 protein to FN1 gene promoter site 1 and site
2 nucleic acids, and control nucleic acids were detected by CT45A1
specific monoclonal antibody and Dot blot (O). Data are shown as
the mean ± SE of at least three independent replicates. *p < 0.05,
**p < 0.01 in an unpairedt-test
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2.3 CT45A1 boosts cisplatin drug resistance
andapoptosis resistance andisatarget
fordeveloping novel therapeutics
againstcervical cancer
Overexpression of CT45A1 was found to boost cisplatin
resistance and apoptosis resistance (Fig.7) in cervical can-
cer Siha cells. CT45A1 significantly diminished 10μM
cisplatin-induced DNA damage (Fig.7A–E), as evident
by decreases in levels of the DNA damage marker γH2AX
(Fig.7B, C and H) and apoptotic cells (Fig.7F and G). This
suggests that CT45A1 increases cervical cancer cell cispl-
atin drug resistance and apoptosis resistance.
Next, CT45A1-targeted therapeutics were explored and
the small molecule lycorine (MW: 287.31) was found to
markedly reduce CT45A1 levels in cervical cancer cells
(Fig.8A, B). Additionally, lycorine inhibited the phospho-
rylation of oncogenic SRC and ERK in a concentration-
dependent manner (Fig.8A, C, D), reduced cervical cancer
cell colony numbers (Fig.8E, F), and decreased HeLa cell
invasion (Fig.8G, H). Furthermore, we investigated the
effect of lycorine on expression of oncogenic YAP and TAZ
in cervical cancer HeLa cells. The results showed that after
treatment of HeLa cells with lycorine for 72h, YAP levels
in the cells were significantly reduced by lycorine at concen-
trations of 20 and 40μM; meanwhile TAZ levels were also
significantly diminished by lycorine at the concentration of
40μM compared to the control without lycorine treatment
(Supplementary Fig.S5A-S5C). These data indicate that
lycorine reduces CT45A1-induced overexpression of onco-
genic YAP in cervical cancer cells, implying that lycorine is
a new inhibitor of the CT45A1-SRC-YAP signaling pathway.
In the xenograft mouse model, lycorine treatment of
tumor-bearing mice significantly reduced the tumor vol-
ume (Fig.8I) and tumor weight (Fig.8J) and decreased
the number of blood vessels 2.3-fold (Fig.8K and L) as
compared with the saline control. Strikingly, tube formation
assay showed that the tube forming ability of CT45A1-over-
expressing HeLa cells was completely inhibited by 5μM
lycorine (Fig.8M and N). There were no obvious complica-
tions in the mice treated with lycorine at the effective dosage
(Supplementary Fig.S5D and S5E). Together, these data
indicate that CT45A1-enhanced tumorigenesis, neovascu-
larization, cisplatin drug resistance, and apoptosis resistance
can be effectively reduced by lycorine, and lycorine is a new
CT45A1 expression suppressor and a novel cervical cancer
inhibitor. Conceptually, these findings indicate that inhibi-
tion of CT45A1 expression is a new strategy for cervical
cancer therapy.
3 Discussion
In addition to HPV, multiple oncogenes trigger the initia-
tion and progression of cervical cancer [8–11]. However, the
mechanisms underlying HPV-independent cervical cancer
initiation and progression are unclear. The effect of CT45A1
and CT45 family members on cervical cancer progression
has not yet been reported in the literature. We revealed that
CT45A1 was abnormally overexpressed in cervical cancer
and overexpression of CT45A1 was closely associated with
poor prognosis in the cancer patients. CT45A1 enhanced
tumor growth, neovascularization, metastasis, drug resist-
ance, and apoptosis resistance by up-regulation of various
oncogenic genes and activation of key tumorigenic signaling
pathways. CT45A1-mediated carcinogenesis was markedly
inhibited by the small molecule lycorine. These findings
provide new sight into the pathogenesis of cervical cancer
and offer a new biomarker for cervical cancer diagnosis and
prognostic prediction, and targeted therapy.
