(2023) 18:183
Zhou et al. Radiation Oncology
https://doi.org/10.1186/s13014-023-02369-3

Radiation Oncology

Open Access

RESEARCH

Hypoxic tumor cell‑derived small
extracellular vesicle miR‑152‑3p promotes
cervical cancer radioresistance through KLF15
protein
Junying Zhou1†, Ningjing Lei2†, Wanjia Tian1, Ruixia Guo1, Feng Gao3,4, Hanlin Fu1, Jing Zhang3, Shiliang Dong5,
Mengyu Chen1, Qian Ma1, Yong Li6,7* and Lei Chang1*

Abstract
Background Radiotherapy is widely used in treating cervical cancer patients, however, radioresistance unavoidably
occurs and seriously affects the treatment effect. It is well known that hypoxia plays an important role in promoting
radioresistance in tumor microenvironment, yet our understanding of the effect of small extracellular vesicles miRNAon cervical cancer radiosensitivity in hypoxic environment is still limited.
Methods Small extracellular vesicles extracted from hypoxic and normoxic cultured cervical cancer cells were evaluated for their effects on radioresistance. miR-152-3p was found to be a potential effector in hypoxia-derived extracellular vesicles by searching the GEO database. Its downstream substrate was confirmed by double luciferase report,
which was KLF15. The role of miR-152-3p and KLF15 in regulating cervical cancer radioresistance was detected by cell
activity assays. The findings were confirmed in vivo by animal models. The expression of miR-152-3p was quantified
by qRT-PCR and its prognostic significance was evaluated.
Results Hypoxic environment promoted the secretion of small extracellular vesicles, and reduced the apoptosis
and DNA damage caused by radiation, accompanied by increased expression of small extracellular vesicles miR152-3p from hypoxic cervical cancer cells. Furthermore, small extracellular vesicles miR-152-3p promoted Hela xenograft growth and reduced the radiosensitivity vivo. Mechanism studies revealed that KLF15 protein was the downstream target of miR-152-3p in regulating radioresistance.
Conclusion Our findings suggest that small extracellular vesicles miR-152-3p affects the therapeutic effect of radiotherapy and holds potential as a biomarker or therapeutic target for cervical cancer prognosis and improving
radiotherapy.
Keywords Cervical cancer, Radioresistance, Hypoxia, Extracellular vesicle, miR-152-3p†

Junying Zhou and Ningjing Lei contributed equally to this work.

*Correspondence:
Yong Liy.li@unsw.edu.au
Lei Chang
fccchangl@zzu.edu.cn
Full list of author information is available at the end of the article© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother 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 thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Zhou et al. Radiation Oncology(2023) 18:183

Introduction
Cervical cancer (CC) brings a huge disease burden on
women due to its high morbidity and mortality all over
the world [1]. Currently, various therapeutic strategies
including surgery, radiotherapy (RT), chemotherapy, andimmunotherapy are applied in treating CC. For locallyadvanced CC, simultaneous RT and chemotherapy haveshown promising effects [2, 3]. However, radioresistancehas become one of the main factors limiting the efficacy
of RT [4]. Therefore, enhancing the effectiveness of RT
for the treatment of CC patients requires a deeper knowledge of the mechanisms underlying radioresistance.
It is well known that hypoxia is associated with radioresistance since free radicals come from oxygen and arenecessary to kill tumor cells for ionizing radiation (IR)[5, 6]. In addition, the hypoxic environment also produces other biological changes in tumors. For instance,
decreased cell oxygen content in the tumor microenvironment (TME) interferes with radiation-induced cell
apoptosis, DNA damage and other biological processes
that are beneficial to curative effects [7]. Therefore,
whether and how the hypoxia environment affects theefficacy of RT requires in-depth research.
Most of the research on the interaction between
hypoxia and radioresistance focuses on hypoxic tumor
cells [8, 9], but the communication between cells in
the TME is not well investigated. Areas of hypoxia areusually distributed throughout the surviving tumor mass.
Extracellular vesicles (EVs) are a general term for a variety of membrane-enclosed nanosized vesicles carrying
cell-derived cargo. These vesicles range in diameter from50 to 1000 nm and are released from all types of cells
under normal physiological conditions and pathologicalprocesses [10, 11]. Two prominent kinds of EVs that havebeen long recognized are small EVs (sEV, i.e. exosomes,
40–150 nm in diameter) and large EVs (lEV, i.e. microvesicles, 150–1000 nm in diameter) [12–14]. In this study, allexosomes quoted from references are referred to as EVs
because EVs constitute a diverse population of vesicles,
according to the standards of the International Society
for Extracellular Vesicles (ISEV) [15]. EVs released byhypoxic tumor cells promote intercellular communication and thus play various roles in different biologicalprocesses [16]. EVs usually play their biological roles bywrapping microRNA, mRNA or protein in a cargo [17,18]. The stable miRNA encapsulated by EVs was reported
to be used as an effector molecule, conferring biologicalchanges induced by hypoxia, such as radioresistance [19].
From the perspective of the hypoxic environment,
EVs in the TME may provide a novel outlook for the
research of CC radioresistance. In the study, we foundthat EVs induced by hypoxia enhanced CC radioresistance by reducing apoptosis and DNA damage caused byPage 2 of 16

radiation. In addition, we also for the first time demonstrated sEV miR-152-3p was highly expressed in hypoxic
CC cells, which could be transferred to normoxic cells
and affect radioresistance by targeting its downstream
substrate Kruppel-like factor 15 (KLF15) in vitro. The
regulatory effect of miR-152-3p on the progression of
prostate cancer and liver cancer was studied previously. It
was reported that miR-152-3p inhibited the proliferationof prostate cancer cells by targeting KLF4 [20]. Anotherstudy showed that miR-152-3p inhibited the proliferation and promoted the apoptosis of hepatocellular carcinoma cells by repressing roundabout guidance receptor
1 [21]. However, miR-152-3p has not been reported in
the regulatory mechanism of cervical cancer progression.
So we detected that increased expression of sEV miR152-3p reduced the killing effect of irradiation on tumor
in a xenograft tumor experiment in vivo, associated with
inhibiting cell apoptosisin cervical cancer cell lines SiHa
and Hela. Furthermore, we showed that the high expression of plasma sEV miR-152-3p was associated with therisk of recurrence in clinical CC patients. Our findings
suggest that sEV miR-152-3p is a useful biomarker forpredicting CC radioresistance as well as a potential therapeutic target for CC treatment.

