Cancer Medicine - 2023 - Fan - BIRC5 Facilitates Cisplatin Chemoresistance in A M6a Dependent Manner in Ovarian Cancer

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Cancer Medicine - 2023 - Fan - BIRC5 Facilitates Cisplatin Chemoresistance in A M6a Dependent Manner in Ovarian Cancer

cancer med

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Received: 18 February 2023

| Revised: 26 August 2023

| Accepted: 26 September 2023

DOI: 10.1002/cam4.6811

RESEARCH ARTICLE

BIRC5 facilitates cisplatin-chemoresistance in a

m6A-dependent manner in ovarian cancer

Yadan Fan1 | Yinglian Pan2 | Liping Jia1 | Shuzhen Gu1 | Binxin Liu1 |
Ziman Mei1 | Chunyan Lv1 | Haohao Huang3 | Genhai Zhu4 | Qingchun Deng1
1
Department of Gynecology, The Second Affiliated Hospital of Hainan Medical University, Haikou, China
2
Department of Oncology, The First Affiliated Hospital of Hainan Medical College, Haikou, China
3
Department of Neurosurgery, General Hospital of Central Theater Command of Chinese People's Liberation Army, Wuhan, China
4
Department of Gynecology, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou, China

Correspondence
Haohao Huang, Department of Abstract
Neurosurgery, General Hospital of Cisplatin-based chemotherapy is the standard treatment for metastatic ovar-
Central Theater Command of Chinese
ian cancer (OC). However, chemoresistance continues to pose significant clini-
People's Liberation Army, Wuhan
430070, China. cal challenges. Recent research has highlighted the baculoviral inhibitor of the
Email: [email protected] apoptosis protein repeat-containing 5 (BIRC5) as a member of the inhibitor
Genhai Zhu, Department of of the apoptosis protein (IAP) family. Notably, BIRC5, which has robust anti-
Gynecology, Hainan General Hospital, apoptotic capabilities, is overexpressed in numerous cancers. Its dysfunction
Hainan Affiliated Hospital of Hainan
Medical University, Haikou, 570011,
has been linked to challenges in cancer treatment. Yet, the role of BIRC5 in the
China. chemoresistance of OC remains elusive. In our present study, we observed an
Email: [email protected] upregulation of BIRC5 in cisplatin-resistant cell lines. This upregulation was as-
Qingchun Deng, Department of sociated with enhanced chemoresistance, which was diminished when the ex-
Gynecology, The Second Affiliated pression of BIRC5 was silenced. Intriguingly, BIRC5 exhibited a high number of
Hospital of Hainan Medical University,
Haikou, 570216, China. N6-methyladenosine (m6A) binding sites. The modification of m6A was found
Email: [email protected] to enhance the expression of BIRC5 by recognizing and binding to the 3′-UTR
of mRNA. Additionally, the insulin-like growth factor 2 mRNA-binding protein
Funding information
General Hospital of Central Theater 1 (IGF2BP1) was shown to stabilize BIRC5 mRNA, synergizing with METTL3
Command of Chinese People's and intensifying chemoresistance. Supporting these in vitro findings, our in vivo
Liberation Army Foundation, Grant/
experiments revealed that tumors were significantly smaller in size and vol-
Award Number: ZZYCZ202107;
Nature Science Foundation of Hubei ume when BIRC5 was silenced. This reduction was notably counteracted by
Province, Grant/Award Number: co-silencing BIRC5 and overexpressing IGF2BP1. Our results underscored the
2022CFB883; the China Postdoctoral
Science Foundation, Grant/Award
pivotal role of BIRC5 in chemoresistance. The regulation of its expression and
Number: 2021MD703960; The Hainan the stability of its mRNA were influenced by m6A modifications involving both
Health Commission Project, Grant/ METTL3 and IGF2BP1. These insights presented BIRC5 as a promising potential
Award Number: 20A200247; The
Hainan Provincial Key Research therapeutic target for addressing cisplatin resistance in OC.
and Development Program Project

Yadan Fan, Yinglian Pan, Liping Jia have contributed equally to this work and co-first authors.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

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|

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Fund, Grant/Award Number:

KEYWORDS
ZDYF2022SHFZ068; The High-level
Talents Program of Hainan Province BIRC5, chemoresistance, IGF2BP1, METTL3, ovarian cancer
Natural Science Foundation, Grant/
Award Number: 821RC712; The
National Natural Science Foundation of
China, Grant/Award Number: 82203879