Overexpression and/or activation of tumorigenic signal-
ing proteins, including FN1 [32, 33], SRC [34–36], CREB
[37, 38], YAP [39–41], and TAZ [42–44], play a critical
role in the progression of cervical cancer. The mechanisms
underlying HPV-independent cervical cancer carcinogen-
esis and progression are unknown. In the current study,
novel tumorigenic signaling pathways in cervical cancer
were identified, including the CT45A1-FN1-SRC-CREB,
CT45A1-SRC-ERK, and CT45A1-SRC-YAP/TAZ signal-
ing pathways. We found that CT45A1 strongly up-regu-
lated expression of the oncogenic FN1 by enhancement
of FN1 gene promoter activity. CT45A1 interacted with
the key signaling protein kinase SRC and constitutively
activated the oncoprotein protein in the absence of HPV
and growth factors, resulting in activation of downstream
tumorigenic CREB and YAP/TAZ signaling proteins.
The transcription factor CREB and the transcription co-
activators YAP/TAZ promote overexpression of a large
numbers of oncogenic genes, consequently driving cervi-
cal cancer progression [40–45]. However, the mechanisms
Fig. 4 CT45A1 activates the oncogene SRC via interaction with
the protein in cervical cancer cells. Caski cells barely expresses
CT45A1. We first transfected Caski cells with CT45A1 cDNA-
vector and empty vector, respectively, to produce CT45A1 overex-
pressed Caski cells, then CT45A1-overexpressed Caski cells were
silenced by shRNA. Western blot indicated that the overexpression of
CT45A1 triggered SRC phosphorylation in cervical cancer cells (A,
B). Silencing of fibronectin 1 (FN1) notably reduced SRC phospho-
rylation (C, D). Co-immunoprecipitation showed that CT45A1 inter-
acted with SRC (E, F). Immunofluorescence staining revealed co-
localization of SRC with CT45A1 in the nucleus; red refers to SRC,
green refers to CT45A1, and light blue arrows refer to changes in the
SRC location (Gand H, 600 ×). Pull down of HeLa cell lysate with
CT45A1-specific antibody further confirmed the interaction between
CT45A1 and SRC (I). In vitro protein kinase assay showed that
CT45A1 induced SRC phosphorylation in an ATP-dependent manner
(J, K). Data are shown as the mean ± SE of three independent repli-
cates. *p < 0.05, **p < 0.01 in an unpairedt-test
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underlying activation of these transcription factor and co-
activators in cancer are enigmatic. In this study, we found
that CT45A1 acts as an activator of various tumorigenic
signaling pathways. These findings provide new sight into
the mechanisms underlying cervical cancer tumorigenesis,
neovascularization, metastasis, and progression.
Based on these findings, we think that CT45A1 is a
potential new target for cervical cancer therapy and
explored small molecules that inhibit CT45A1-mediated
carcinogenesis. In the current investigation, lycorine was
found to suppress CT45A1-mediated tumorigenesis by
inhibiting the expression of CT45A1 and suppressing the
oncogenic SRC, ERK, and YAP/TAZ signaling pathways.
Inhibition of CT45A1 expression by lycorine markedly
diminished cisplatin drug resistance and apoptosis resist-
ance in cervical cancer. Thus, inhibition of oncogenic
CT45A1 expression is a new strategy for tumor suppres-
sion. Our findings offer a new platform and a drug can-
didate for the development of novel therapeutics against
cervical cancer.
There are several limitations in this study. First, although
we found that CT45A1 activated oncogenic SRC in cervi-
cal cancer cells and invitro as well, our CT45A1 protein
functional domain analysis show that there is no protein
kinase functional domain in CT45A1, hence whether the
binding of CT45A1 to SRC changes the conformation of
SRC protein that results in SRC self-activation remains to
be further investigated. Second, our CT45A1 protein func-
tional domain analysis show that CT45A1 has two functional
domains, one is a nuclear localization signature (NLS) that
enables the protein to bind to DNA, the other is a D/HEAD
domain that interacts with RNA polymerase II. However,
whether CT45A1 functions as a gene transcription activator
needs to be deeply studied.