Methods
Patients and samples

Our study recruited 40 patients with CC who underwent
surgery from 2017 to 2019. Paraffin-embedded tumor
tissues and adjacent normal tissues (> 5 cm from tumor
tissue) were collected during the surgery for detecting
the level of miR-152-3p. In addition, blood (10 mL/tube)
of 20 CC patients who did not relapse after RT and 20
CC patients who relapsed after RT were collected forprognostic analysis. Patients included in this study were
followed up every 3 months until death or April 2021.
All cases were confirmed by experienced pathologists,
and none of them had received chemotherapy or RT
before surgery. Prior to the study, the written informed
consent of all patients was obtained. The study protocol was approved by the Ethics Committee (No.2022ky-1371-002) and was based on the ethical principles of
the Declaration of Helsinki for medical research involving human beings.
Cell lines and cell culture

Normal cervical epithelial cell line (Ect1/E6E7) was purchased from BeiNa biological company (China) and CC
cell lines (SiHa, C33A and Hela) were purchased from
Procell biological company (China). Normal cervicalepithelial cell line was cultured with DMEM medium(Gibco, MA, USA) and CC cell lines (SiHa, C33A andHela) were cultured with MEM medium (Gibco, MA,

Zhou et al. Radiation Oncology(2023) 18:183

USA). All media contain 10% fetal bovine serum (FBS)(PAA, Bentley, Australia) and 1% penicillin–streptomycin (Procell, Wuhan, China). All cell lines were kept at
37 °C in a humidified environment with indicated C­ O2
concentrations.
Hypoxia treatment

In this study, the normal oxygen cells were cultured in an
incubator at 37 °C with 5% C
­ O2, while the hypoxic cells
were cultured in another incubator at 37 °C with 1% ­O2,
5% ­CO2 and 94% ­N2. Hypoxic cells were confirmed bywestern blot analysis to detect levels of hypoxia-inducible
factor 1α (HIF-1α) in cultured cells [22, 23].
Cell transfection

KLF15 overexpression plasmid and its negative control, miR-152-3p mimic or inhibitor and their negative
controls were purchased from GenePharma (Shanghai,
China). The stable knockdown of KLF15 was performed
with lentiviral short hairpin RNA targeting KLF15 (shKLF15), and its negative control were produced by
Genechem (Shanghai, China). The transfection was constructed with Lipofectamine 2000 (Invitrogen, Ma, USA)according to the manufacturer’s protocols. The information about plasmids is listed in Additional file 1: Table S1.
Quantitative real‑time PCR (qRT‑PCR)

Total RNA was extracted using the High Pure RNA Isolation Kit (Ambion, USA) according to the manufacturer’s instruction. The qRT-PCR was performed using
our previously established method [24]. GAPDH and U6
were used as internal references, and the relative gene
expression was calculated using the 2­ −ΔΔCt method. The
information about plasmids is listed in Additional file 1:
Table S2.
sEV isolation from cell lines

After the cells (SiHa and Hela) were incubated in 20 mL
fresh sEV-depleted medium for 48 h, the supernatantfrom each cell line was collected. The supernatant wascentrifuged at 2000 g for 20 min at 4 °C to remove deadcells and followed by 10,000 g for 45 min. The samples
were then filtered using a sterile filter (0.22 μm, Millipore, USA). Centrifugation was conducted at 100,000 g
for 80 min, and the supernatant was discarded, and thenresuspended with PBS. After the last centrifugation(100,000 g, 80 min), the sample was dissolved with PBS
and added with RNase I. Purified EVs were collected andstored at − 80 °C.
sEV isolation from blood sample

Whole blood (10 mL/tube) from patients was collected
in tubes containing EDTA, and the plasma was separated

Page 3 of 16

by centrifuging the tubes at 3000 g for 15 min at 4 °C.
Plasma samples were mixed with ExoQuick ULTRA kit(System Biosciences, Palo Alto, CA, USA) according to
the manufacturer’s protocol, co-incubated at 4 °C for
30 min and then centrifuged at 3000 g for 10 min. Afterdiscarding the supernatant, 200 μL of ExoQuick ULTRA
buffer was added for resuspension and a further 200 μL
of buffer A was added for resuspension. The suspension
was added to the resin column and purified by centrifugation at 1000 g for 2 min. Purified EVs were collected
and stored at − 80 °C.
sEV identification

TEM was used to determine the morphology of sEVsamples. The NanoSight NS300 system (Malvern Instruments, Malvern, UK) was used to find the size distribution and concentrations of sEVs. A detailed description of
the method is included in Additional file 2.
Western blot (WB) analysis

Total protein from sEVs or cell lines was obtained using
the RIPA lysis buffer containing 1 × Halt™ protease andphosphatase inhibitor cocktail (Beyotime, China) andtheir concentrations were then assessed using a BCA
assay (Thermo Fisher Scientific, USA). The western blotting analysis was carried out as previously described [24].
Antibodies used are listed in Additional file 1: Table S3.
CD63, CD81, Alix and Calnexin were used to identify
sEV characterization.
External miRNAs inhibition of EVs

To eliminate the interference of the external EV miRNAsof EVs, RNase R (5 µg/mL) was added to the EVs suspension from cell suspension, incubated for 15 min, and thelevel of EV miR-152-3p was detected by qRT-PCR. 0.1%
Triton X-100 and RNase R were added to the EV suspension for 30 min, and the level of miR-152-3p was detected
by qRT-PCR to eliminate interference from nonspecificprecipitates.
Cell proliferation

Proliferating capacity of transfected CC cell lines
SiHa and Hela, and the control cell line Ect1/E6E7 wasdetected by the Cell Counting Kit-8 (DOJINDO, Japan)
according to the instruction. A detailed description of themethod is included in Additional file 2.
Immunofluorometric assay

5 × ­104 cells were inoculated into 24-well plates with glass
cover-slips and irradiated after 24 h. After 4 h, the cells
were fixed with 4% paraformaldehyde for 30 min, andinfiltrated with 0.1% Triton X-100 for 2 h, respectively.
After sealing with 5% BSA for 90 min, washing with PBS