1 | I N T RO DU CT ION family.16 Although it has a low expression level in healthy

adult tissues, BIRC5 is markedly upregulated in a variety
Ovarian cancer (OC) is the leading gynecological malig- of tumors, where it assumes an oncogenic role. Its ex-
nancy worldwide and poses a significant threat to wom- pression is modulated by circadian rhythms and is acti-
en's health, accounting for the highest mortality rates vated by transcription factors such as STAT, NF-κB, and
among gynecological cancers.1 In China, OC ranks as the other signal transducers.17 Several studies have reported
third most common cancer affecting the female reproduc- that miRNAs, small-molecule inhibitors, antisense oligo-
tive system. In its early stages, OC often remains asymp- nucleotides, and peptide-based immunotherapy can sig-
tomatic. By the time symptoms such as abdominal pain, nificantly downregulate BIRC5 expression, consequently
bloating, and irregular vaginal bleeding manifest, the dis- inhibiting tumor cell growth.18 Nonetheless, the biological
ease is frequently in an advanced stage, correlating with significance of BIRC5 and its regulatory mechanisms re-
a poor prognosis.2 The primary therapeutic approach for lated to drug resistance in OC remain unclear.
OC combines cytoreductive surgery with platinum-based In this study, we conducted a series of in vitro and
chemotherapy.3 However, despite this treatment regimen, in vivo functional experiments to investigate the role and
recurrence rates soar as high as 70% within 3 years.4 While regulation of the BIRC5 gene in OC with cisplatin resis-
many patients initially respond well to chemotherapy, oth- tance. Our findings illuminated the biological significance
ers face recurrence after standard treatment. Moreover, of m6A modifications mediated by METTL3 and IGF2BP1.
with each successive chemotherapy treatment and in- These entities controlled the expression of BIRC5 and its
creased dosage, the intervals between treatments shorten, mRNA stability, respectively. Consequently, our results
leading to heightened chemoresistance and a deteriorating suggested that BIRC5 might serve as a potential therapeu-
prognosis.5 Research suggests that heightened antioxidant tic target for addressing chemoresistance in OC.
capacities within cancer cells and increased capabilities
to expel drugs are primary contributors to chemoresis-
tance.6–9 Additionally, an enhanced ability to repair DNA 2 | METHODS
damage and the inactivation of tumor cell apoptosis are
other prevalent factors.10,11 Epigenetic changes also play a 2.1 | Establishment of stable cell lines
pivotal role, often leading to aberrant gene expression.12,13
Despite these insights, the specific mechanisms underly- Ovarian carcinoma cell lines SKOV3 and A2780 were ob-
ing chemoresistance in OC remain elusive. tained from American Type Culture Collection (ATCC).
N6-methyladenosine (m6A) methylation is the most Cells cocultured with gradually increase the concentra-
prevalent modification in mRNA and is dynamically re- tion of cisplatin, and then obtained SKOV3/DDP and
versible, contingent upon specific enzymes. The implica- A2780/DDP after 6 months. To identify sensitive and re-
tions of m6A span a wide range, including the regulation sistant cells using CCK8 and the median inhibitory con-
of gene expression, modulation of mRNA stability or centrations (IC50). SKOV3 and SKOV3/DDP cultured in
degradation, splicing, and interactions between RNA and McCoy's 5A medium, while A2780 and A2780/DDP cul-
protein. The m6A modification is introduced by enzymes tured in DMEM medium, supplemented with 10% FBS
known as methyltransferases, referred to as “writers,” and (Gibco, ThermoFisher, USA), 100 μg penicillin and 100 U/
removed by demethylases, termed “erasers.” These are mL streptomycin (Pricella, China). All cells were cultured
RNA-binding proteins that either directly or indirectly at 37°C in a humidified atmosphere of 5% CO2.
recognize the m6A motif and play roles in RNA stabiliza-
tion, degradation, and translation.14,15 While prior studies
have identified the influence of m6A in OC progression, its 2.2 | Cell viability and IC50
role in OC chemoresistance remains elusive. Baculoviral
IAP repeat containing 5 (BIRC5) has been identified as a Cell viability detected by CCK8 assay. SKOV3, SKOV3/
member of the inhibitor of the apoptosis proteins (IAP) DDP, A2780, A2780/DDP were seeded 2000 cells into
|

96-well templates for 24 h, and treated with different doses YTHDF1, YTHDF2, YTHDF3, IGF2BP1, IGF2BP2, and
(0, 5, 10, 20, 40, and 80 μM) of cisplatin for another 24 h, IGF2BP3 were listed in Table S1.
then added 10 μL CCK8 to incubate for 3 h. Absorbance
was examined at 450 nm using a microplate reader
(BioTek, USA). 2.5 | Cell transfection