In conclusion, CT45A1 is aberrantly overexpressed in
cervical cancer patients and overexpression of CT45A1 is
closely associated with poor prognosis in these patients.
CT45A1 promotes tumor growth, neovascularization, and
metastasis by promoting the expression of many tumori-
genic genes and activating oncogenic signaling pathways.
The small molecule lycorine effectively inhibits CT45A1
expression and reduces cervical cancer cell tumorigenesis,
neovascularization, cisplatin drug resistance and apoptosis
resistance. Thus, CT45A1 is a new biomarker for the diag-
nosis, prognostic prediction, and targeted therapy of cervi-
cal cancer (Fig.9).
4 Methods
4.1 Cervical cancer patients andthecollection
ofcancer tissue samples
This study was approved by the Ethical Committee of Soo-
chow University prior to sample analysis. After written
informed consent was obtained, tissue samples were col-
lected from cervical cancer patients undergoing surgical
resection for cervical cancer and fibroids. Human cervical
cancer tissue arrays, including 119 primary tumor tissues
and 29 paired para-cancerous tissues, were obtained from
Shanghai Outdo Biotech (Shanghai, China) and Shanghai
Zhuoli Biotechnology Co., Ltd (Zhuoli Biotechnology Co,
Shanghai, China). In addition, 20 fibroid tissues were pro-
vided by Suzhou Ninth Hospital Affiliated with Soochow
University. The clinical pathological characteristics of the
patients are summarized in Supplementary TableS1.
4.2 Immunohistochemistry
Immunohistochemistry was carried out using the 2-STEP
protocol (Van Gieson, abs9349). After tissue sectioning,
dewaxing, hydration, and endogenous peroxidase blocking
were carried out (0.3% H2O2 for 10min). Antigen retrieval
was performed by steaming sections in EDTA buffer for
10min. The sections were incubated overnight at 4°C with
mouse anti-human CT45A1, while isotype antibody stain-
ing was used as the negative control. Then, the sections
were incubated with horseradish peroxidase-conjugated
anti-mouse and anti-rabbit secondary antibody, developed
with 3,3′-diaminobenzidine (DAB), and imaged using a
Leica microscope. To define the positive signal of CT45A1
in tumor tissues, we first determined CT45A1 expression
in the tissues by IHC, then used a traditional pathological
analysis by a score system which consists of IHC staining
intensity and positive cell percentage. In the current inves-
tigation, CT45A1 staining intensity was scored as follows:
Negative staining, 0; light yellow, 1; yellow, 2; brown, 3;
the positive cell percentage was counted and ranged from
0–100%. Overall score = (IHC staining intensity score) x
(positive staining cell percentage), which is in the range of
0–300%. The CT45A1 signal was detected and evaluated in
cervical cancers without bias. Histoscores were computed
based on the intensity and tissue area of positive staining.
Fig. 5 The CT45A1-SRC-ERK-CREB axis controls the migration of
cervical cancer cells. The SRC inhibitor PP2 (5μM) and ERK inhibi-
tor SCH772984 (5μM) reduced the phosphorylation of SRC, ERK,
and CREB (A-D). CT45A1-induced cervical cancer cell migration
was significantly inhibited by the SRC inhibitor PP2 (E, F). Schemes
of the mechanism underlying the regulation of the migration by
the newly identified CT45A1-SRC-ERK-CREB axis (G). Data are
shown as the mean ± SE of three independent replicates. *p < 0.05,
**p < 0.01 in an unpairedt-test
◂
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4.3 Cell lines andcell culture
The human cervical cancer cell lines Caski, Siha, and
C33A were obtained from the Cell Bank of the Shang-
hai Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences (Shanghai, China). HeLa and other
cells were from ATCC. The cells were mycoplasma-free
and were cultured in RPMI-1640 (Gibco BRL, San Fran-
cisco, CA, USA) or Dulbecco’s Modified Eagle Medium
(DMEM) (high glucose) supplemented with 10% heat-inac-
tivated fetal bovine serum (FBS) (HyClone, Logan, Utah,
USA), 100 U/mL penicillin G, and 100μg/mL streptomy-
cin (complete medium) under a humidified atmosphere of
5% CO2 at 37°C, as previously described [26, 46, 47].