Zhou et al. Radiation Oncology(2023) 18:183

and incubation overnight (o/n) with anti-γ-H2AX primary antibody (1:400; Abcam, USA). The primary antibody was removed with PBS, followed by incubation with
Alexa fluor 555 conjugated secondary antibody (Beyotime, China) for 90 min at room temperature. DAPI wasused to stain the nuclei of cells and then visualized under
the confocal microscope (Leica, Germany).
Flow cytometry for cell apoptosis detection

SiHa and Hela cells were cultured in 6-well plates for 24 hand then irradiated at a dose of 4 Gy [25]. For apoptosis assays, cells were collected after centrifugation andresuspended in the binding buffer, then stained using
Annexin V and propidium iodide (PI) (BestBio, China).
At last, measurements were made using flow cytometry(Beckman Coulter, USA). The results were subsequently
calculated with FlowJo software (Tree Star Inc., Ashland,
OR, USA).
Invasion assay

Commercial Matrigel and transwell chambers were used
to measure the incasiveness of the transfected CC cells(BD Bioscience, CA, US). A detailed description of themethod is included in Additional file 2.
Mouse xenograft experiment

Seventy female BALB/c nude mice (5 weeks old) wereobtained from Charles River Laboratories (China).
Briefly, Hela cells (2 × ­106) were collected and subcutaneously injected into the right lower abdomen of
the mice. The tumor size was measured every 5 days
after injection. Tumor volume was calculated: volume = ­(width2 × length)/2. When the tumor volume
reached 50 ­mm3, 10 μg/50 μL EVs or 50 μL PBS was
injected into the tumor tissue once a day for 5 days. In
addition, the tumor was irradiated locally at a dose of
4 Gy per day for 4 days after the second day of EVs injection and other part of animal was shielded with lead during irradiation. In addition, in the same xenograft tumor
model, PBS or miR-152-3p mimics was injected into
nude mice, respectively. Two weeks after irradiation,
the mice were euthanized and tumor xenograft tissues
were collected for further analysis. The animal treatment was grouped as follows: PBS + IR (n = 6), N-EV + IR
(n = 6) and H-EV + IR (n = 6); NC + IR (n = 6) and miR152-3p + IR (n = 6).
Immunohistochemistry (IHC)

At the end of the experiment, the fresh tumor xenograft
tissue was quickly frozen or fixed by 4% formalin, thenembedded in paraffin and sliced for IHC and TUNEL staining, respectively. Hematoxylin and eosin (H&E)-stained
sections was used to determine the tumor structure. A

Page 4 of 16

detailed description of the method and assessment of
immunostaining was included in Additional file 2. Antibodies used were listed in Additional file 1: Table S3. The
assessment criteria were as previously reported [26].
TUNEL assay for apoptotic cells in vivo

To evaluate apoptosis in animal xenograft tumors with different treatments, TUNEL assay was performed in paraffin-embedded sections using a Tunel analysis kit (Roche,
Switzerland) according to the manufacturer’s protocol.
Images were recorded by a light microscope (Nexcope
NE900, China) (Magnification, × 200). The staining results
were assessed using above-mentioned method.
Dual luciferase reporter assay

The miR-152-3p target binding site on KLF15 was determined by Starbase online software (ENCORI). The predicted miR-152-3p binding region was amplified by PCR
and cloned into the pMIR reporter plasmid in either thewild-type or mutant KLF15 3’UTR. After inoculation of
SiHa and Hela cells into 24-well plates and incubation for
24 h, two luciferase reporter plasmids containing KLF15
and miR-152-3p simulant were co-transfected into cells.
After 48 h, the Dual Luciferase Assay Kit (Promega, USA)was used to detect luciferase activity. Three independent
experiments were performed.
Ribonucleoprotein immunoprecipitation (RIP) assay

To find out the downstream target protein of miR-152-3p,
5 × ­107 cultured cells treated with miR-152-3p mimics andcontrol plasmids were collected and lysed, and the lysates
were incubated with anti-AGO G magnetic beads (Thermo
Scientific, Massachusetts, USA) o/n. The magnetic beads
were collected, washed with RIPA buffer and resuspended
in 50 mmol/L Tris HCl at pH 7.0. Finally, RNA was isolated
and enriched from magnetic beads and analyzed quantitatively by qRT-PCR.
Statistical analysis

Date were analyzed using SPSS 22.0 and GraphPad prism
9 software and shown as the mean ± standard deviation.
Unpaired student’s t test was used to analyze the differences between the two groups of data, and one-way
analysis of variance (ANOVA) was used to analyze thedifferences of data in three or more groups. *P < 0.05,**P < 0.01, ***P < 0.001.

Results
Hypoxic CC cell‑derived sEVs reduce cell damage inducedby IR and enhance radioresistance

To study the effect of sEVs derived from the hypoxia
environment on CC radiation response, we first isolatedsEVs from supernatants secreted by CC cells treatedZhou et al. Radiation Oncology(2023) 18:183

in a hypoxia environment (HE) or a normoxia environment (NE). The isolated sEVs were detected by transmission electron microscopy (TEM), nanoparticle tracking
analysis (NTA) and western blot analysis. We discoveredthat 150 nm was the particle size that was most widely
disseminated from SiHa and Hela cell lines (Additional
file 1: Fig. S1A). In addition, NTA also demonstrated thathypoxia increased the number of sEVs secreted by both
Hela and SiHa cells in HE compared to cells treated in NE(Additional file 1: Fig. S1B). TEM results showed different
oxygen concentrations did not change the morphology ofsEVs from HE and NE conditions (Additional file 1: Fig.
S1C). The expression of sEV markers CD63, CD81 andAlix were considerably enhanced in HE-treated SiHa andHela cells, and the negative control marker, Calnexin,
showed deletion of expression, which verified the identity of sEVs (Additional file 1: Fig. S1D, E). Furthermore,
we showed total sEV proteins and RNAs were increased
in HE compared to those observed in NE (Fig. 1A, B),
indicating that HE led to an increased sEV secretion. To
confirm the accurate construction of the hypoxia model,
we tested HIF-1α by western blot analysis in our modeland demonstrated increased expression of HIF-1α in HE
model compared to NE one (Additional file 1: Fig. S1F).
Finally, we used PKH67 to label and detect sEVs andshowed that the labelled sEVs could successfully enter
CC cells (Additional file 1: Fig. S1G).
We then co-cultured SiHa and Hela CC cells with HEsEVs and NE-sEVs to find the difference of effects of sEVs
from two models on CC radiation response. Our results
indicated that HE-sEVs produced an anti-radiation effectand significantly reduced cell apoptosis after IR (Fig. 1C),
while the expression of DNA damage protein γ-H2AX
in two CC cell lines after HE-sEV treatment was significantly lower than that in untreated group or NE-sEV
treatment group. And this protective effect was more
obvious with the increase of sEV concentration (Fig. 1D,
E). Cell apoptosis assay further confirmed that HE-sEV