Short hairpin RNAs specific for BIRC5, METTL3, IGF2BP1

2.3 | Western blotting and negative control, and the overexpressed plasmids
(BIRC5, METTL3, METTL5, METTL14, WTAP, RBM15,
OC cells were washed twice in pre-chilled PBS, and RBMX, VIRMA, IGF2BP1, IGF2BP2, IGF2BP3, YTHDF1,
lytic cells in RIPA lysis buffer with protease and phos- YTHDF2, YTHDF3, and empty vector plasmid) were con-
phatase inhibitors. Protein lysate were centrifuged at structed by GeneChem (Shanghai, China). SKOV3/DDP and
10,000 g, and supernatants were denatured in 100°C A2780/DDP cells were seeded into 6-well plates with 3 × 105
with 1 × loading buffer (Biosharp, China), and the cells/well concentration and transfected by Lipofectamine
protein concentration was determined using BCA 3000 (Invitrogen, UA) for 48 h according to the manufactur-
method. Protein samples were separated by sodium do- er's protocol. In addition, cells were infected by lentivirus and
decyl sulfate-polyacrylamide gel electrophoresis (SDS- selected by 1 μg/mL puromycin (Solarbio, China). The trans-
PAGE) and transferred to PVDF membranes. These fection efficiency was identified by RT-qPCR and Western
PVDF membranes were closed in 5% nonfat dried blotting. The sequences of shRNAs are shown in Table S2.
milk (Biosharp, China), and hatch to primary antibod-
ies overnight at 4°C, which including: BIRC5 (1:1000,
Abcm, ab134170), METTL3 (1:1000, Abcam, ab195352), 2.6 | RNA stability assay
IGF2BP1 (1:1000, Abcam, ab184305), GAPDH (1:1000,
Abcam, ab128915), METTL14 (1:1000, Abcam, To evaluate RNA stability, OC cells who knockdown and
300,104), METTL5 (1:1000, Proteintech, 16791-1-AP), overexpress IGF2BP1 were seeded in 12-well plates, and
METTL16 (1:1000, Proteintech, 19924- 1-
AP), WTAP treated with actinomycin D (MedChemExpress, USA) in
(1:1000, Proteintech, 10200- 1-
AP), RBM15 (1:1000, 5 μg/mL. Total RNA was isolated after culturing for in-
Proteintech, 10587-1-AP), VIRMA (1:1000, Proteintech, dicted times (0, 1, 3, and 6 h), and quantify the relative
25712- 1-
AP), IGF2BP2 (1:1000, Proteintech, 11601- level of BIRC5 mRNA.
1-
AP), IGF2BP3 (1:1000, Proteintech, 14642- 1-
AP), YTHDC1 (1:1000, Proteintech, 14392- 1-
AP),
YTHDF1 (1:1000, Proteintech, 26787-1-AP), YTHDF2 2.7 | Dual luciferase report assay
(1:1000, Proteintech, 24744- 1-
AP), YTHDF3 (1:1000,
Proteintech, 25537-1-AP).Then further incubated with We constructed the BIRC5 wild-type 3′-UTR and bind-
secondary antibodies for 1 h at room temperature, then ing motif-mutated 3′ vector in psiCHECK- 2 plasmid
ECL Western blotting detection regents (Biosharp, (HonorGene, China). SKOV3/DDP and A2780/DDP cells
China) were used to detect proteins in imagining sys- were seeded in 96-well plates separately, and co-transfected
tem (SageCreation, China). with wild-type and mutated plasmids when the density
reached to 70%. After incubating 48 h, we collect cells to test
in Promega dual-luciferase system (USA). The luciferase
2.4 | RNA isolation, reverse activity was normalized to the Renilla luciferase activity.
transcription, and quantitative
real-time PCR
2.8 | Me-RIP
Total RNA was isolated from tissues and cells using
TRIzol reagent (TaKaRa, Japan), then reverse transcribed The RNA extracted in cell line using Total RNA Miniprep
to cDNA by PrimeScript™ RT reagent Kit (TaKaRa, Kit (Monarch, USA), and fragmented into 300 bp with
Japan). Quantitative reverse transcription- PCR (RT- fragment reagents. Then added m6A anti-body and IgG
qPCR) reactions were conducted using the Tli RNaseH anti-body, and added to fragment RNA into magnetic
Plus kit (TaKaRa, Japan) according to manufacturers' beads at 4°C for 1 h. We added RNA wash buffer to it and
protocols. The primers of BIRC5, METTL3, METTL14, extracted RNA, and expression of related gene was de-
WTAP, METTL5, METTL16, VIRMA, RBM15, YTHDC1, tected by RT-qPCR.
|