4.4 RNA extraction andqRT‑PCR
Total RNA was extracted from the cultured cells and fresh
frozen cervical tissues using Trizol reagent (Vazyme Bio-
tech, Nanjing, China), according to the manufacturer’s
instructions. Reverse transcriptase reactions were per-
formed according to the manufacturer’s protocol using
HiScript II Q Select RT SuperMix as the qPCR reverse
transcriptase reagent (Vazyme Biotech, Nan Jing, China).
Gene expression levels were normalized to the house-keep-
ing gene β-actin. Reactions were performed in triplicate
with ABI QuantStudio6 Q6 (Applied Biosystems, USA).
Primer sequences are listed in Supplementary TableS5.
4.5 DNA microarray andgene expression profile
analysis
The effect of CT45A1 on the gene expression profile in cer-
vical cancer Caski cells was analyzed by DNA microarray as
we previously reported [26]. The top 30 signaling pathways
regulated by CT45A1 were assessed by KEGG pathway
enrichment analysis.
4.6 Western blotting
Proteins were extracted using protein extraction lysis
buffer (Merck, 20-188, USA). Protein samples were
treated with RPMI Buffer (Merck) containing reducing
agent at 95°C for 10min, resolved on 10% Tris–HCl
polyacrylamide gels, and transferred to a nitrocellulose
blotting membrane (GE, Germany). Overnight incubation
(4°C) with the primary antibody was followed by incuba-
tion with HRP-conjugated antibody and Chemilumines-
cent HRP Substrate (JacksonImmuno Research, USA),
as previously described [26, 46, 47]. Detailed antibody
information is provided in Supplementary TableS6.
4.7 RNA interference
The expression of CT45A1 and FN1 was silenced by the
shRNA method, as previously described [26, 47]. shRNAs
designed specifically against CT45A1 and FN1 were pur-
chased from Genechem (Shanghai, China). For transfec-
tion, 4μg of shRNA was dissolved in 250μl of Optimem
medium (Life Technology). In another tube, the transfec-
tion medium Lipofectamine 2000 reagent (Life Technol-
ogy) was dissolved in 250μl of Optimem medium. These
two solutions were then mixed and incubated for 20min
at room temperature. The mixture was added to 5 × 105
cells in 2ml of serum-free media, and the cells were incu-
bated for 5h at 37°C. After transfection, the medium
was replaced with normal growth medium containing 10%
FBS. The cells were selected with puromycin for 7days
(10μg/ml) to obtain stable cell lines. The gene expression
levels were examined by western blotting.
4.8 Plasmid constructs andlentivirus infection
The CT45A1 cDNA and CT45A1 promoter DNA
(− 1354 ~ + 247) were synthesized by Synbio Technolo-
gies, Suzhou and cloned into Venus-GFP and pGL4
vectors, respectively. The CT45A1-shRNA-1, CT45A1-
shRNA-2, CT45A1-shRNA-3, FN1-shRNA-1, FN1-
shRNA-2, FN1-shRNA-3, and U6-MCS-Ubiquitin-
Cherry-IRES-puromycin-plasmids and control plasmids
were purchased from Genechem (Genechem, Shanghai,
China). These constructs were used to transfect the pack-
age cell line 293T using Lipofectamine 2000 reagent, as
previously described [26, 46]. Virus-containing superna-
tants from 293T cells were collected and then filtered
using 0.45μm filters. The filtered infectious virus was
added to 70% confluent cervical cancer cells. After 48h,
stably transfected cells were selected by puromycin.