Page 5 of 16

treatment significantly reduced SiHa and Hela cell death
compared with that from NE-sEV treatment (Fig. 1F).
To further investigate the radioresistant effects of HEsEVs in vivo, we established tumor xenograft mousemodels and injected 10 μg/50 μL HE-sEVs or NE-sEVs
or 50 μL PBS as negative control into the tumor sites
when tumor volume reached about 50 ­mm3 (Fig. 1G).
After injection, we found that the growth of tumor volume and weight in HE-sEVs injection group was significantly increased compared with NE-sEVs injection group
and PBS control group after IR, respectively (Fig. 1H).
In tumor xenograft examination after RT, we foundincreased expression of Ki67 (1.58 ± 0.14) and reduced
TUNEL-positive cells (0.23 ± 0.13) in HE-sEVs + IR treatment group in comparison with that in NE-sEVs + IR
group or PBS + IR group with Ki67 (0.98 ± 0.12),(0.99 ± 0.21) and TUNEL-positive cells (1.06 ± 0.14) and(1.12 ± 0.10), respectively, indicating that HE-sEVs + IR
treatment significantly reduced radiation-induced damage by increasing the cell proliferation and inhibiting
apoptosis of the two CC cells ( Fig. 1I, J). These results
demonstrate that sEVs derived from hypoxic tumor cells
reduce IR-induced cell damage and promote CC radioresistance in vitro and in vivo.
MiR‑152‑3p affects the radiosensitivity of CC cells

To find out if the expression level of miRNAs is associated with CC RT, we first analyzed the NCBI database(GSE81137) and found that miR-152-3p was significantly
upregulated in CC tissues (Fig. 2A). We then examined
miR-152-3p expression in CC tissues using our existingtissue archive and found higher expression level of miR152-3p was detected in CC tissues compared to the adjacent normal tissues (Fig. 2B). Furthermore, we also foundthe expression of miR-152-3p in HE-sEVs is higher thanthat in NE-sEVs derived from both SiHa and Hela cells(Fig. 2C). To explore whether miR-152-3p affects radiosensitivity of CC cells, we transfected miR-152-3p mimics(See figure on next page.)
Fig. 1 Hypoxic tumor cell-derived sEVs alleviate cell damage induced by IR and enhance radioresistance. A The expression of total protein in sEVs
derived from hypoxic and normoxic CC cells was detected by BCA method. *P < 0.05 versus normoxic tumor cells. B Nano was used to detect
the total RNA content in sEVs derived from hypoxic and normoxic tumor cells. **P < 0.01 versus normoxic tumor cells. C SiHa and Hela CC cells
were co-cultured with PBS, NE-sEVs, HE-sEVs, tumor cell apoptosis was measured after 4 Gy radiation treatment by flow cytometry. D Expressionof γ-H2AX protein in blank control cells, HE-sEVs or NE-sEVs-treated cells after 4 Gy radiation treatment was detected by Immunofluorometricassay. **P < 0.01 versus normoxic tumor cells with PBS. E Expression of γ-H2AX protein after different doses of HE-sEV treatment was detectedby Immunofluorometric assay. *P < 0.05 and **P < 0.01 versus normoxic tumor cells with PBS. F The apoptotic cells treated with different dosesof HE-sEVs in SiHa and Hela CC cells was detected and quantitatively analyzed by flow cytometry. *P < 0.05 and **P < 0.01 versus normoxic tumor
cells with PBS. G The flow chart of animal study. H The tumor volume and weight in mice after different treatments with PBS + IR, NE-sEVs + IR,
HE-sEVs + IR was measured (n = 6 in each treatment group). **P < 0.01 versus PBS + IR group and HE-sEVs + IR group. I The representative imagesof HE, Ki67 and TUNEL staining in PBS + IR, NE-sEVs + IR, HE-sEVs + IR groups. In HE, less damage is seen in HE-sEVs treatment; in Ki67 staining, brown
indicates positive stating while blue indicates nuclear; in TUNEL staining, green indicates positive staining. Scale bar: 20 μm. All irradiation treatment
was performed with a single 4 Gy. J Right panel shows the quantification of Ki67-positive and TUNEL-positive cells in PBS + IR, NE-sEVs + IR,
HE-sEVs + IR groups. **P < 0.01 and ***P < 0.001 versus PBS + IR group and NE-sEVs + IR group

Zhou et al. Radiation Oncology(2023) 18:183

Fig. 1 (See legend on previous page.)