Fig. 6 CT45A1 activates the oncogenic proteins YAP and TAZ in
cervical cancer cells. Western blot revealed that CT45A1 overexpres-
sion increased the levels of the oncogenic YAP and Tafazzin (TAZ)
proteins in Siha cells (A-C). By contrast, silencing of CT45A1 in
HeLa cells diminished the expression of YAP and TAZ (D-F). Addi-
tionally, the SRC inhibitor PP2 significantly suppressed CT45A1-
induced expression of YAP (G, H). Co-IP (I, J) and pull-down assays
(K) revealed that CT45A1 interacted with YAP and TAZ. Immuno-
fluorescence staining and confocal microscopy techniques confirmed
that YAP and CT45A1 were co-localized in the nucleus; red refers to
YAP and green refers to CT45A1 (L, 1200 ×). Data are shown as the
mean ± SE of three independent replicates. *p < 0.05, **p < 0.01 in an
unpairedt-test
◂
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Cancer/testis‑45A1 promotes cervical cancer cell tumorigenesis anddrug resistance by…
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4.9 Cell migration andinvasion assays
Tumor cell migration assays were performed as previ-
ously reported [26, 47]. Briefly, Caski and Siha cells were
seeded in six-well plates for 24h to reach confluence and
were then wounded using a plastic tip. The wounded mon-
olayer was then incubated in RPMI-1640 or DMEM sup-
plemented with 2% FBS for either 0 or 24h. The migrated
tumor cells were stained with Wright–Giemsa solution,
imaged under a microscope using five randomly chosen
fields for each well line, and statistically analyzed. For
HeLa cell migration assays, 4 × 104 cells were placed in
the top chamber of each insert (BD, Durham, NC, USA).
For the cell invasion assays, 4 × 104 cells were plated in
a 24-well culture plate and placed in a Transwell chamber
coated on the inside with 1:4 diluted Matrigel (BD Bio-
sciences, Bedford, Massachusetts, USA). Medium contain-
ing 10% FBS was added to the lower chamber as a chem-
oattractant. After incubation in a CO2 incubator for 24h,
the cells inside the chamber were gently removed with a
cotton swab. Migrated cells located on the lower side of
the chamber were stained with crystal violet, air-dried,
and photographed. Three independent experiments were
performed, and the data are presented as the mean ± SE.
4.10 Chromatin immunoprecipitation
Cells were grown to 90% confluence and then treated with
10% formaldehyde to cross-link the proteins to DNA. The
crosslinking, immunoprecipitation, washing, elution,
reverse crosslinking, and proteinases K treatment were
performed according to the Simple ChIP Enzymatic Chro-
matin IP Kit 102,026 (Active Motif, US) manufacturer’s
instructions. Antibody information is listed in Supplemen-
tary TableS6. Purified immunoprecipitated DNA was used
for RT qPCR. The primers for ChIP PCR are shown in
Supplementary TableS5.
4.11 Luciferase assay
5 × 104 cells per plate were seeded in 12-well plates in trip-
licate and incubated for 24h. Caski cells with CT45A1
expression were transfected with pGL4.17-basic, pGL4.17-
ctrl, or pGL4.17-CT45A1 promoter DNA fragments using
Lipofectamine 2000 reagent, and the positive control was
pTK-Renilla. Luciferase and Renilla signals were measured
48h after transfection using a Dual-Luciferase Reporter
Assay Kit (Promega Corporation), as previously described
[47].
4.12 Tumor xenograft mice
Tumor xenograft mice were treated in accordance with the
protocols approved by the Institutional Animal Care and Use
Committee (IACUC) of Soochow University, as previously
reported [47]. In brief, BALB/c nude mice were randomly
divided into four groups (n = 6/group) and injected with
5 × 106 cells (Caski-vector/ Caski-CT45A1) into the sub-
cutaneous abdomen. The tumor volume was measured and
calculated according to the following formula: tumor vol-
ume = 0.5 × length × width2. After 45days, the tumor tissues
were isolated, imaged, and paraffin-embedded for further
routine histology examination by H&E staining.
4.13 Co‑immunoprecipitation (Co‑IP)
Co-IP was carried out as previously described [26, 47].