Page 6 of 16

Zhou et al. Radiation Oncology(2023) 18:183

into SiHa and Hela cells (Fig. 2D). Our results showedthat IR-induced apoptosis was significantly reduced after
the overexpression of miR-152-3p (Fig. 2E). Meanwhile,
miR-152-3p mimics significantly reduced the DNA damage of the transfected CC cells compared to control cells
after irradiation (Fig. 2F, G). To further investigate therole of tumorigenicity of miR-152-3p in CC radiation, we
conducted a subcutaneous tumor xenograft experiment.
Similarly, we observed that injection of miR-152-3p mimics significantly promoted tumor growth and increased
tumor weight in the IR group compared with control
group (Fig. 2H). In addition, overexpression of miR152-3p protected IR-induced cell damage by increasingcell proliferation (Ki67 positive cells) and reducing cell
apoptosis (TUNEL-positive cells) compared with negative control (Fig. 2I). These results show that miR-152-3p
plays an important role in the sensitivity of RT for CC.
It reduces the apoptosis and DNA damage of CC cells
caused by IR treatment, therefore enhancing the radiation tolerance of CC cells.
Hypoxia cell‑derived sEV miR‑152‑3p is an important cargomolecule mediating CC radioresistanceOur results showed that the expression of miR-152-3p in
HE-sEV treatment group was significantly higher thanthat in PBS control or NE-sEV treatment group, but nosignificant changes were observed between PBS control
group and NE-sEV treatment group in both SiHa andHela cell lines (Fig. 3A). Using RNase R to destroy theresidual RNA outside sEVs to eliminate its interference
to the results, we found similar result that the expression of miR-152-3p in HE-sEV treatment group was
significantly higher than that in PBS control or NE-sEV
treatment group. Our result verified that miR-152-3p
was derived from hypoxia cells-derived sEVs (Fig. 3B).
We then used Triton X-100 to destroy sEVs followed by
RNase R to destroy the RNAs encapsulated in sEVs, andthe result showed a reversion of the increased level ofPage 7 of 16

miR-152-3p (Fig. 3C). Moreover, the increased expression level of miR-152-3p was found in a time-dependentand dose-dependent manner after culturing cells with
HE-sEVs (Fig. 3D). These data suggest that miR-152-3p
was wrapped in HE-sEVs derived CC cells to play a role.
To further explore the function of HE-sEV miR-152-3p
on CC cell radioresistance, HE-sEV miR-152-3p was suppressed by transfecting CC cell lines with sh-miR-152-3p(Fig. 3E). After RT, the cell viability and invasion ability
were significantly reduced in both SiHa and Hela celllines incubated with sh-miR-152-3p transfected HE-sEVs
compared with HE-sEVs (without transfection) incubations (Fig. 3F, G). The inhibition of HE-sEV miR-152-3p
was found to be associated with increased Annexin V
and γ-H2AX expressions in both SiHa and Hela cell lines
compared with HE-sEVs (without transfection) incubations, demonstrating this inhibition induces more apoptosis and DNA damage and reverses radioresistanceinduced by hypoxia treatment (Fig. 3H, I). Our findings
confirm the importance of sEV miR-152-3p induced byhypoxic environment for CC radiation response.
KLF15 is the downstream target of miR‑152‑3p

To investigate the mechanism of miR-152-3p in the regulation of CC radioresistance and identify the downstream
targets of miR-152-3p, we first screened significantly
down-regulated mRNAs from the TCGA database andcross-selected candidates with the target genes of miR152-3p. We found that miR-152-3p was associated withits downstream targets KLF15, KLF2, KLF8 and KLF9
(Additional file 1: Fig. S2A-C). After validation through
GEPIA and Starbase databases, KLF15 was found to havethe most significant difference between CC tissues andcontrol tissues (Additional file 1: Fig. S 2D, E). In comparison of human CC tissues with adjacent control tissues, the low-level expression of KLF15 was found in CC
tissues (Fig. 4A). Similarly, in comparison of human CC
cell lines with a cervical control cell line, the low-level(See figure on next page.)
Fig. 2 The role of miR-152-3p in CC radiation response. A The differentially expressed miRNAs from GSE81137 database were shown by volcanic
map. B The level of miR-152-3p expression in cervical tumor tissues compared to adjacent tissues was examined by qRT-PCR, n = 20 in eachgroup. *P < 0.05 versus adjacent tissues. C The significant difference of sEVs miR-152-3p expression was found in SiHa and Hela cells with different
oxygen contents (normoxia and hypoxia) by qRT-PCR. **P < 0.01 versus CC cells with normoxia. D A significant difference of miR-152-3p expression
was found after miR-152-3p mimic treatment on SiHa and Hela CC cells by qRT-PCR, **P < 0.01 versus negative control CC cells. E Flow cytometry
was conducted to detect the effect of miR-152-3p mimics on radiation-induced apoptosis in SiHa and Hela cells. **P < 0.01 versus negative control
CC cells. F The significant difference of expression of the DNA damage markers γ-H2AX was shown by immunofluorometric assay after miR-152-3p
mimic treatment on SiHa and Hela CC cells. **P < 0.01 and ***P < 0.001 versus negative control CC cells. G The reduced expression of γ-H2AX
and p-DNApkcs was shown by western blot in SiHa and Hela cells treated with miR-152-3p mimic or negative control (NC). H Hela cells transfectedwith miR-152-3p mimics and negative control (NC) plasmid were injected into nude mice, respectively, tumor volume and weight after irradiation
were measured, n = 6 in each group. **P < 0.01 versus negative control CC cells. I Representative images of HE, Ki67 and TUNEL staining with NC + IR
and miR-152-3p + IR, scale bar: 20 μm. All irradiation treatment doses used were 4 Gy. Right panel shows the quantification of Ki67 and TUNEL
positive cells in two treatment groups. *P < 0.05 and **P < 0.01 versus negative control CC cells

Zhou et al. Radiation Oncology(2023) 18:183

Fig. 2 (See legend on previous page.)

Page 8 of 16

Zhou et al. Radiation Oncology(2023) 18:183

expression of KLF15 was also found in CC cell lines(Fig. 4B). qRT-PCR analysis showed that KLF15 level was
decreased in CC cells with miR-152-3p overexpression
compared with control cells (Fig. 4C). In consistence,
IHC results showed that the number of KLF15-positive
cells in animal xenograft tissues significantly decreasedin the treatment with miR-152-3p mimics comparedwith control (Fig. 4D). Double luciferase report analysis
showed that the relative luciferase activity of KLF15-WT
in SiHa and Hela cells was limited after co-transfection
with miR-152-3p mimics, but the luciferase activity of
KLF15-mutant plasmid did not change, indicating that
KLF15 was the direct target of miR-152-3p (Fig. 4E). RIP
results indicated that KLF15 was upregulated in miR152-3p mimics and down-regulated in miR-152-3p inhibitor in SiHa and Hela CC cell lines (Fig. 4F). These results
suggest that KLF15 is a direct target of miR-152-3p.
miR‑152‑3p regulates CC radioresistance by targeting
KLF15

To determine the biological role of KLF15, we constructed a lentiviral vector of KLF15 inhibitor and transfected it into SiHa and Hela cells (Fig. 5A). The results
showed that silencing KLF15 significantly reduced apoptosis after IR treatment (Fig. 5B). In addition, inhibitionof KLF15 expression also reduced DNA damage causedby IR (Fig. 5C). These findings indicated that KLF15 wasa tumor suppressor and played an important role in cell
damage after radiation treatment. In addition, we overexpressed KLF15 in SiHa and Hela cells by transfection of
miR-152-3p to examine whether KLF15 was the substratethat affected cell radiosensitivity. The results showedthat the overexpression of KLF15 reversed the increased
proliferation and migration of CC cells caused by miR152-3p overexpression (Fig. 5D, F). In addition, KLF15
expression rescinded the changes in key protein levels
during miR-152-3p induced apoptosis and DNA damage

Page 9 of 16

repair (Fig. 5G, H). These results suggest that miR-152-3p
promotes radioresistance by directly targeting KLF15.
Expression of plasma sEV miR‑152‑3p and KLF15
is associated with CC prognosis in RT

The expression of plasma sEV miR-152-3p and KLF15
was analyzed in 20 CC patients who received explicit RT.
A high level of sEV miR-152-3p expression in patients
who relapsed within 3 years after RT was identified(Fig. 6A). Further detection of KLF15 expression by qRTPCR showed that sEV KLF15 expression was significantly
lower in the recurrence patients than that in the nonrecurrence group (Fig. 6B). In addition, qPCR analysis
confirmed that the expression of KLF15 mRNA in CC
tissues of these patients was negatively correlated withthe level of plasma sEV miR-152-3p (­ R2 = 0.614, P < 0.001)(Fig. 6C). Furthermore, patients with lower miR-152-3p
level had worse overall survivals by Kaplan–Meier analysis (Fig. 6D). Taken together, our findings indicate thatboth sEV miR-152-3p and KLF15 mRNA markers can beused to predict CC radioresistance.

Discussion
RT is widely recognized in the treatment of CC witheffective consequences. However, the gradual increase ofradioresistance is a major problem limiting the clinicaltreatment efficacy of CC patients [27]. Therefore, further
research investigating the molecular mechanism of radioresistance to increase CC cell sensitivity is an important
step for a better curative effect to improve the prognosis
and survival of CC patients. Hypoxia has been found to
be one of the important factors in radioresistance [5, 7].
The mechanism of radioresistance of hypoxic cells has
been extensively studied in CC such as in cancer stem
cell-like phenotype and glucose metabolism [28, 29].
However, the TME and cell communication under HE
has not been elucidated yet in CC, which is still needed
to be further explored in this area [30].

(See figure on next page.)
Fig. 3 MiR-152-3p acts as an effector cargo molecule in hypoxic cells-derives sEVs in CC radiation response. A The expression levels of miR-152-3p
in SiHa and Hela CC cells after HE-sEVs or NE-sEVs treatment was examined by qRT-PCR. ***P < 0.001 versus negative control CC or NE-sEV cells. B
The different expressions of sEV miR-152-3p in SiHa and Hela CC cells treated with NC + RNase R (0.1 mg/mL), HE-sEVs + RNase R or NE-sEVs + RNase
R were shown by qRT-PCR. ***P < 0.001 versus CC cells sEVs treated with RNase R. C qRT-PCR was used to detect the expression of miR-152-3p
in sEVs treated with different oxygen concentrations and destroyed with TritonX-100 (0.3%) and RNase R (0.1 mg/ml). D The level of miR-152-3p
in SiHa and Hela cells is correlated with the time and dose substitution of HE-sEVs treatment. E Inhibition of miR-152-3p by sh-miR-152-3ptransfection in HE-sEVs was confirmed by qRT-PCR. ***P < 0.001 versus NC group, ###P < 0.001 versus HE-sEVs group. F Effects of different treatments
on proliferation of SiHa and HeLa cells was measured by CCK-8 assay. **P < 0.01, ***P < 0.001 versus NC group, ##P < 0.01, ###P < 0.001 versus H-EV
group. G Transwell assay of SiHa and Hela cells with different treatments was conducted, and the representative images were shown. *P < 0.05,**P < 0.01 versus NC group, #P < 0.05, ##P < 0.01 vs HE-sEVs group. H The effects of different treatments on apoptosis of SiHa and HeLa cells wereanalyzed by flow cytometry. ***P < 0.001 versus NC group, ###P < 0.001 versus HE-EVs group. I The level of γ-H2AX expression in SiHa and Helacells with different treatments was quantified. All irradiation treatment doses used were 4 Gy. *P < 0.05, **P < 0.01 versus NC group, #P < 0.05
versus HE-sEVs group

Zhou et al. Radiation Oncology(2023) 18:183

Page 10 of 16

Fig. 3 (See legend on previous page.)EVs derived from cancer cells are important mediators in cell communication, which play various roles inregulating cell proliferation, progression, drug resistance, immune escape and metastasis [31]. Several lines

of evidence demonstrate that increased number of EVs
is produced in tumor hypoxic environment [32]. In
addition, recent reports also showed EV cargos fromhypoxic cells were transferred to normoxic cells throughZhou et al. Radiation Oncology(2023) 18:183