400μl of cervical cancer cell lysate was incubated with pri-
mary monoclonal antibody (1:500) or normal IgG as a con-
trol at 4°C for 4h. Then, further incubation was performed
with 20μl of prewashed magneticA/G beads (MedChem
Express, China) at 4°C overnight with rotation. The immune
complexes were released from the beads in SDS loading
buffer. The proteins were detected by western blotting as
mentioned above.
4.14 Nuclear/cytosol fractionation andprotein
assay
Nuclear and cytosolic fractions were extracted using a
Nuclear/Cytosol Fractionation Kit, according to the manu-
facturer’s instructions (Nuclear/Cytosol Fractionation Kit,
Beyotime Biotechnology, Shanghai, China.). 3 × 106 HeLa
cells were harvested. One-tenth of these cells were lyzed
by SDS lysis buffer as the input for the protein expression
western blotting analyses, and nine-tenths of the cells were
extracted using the Nuclear/Cytosol Fractionation Kit. One-
fifth of both the nuclear and cytoplasmic fractions were used
for the detection of CT45A1 and other proteins by western
blotting, as mentioned above.
Fig. 7 CT45A1 enhances cisplatin drug resistance and apoptosis
resistance in cervical cancer cells. CT45A1-expressing Siha cells
were treated with 0–20μM of the anti-cancer drug cisplatin for 48h
and cell proliferation was assessed with Alarm blue assays (A). The
levels of cleaved PARP, γH2AX, and caspase 3 (cas3) in CT45A1-
expressing Siha cells were compared to the control Siha cells with-
out expression of CT45A1 (C-E). The apoptotic assay indicated that
apoptosis in CT45A1-expressing Siha cells was significantly reduced
when the cells were treated with cisplatin for 48h (F, G). Immuno-
fluorescence staining and confocal microscopy techniques confirmed
that the expression of γH2AX was diminished in CT45A1-expressing
cells as compared to control cells without expression of CT45A1;
red refers to γH2AX and blue refers to DAPI (H, 2500 ×). Data are
shown as the mean ± SE of three independent replicates. *p < 0.05,
**p < 0.01, and ***p < 0.001 in an unpairedt-test
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4.15 Tube formation assay
The Matrigel gel solution was first spread in a 48-well
plate and placed in a 37℃ cell culture incubator for
30min, 4 × 104 cervical cancer cells were added to the sur-
face of Matrigel gel and incubated for 12h, the tube-like
structures in the randomized fields were imaged, counted,
and statistically analyzed.
4.16 Statistical analysis
All results are presented as the mean ± SE. Differ-
ences between the groups were assessed by one-way
ANOVA using GraphPad Prism 8. Statistical compari-
sons were performed using the Unpaired Student’s t-test.
Fig. 8 Reduction of CT45A1 expression by lycorine suppresses
tumor growth of cervical cancer cells. CT45A1-overexpressing
HeLa cells were treated with the small molecule lycorine (LH) at
concentrations of 0–40μM for 72h. The protein levels of CT45A1,
phosphorylated-SRC (p-SRC), and phosphorylated-ERK (p-ERK)
were measured by western blot (A) and were statistically analyzed
(B-D). HeLa cell-forming colonies were counted (E, F). Lycorine
significantly inhibited HeLa cell invasion (G, H) and tumor growth
in tumor-bearing nude mice (I, J, n = 6 in each group). H&E stain-
ing showed that the number of tumor blood vessels was significantly
reduced (K); the red arrows refer to the blood vessels, and the data
represent the tumor vascular diameter mean (± SE) of six mice (L).
Additionally, CT45A1-overexpressing HeLa cells were first treated
with lycorine at the concentration of 5μM for 72h, the living cells
were counted and added to the 48-well plates coated with Matrigel.
The tube-like structures in the randomized fields were imaged
(M), counted and statistically analyzed (N). Data are shown as the
mean ± SE of three independent replicates. *p < 0.05, **p < 0.01 in an
unpairedt-test
◂
Fig. 9 CT45A1 induces tumorigenesis and is a new biomarker for the
diagnosis, prognostic prediction, and targeted therapy of cervical can-
cer. CT45A1 was abnormally overexpressed in cervical cancer with
high specificity and sensitivity, and the overexpression of CT45A1
was closely associated with poor prognosis in these cancer patients.