Page 11 of 16

Fig. 4 KLF15 is a downstream target of miR-152-3p in CC cells. A The reduced expression of KLF15 was found in cervical tumor tissues comparedwith adjacent tissues from CC patients by qRT-PCR (n = 20 in each group), **P < 0.01 versus adjacent tissues. B The reduced expression of KLF15
was shown in CC cells (C33A, SiHa and Hela) compared with normal cervical epithelial cell (Ect1/E6E7) by qRT-PCR. **P < 0.01 versus Ect1/E6E7
cells. C The expression of KLF15 in SiHa and Hela cells was reduced after treatment with miR-152-3p mimics compared with untreated control
by qRT-PCR. **P < 0.01, ***P < 0.001 versus NC group. D The expression of KLF15 was reduced in animal tumor xenografts treated with miR-152-3p
mimics compared with control. Representative images are shown for KLF15 expression in treated and control groups by IHC. The brown indicates
positive staining while the blue indicates nuclei, scale bar: 20 μm. E The direct binding of miR-152-3p to the 3’-UTR region of KLF15 was shownby dual luciferase report assay. The significant difference was found between KLF15-WT and KLF15-MUT after miR-152-3p mimics treatments**P < 0.01 versus NC group. F RIP assay was shown the binding status between miR-152-3p and KLF15 in SiHa and Hela cells, confirming
the miR-152-3p specifically binding KLF15. **P < 0.01 versus miR-152-3p mimics IgG groupinternalization, so the cargo molecules contained in the
EVs were also transferable to normoxic cells [19]. In thisstudy, we tested NTA, total proteins and RNAs to verify
that the HE led to an increased number of sEVs secreted
by CC cells. Accumulating reports show that sEV miRNAs contribute to the cancer progression with implications for both diagnosis and treatment [33]. sEV cargos
from hypoxic cells could be transferred to normoxic
cells to play a biological role [19]. Tumor cell-derivedsEV miRNAs from hypoxia usually further aggravatethe malignant transformation of normoxic cells. In thecurrent study, we demonstrate that the EVs secreted byhypoxic CC cells are higher than normoxic cells, and EVs
derived from hypoxic CC cells can reduce DNA damage
induced by RT.
The in vivo and in vitro results of this study suggest thatafter incubation with hypoxic-derived sEVs, the proliferation and radioresistance of CC cells were significantly
increased, indicating that they have a cancer-promoting
effect. In the meanwhile, we found that miR-152-3p wasZhou et al. Radiation Oncology(2023) 18:183

Page 12 of 16

Fig. 5 MiR-152-3p exerts it’s radioresistant effect on CC cells by targeting KLF15. A qRT-PCR analysis verified the expression of KLF15 knockdownby transfecting shRNA in SiHa and Hela cells. **P < 0.01 versus NC group. B The apoptosis in KLF15 knockdown CC cells was significantly reduced
compared with control after 72 h of radiotherapy by flow cytometry. **P < 0.01 versus NC group. C The expression of γ-H2AX after transfectionin KLF15 knockdown CC cells was significantly reduced compared with control by qRT-PCR. *P < 0.05 and **P < 0.01 versus NC group. D
qRT-PCR verified the expression of KLF15 in SiHa and Hela cells after different transfections as indicated and significant difference was foundbetween miR-152-3p overexpression group and NC group. **P < 0.01 and ***P < 0.001 versus NC group. E The proliferation of SiHa and Helacells after different transfections was shown by CCK-8 assay and significant difference was found between miR-152-3p overexpression group
and NC group. **P < 0.01 versus NC group. F Cell migration in SiHa and Hela cells was measured by transwell assay after different transfections.
Representative images are shown for NC, miR152-3p and miR152-3p + KLF15 from 3 independent experiments (n = 3), scale bar: 20 μm. G
The apoptosis assay of SiHa and Hela cells after different transfections was shown by flow cytometry, and the significant difference was foundbetween miR-152-3p overexpression group and NC group. **P < 0.01 and ***P < 0.001 versus NC group. H The expression of γ-H2AX in SiHa and Helacells after different transfections was quantified by qRT-PCR and the significant difference was found between miR-152-3p overexpression group
and NC group. *P < 0.05 versus NC group. *P < 0.05 and **P < 0.01 versus NC group

Zhou et al. Radiation Oncology(2023) 18:183

Page 13 of 16

Fig. 6 Expression of plasma sEV miR-152-3p and KLF15, and its prognostic value. A The level of sEV miR-152-3p in CC patients with or without
recurrence after radiotherapy was analyzed by qRT-PCR (n = 20 in each group), indicating increased sEV miR-152-3p was found in recurrent CC
patients compared with non-recurrent CC patients. **P < 0.01 vs non-recurrence. B The expression of KLF15 in the above patients (n = 20 in eachgroup) was shown by qRT-PCR verifies, indicating reduced sEV KLF15 was found in recurrent CC patients compared with non-recurrent CC patients.
**P < 0.01 vs non-recurrence. C Correlation analysis between miR-152-3p and KLF15 in the same patients was performed qRT-PCR, the expressionof KLF15 mRNA in CC tissues of these patients was negatively correlated with the level of plasma sEV miR-152-3p. **P < 0.01 vs non-recurrence. D
Kaplan–Meier survival rate analysis of the overall survival rate of these patients by miR-152-3p expression level in tissues was analyzed by Kaplan–
Meier analysis. **P < 0.01 vs non-recurrence

highly expressed in hypoxic CC cell-derived sEV. The
expression of miR-152-3p was significantly different invarious cancer tissues and cells. For example, the upregulation of miR-152-3p was reported to play an anti-tumor
role by negatively regulating PI3KCA to inhibit the proliferation of breast cancer cell HCC1806 [34]. However,miR-152-3p was found to be overexpressed in chronic
myeloid leukemia (CML), which promoted K562 cell proliferation by inhibiting P27, thereby increasing its malignant potential [35]. The data of this study showed that theexpression of sEV miR-152-3p was increased under HE,
which is essential for HE-sEV-induced radioresistance for
CC. Using bioinformatics analysis, we found KLF15 was a
direct target of miR-152-3p that contributed to CC radioresistance. The KLF family is engaged in numerous cellbiological processes, including proliferation and apoptosis, according to earlier research [36]. The level of KLF15
in ovarian cancer tissues and cells is lower than normal.
Overexpression of KLF15 suppressed the proliferationand migration of ovarian cancer cells [37]. In addition,

low KLF15 expression level indicates a poor prognosis ofgastric cancer patients [38]. However, the role of KLF15
in CC has not been reported. We explored for the firsttime the impact of KLF15 in proliferation, apoptosis and
DNA damage for CC. KLF15 overexpression reverses
the inhibition of apoptosis and DNA damage by miR152-3p in CC cells. KLF15 improves radiosensitivity byenhancing apoptosis of CC cells. sEV miR-152-3p andKLF15 are potential therapeutic targets to improve CCradiosensitivity.
Finally, we verified the expression of plasma sEVs miR152-3p increased in CC patients who relapsed after RT.
In addition, the levels of plasma sEV miR-152-3p andKLF15 have shown encouraging results in predicting
CC prognosis in RT. At present, liquid biopsy of EV biomarkers such as miRNAs, mRNAs and proteins is a new
developing area for the early diagnosis, predicting prognosis and monitoring cancer progression [39, 40]. Since
increased sEV miR-152-3p and reduced sEV KLF15
were found in cervical patients’ plasma after recurrenceZhou et al. Radiation Oncology(2023) 18:183

after RT, these two EV markers hold promise for liquidbiopsy to predict CC radioresistance or recurrence after
RT. However, more clinical samples are needed to further
validate their values [41].