CT45A1 enhanced cervical cancer cell tumorigenesis, migration,
invasion, metastasis, drug resistance, and apoptosis resistance by
promoting the expression of many oncogenic genes and activating
multiple tumorigenic signaling pathways. CT45A1-mediated cervical
cancer tumorigenesis and progression were effectively inhibited by
the small molecule lycorine. Collectively, these findings indicate that
CT45A1 is a new biomarker for cervical cancer screening, diagnosis,
prognostic prediction, and therapy
M.Meng et al.
1 3
The significance of differences is indicated as follows:
*p < 0.05, **p < 0.01, ***p < 0.001.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s13402- 023- 00891-w .
Author contributions M.M. and Y.G.: data curation, formal analysis,
funding acquisition, investigation, methodology, writing-original draft.
B.Z., X. Li., Y.C., Z. X., J.L., Z.Z., T.Z., B.S., Y. Q., Y. L.: data cura-
tion, investigation, methodology. Q.Z.: Conceptualization, funding
acquisition, writing and editing of the manuscript.
Funding This study was supported by grants from the National Natu-
ral Science Foundation of China (Grants No.81902647, No.82073225,
No.81772535); National Clinical Research Center for Hematologic
Diseases (Grant No. 2020ZKMB04); A project funded by the Priority
Academic Program Development of Jiangsu Higher Education Institu-
tions (PAPD).
Data availability All other relevant data are already available as Sup-
plementary material.
Declarations There are no statements and declarations.
Ethics statement All experimental protocols were approved by the
Suchow University Animal Care Committee and were carried out fol-
lowing the National Institutes of Health Guide for the Care and Use
of Laboratory Animals (NIH Publications No. 8023, revised 2011).
Competing interests The authors declare no conflict of interest.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, 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 changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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1 3
Authors and Aliations
MeiMeng1,2,3,4,5· YanGuo6 · YuChen1· XuLi1· BinZhang7· ZhijiaXie8· JuntaoLiu1· ZheZhao9· YuxiLiu1·
TongZhang1· YingnanQiao1· BingxueShang10,11 · QuanshengZhou1,2,3,4,5
1 Cyrus Tang Hematology Center, Jiangsu Institute
ofHematology, Soochow University, 199 Ren Ai Road,
Suzhou Industrial Park, Suzhou, Jiangsu215123,
People’sRepublicofChina
2 State Key Laboratory ofRadiation Medicine andProtection,
School ofRadiation Medicine andProtection,
Soochow University, Suzhou, Jiangsu215123,
People’sRepublicofChina
3 National Clinical Research Center forHematologic Diseases,
The Affiliated Hospital ofSoochow University, Suzhou,
Jiangsu215123, People’sRepublicofChina
4 2011 Collaborative Innovation Center ofHematology,
Soochow University, Suzhou, Jiangsu215123,
People’sRepublicofChina
5 The Ninth Affiliated Hospital, Soochow University, Suzhou,
Jiangsu215123, People’sRepublicofChina
6 Department ofGynecology andObstetrics, The First
Affiliated Hospital ofSoochow University, Suzhou,
Jiangsu215006, People’sRepublicofChina
7 National Key Laboratory ofImmunity andInflammation,
Suzhou Institute ofSystems Medicine, Chinese Academy
ofMedical Sciences & Peking Union Medical College,
Suzhou215123, Jiangsu, People’sRepublicofChina
8 Department ofObstetrics andGynecology, The Ninth
Affiliated Hospital ofSoochow University, Suzhou,
Jiangsu215123, People’sRepublicofChina
9 CAS Key Laboratory ofNano-Bio Interface, Suzhou
Institute ofNano-Tech andNano-Bionics, Chinese Academy
ofSciences, Suzhou215123, Jiangsu, China
10 Institute ofSystems Medicine, Chinese Academy ofMedical
Sciences andPeking Union Medical College, Beijing, China
11 Suzhou Institute ofSystems Medicine, Suzhou, China