Conclusion
In summary, our findings firstly demonstrate that sEVs
from hypoxic cells enhance the CC radioresistance bytransferring miR-152-3p. The mechanism of sEV miR152-3p involved is associated with blocking IR-induced
apoptosis and DNA repair by targeting KLF15. In addition, increased plasma sEV miR-152-3p and reduced
KLF15 protein have shown promise in predicting CC
prognosis after RT. Our results suggest sEV miR-152-3p
and KLF15 are useful biomarkers for predicting CC radiation response as well as potential therapeutic targets fordeveloping novel EV-based targeted therapy to improve
CC RT and overcome radioresistance.
Abbreviations
ANOVA	Analysis of varianceBcl-2	B-cell lymphoma-2
CD	Cluster of differentiation
DNA	Deoxyribonucleic acidEVs	Extracellular vesicles
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase
GEO	Gene expression omnibus
GEPIA	Gene expression profiling interactive analysis
H2AX	H2A.X variant histoneHE	Hematoxylin and Eosin
HE-EV	Hypoxia environment secreted extracellular vesicle
HIF-1α	Hypoxia-inducible factor 1α
HRP	Horseradish peroxidase
IgG	Immunoglobulin G
IHC	ImmunohistochemistryIR	Ionizing radiation
KLF15	Kruppel-like factor 15
miRNA	MicroRNA
NCBI	National Center for Biotechnology Information
NE-EV	Normoxia environment secreted extracellular vesicle
NTA	Nanoparticle tracking analysis
P21	Cyclin-dependent kinase inhibitor 1A
P56	Cyclin-dependent kinase like 2
PBS	Phosphate buffered saline
PCR	Polymerase chain reaction
PI	Propidium iodide
PVDF	Polyvinylidene fluoride
qRT-PCR	Quantitative reverse transcription polymerase chain reaction
RIPA	Radio-immunoprecipitation assayRNA	Ribonucleic acid
SDS–PAGE	Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
TCGA​	The cancer genome atlas
TME	Tumor microenvironment
UTR​	Untranslated regions

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13014-​023-​02369-3.
Additional file 1: Fig. S1. Verification of sEVs after treatment with different oxygen concentrations (HE and NE). (A, B) The concentration ofsEVs secreted by cells in different treatment groups was measured byPage 14 of 16

NTA. *P<0.05 vs. CC cells treated with normoxia. (C) The morphology ofsEV was observed by transmission electron microscopy (TEM). (D, E) The
expression of sEV markers in different CC cells was detected by western
blot analysis and quantified. **P<0.01 vs. CC cells treated with normoxia.
(F) The expression of HIF-1α after different treatments was detected bywestern blot analysis and quantified. **P<0.01 vs. CC cells treated withnormoxia. (G) Immunofluorescence detects the entry of sEVs into CC cells
after PKH67 labeling, red indicates sEVs and blue indicates cell nucleus,
scale bar: 20 μm.
Additional file 2: Fig. S2. Target gene screening of miR-152-3p. (A, B)
Heat map and volcano map show the differentially expressed mRNAs in
the TCGA database. (C) Venn diagram is used to screen target mRNAs. (D)GEPIA database verifies the differential expression of KLF15 in CC tissues
and normal cervical tissues. (E) The Starbase database (ENCORI) verifies thecorrelation between miR-152-3p and KLF15 in CC tissues.
Acknowledgements
We acknowledge assistance with the access of analytic instruments from theTranslational Medical Center at The First Affiliated Hospital of Zhengzhou
University.
Author contributions
LC, YL, NL and RG conceptualized, designed, and directed the project. JZ,
NL and WT performed the experiments; LC, JZ, YL, FG and HF analyzed thedata and co-wrote the manuscript; JZ, QM, MC, JZ, and SD performed animal
experiments and analyzed the data. All authors edited and approved the final
version of the manuscript.
Funding
This work was supported by Henan Province Colleges and Universities Innovative Talent Support Program (No.21HASTIT044), Henan Province Health Youngand Middle-aged Discipline Leader Training Project, Henan Province Youngand Middle-aged Health Science and Technology Innovative Talent Training
Project (No.YXKC2020039), Central Plains Youth Top Talent Project and Highlevel Talents Return to China for Research Funding Project.
Availability of data and materials
Any individual or group may contact the corresponding author to obtainrelevant research data.

Declarations
Ethics approval and consent to participate
The study about specimen and clinical data was approved by the Ethics Committee of First Affiliated Hospital of Zhengzhou University, with the following
ethics batch number: No.2022-ky-1371-002. All procedures about nude mouse
xenograft model were performed in accordance with the guidelines for laboratory animal ethics issued by Henan Academy of Medical and Pharmaceutical
Sciences and Use Committee.
Consent for publication
All authors have approved the publication of this study.
Competing interests
The authors declare no conflict of interest.
Author details
1
Department of Obstetrics and Gynecology, The First Affiliated Hospitalof Zhengzhou University, No.1 Jianshe East Road, Zhengzhou 450052, Henan,
China. 2 School of Basic Medical Sciences, Zhengzhou University, Zhengzhou,
Henan, China. 3 Department of Neuroimmunology, Henan Institute of Medical
and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan,
China. 4 Henan Engineering Technology Research Center for Accurate Diagnosis Neuroimmunity, Zhengzhou, Henan, China. 5 Department of Radiation
Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou,
Henan, China. 6 Level 2, Research and Education Centre, Cancer Care Centre,
St George Hospital, 4‑10 South St, Kogarah, NSW 2217, Australia. 7 St George

Zhou et al. Radiation Oncology(2023) 18:183

and Sutherland Clinical Campuses, School of Clinical Medicine, UNSW Sydney,
Kensington, NSW, Australia.
Received: 2 September 2023 Accepted: 24 October 2023

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