2.9 | Annexin-V-FITC/propidium parental cell lines by continuously exposing the cells to es-
iodide staining calating concentrations of cisplatin. The resistant pheno-
types were subsequently confirmed using the CCK8 assay.
Annexin-V-FITC/propidium iodide (IP) staining was Upon cisplatin treatment, the viability of the SKOV3/DDP
performed using Annexin-V-FITC/PI apoptosis assay kit and A2780/DDP cells was notably higher compared to the
(Gene-Protein Link, China) according to its manufactures. parental SKOV3 and A2780 cells (Figure 1A). The IC50
Cells were resuspended in binding buffer, and adjusted values for cisplatin in the SKOV3 and A2780 cells stood at
concentration to 1 × 106. Then added 5 μL Annexin-V- 2.164 μM and 1.234 μM, respectively. In contrast, these val-
FITC reagent to incubate for 10 min at room temperature, ues surged to 13.452 μM and 12.242 μM, respectively, in the
then added 10 μL PI reagent and 400 μL PBS to analyze by cisplatin-resistant OC cells (Figure 1B). Concurrently, the
flow cytometry BD FACSCalibur (BD, Biosciences). colony formation capabilities of SKOV3/DDP and A2780/
DDP surpassed those of the parental cells (Figure 1C).
To discern gene expression differences between the
2.10 | Tumor xenograft model SKOV3/DDP and native SKOV3 cells, we executed an
RNA-sequence on total RNA. The resulting data showed
Four weeks old female BALB/c nude mice were purchased fluctuations in mRNA transcript levels. Specifically, 172
from SJA Laboratory Animal Co, Ltd (Changsha, China), RNAs were upregulated, while 206 were downregulated
and acclimated in a specific pathogen free environment for in the cisplatin-resistant cells relative to the parental cells
1 week. SKOV3/DDP cells treated with shBIRC5, or shBIRC5 (with a fold change >2 and a p-value < 0.05). Remarkably,
combined with pcDNA- IGF2BP1 plasmid (IGF2BP1) or the expression of BIRC5 exhibited the most significant in-
corresponding negative control were suspended in 100 μL crease (Figure 1D,E). Existing literature reports an over-
McCoy's 5A medium and implanted subcutaneously into expression of BIRC5 in numerous malignancies. This
the left frank of mice in 1 × 107 cells/mice. After 7 days in- overexpression has implications for modulating carcino-
cubation, three groups of mice received 10 mg/kg cisplatin genesis and chemotherapy resistance. However, the intri-
respectively, sacrificing and tumor collecting after 30 days. cate molecular mechanisms underpinning these effects
The tumor volume measured in V = (L × W2)/2. Care and remain elusive.
handling of the mice were approved by the Institutional
Animal Care and Use Committee of Hainan Medical
University (#HYLL-2022-218, Haikou, China). 3.2 | BIRC5 is upregulated in OC
tissues and promotes chemoresistance of
OC cells in vitro
2.11 | Statistical analysis
To understand the role of BIRC5 in OC, we employed RT-
All experiments were three independent repetitions, qPCR to measure the transcript levels of BIRC5 across 30
presented as mean values ±standard deviation (SD) and primary OC tissues and 30 normal ovarian tissues. As de-
statistically analyzed by GraphPad Prism 9.0. Unpaired picted in Figure 2A, the expression of BIRC5 at the mRNA
student's t-test was used to evaluate the difference be- level was markedly altered in primary OC tissues relative
tween experimental groups and control groups. A one- to normal ovarian tissues. This observation was congru-
way ANOVA test was used for comparison between three ent with the RNA-seq datasets from TCGA and GTEx
groups. Statistical significance is considered as *p < 0.05, (Figure 2B).
**p < 0.01, ***p < 0.001. To investigate the potential role of BIRC5 in cisplatin
resistance in vitro, we assessed its expression levels in
OC cells. Results indicated an upregulation of BIRC5 in
3 | R E S U LTS SKOV3/DDP and A2780/DDP compared to the control
groups (Figure 2C). Subsequently, we employed two spe-
3.1 | Establishment of the cific shRNAs to suppress the expression of BIRC5. The
cisplatin-resistant cells and identification effectiveness of this depletion was validated using both
of their gene expression profile in OC RT-qPCR and Western blotting analysis (Figure 2D).
Functional assays, including CCK8 and EdU, revealed
To unravel the potential mechanisms driving cisplatin that BIRC5 downregulation significantly amplified
resistance in the malignant progression of OC, we devel- the sensitivity of SKOV3/DDP and A2780/DDP cells to
oped stable cisplatin-resistant cell lines: SKOV3/DDP and cisplatin (Figure 2E–G). Furthermore, the IC50 value
A2780/DDP. These were derived from their respective was diminished post- BIRC5 silencing (Figure 2F),
|

F I G U R E 1 Establishment of the cisplatin-resistant cells and identification of their gene expression profile in OC. (A) SKOV3/DDP,
A2780/DDP, SKOV3 and A2780 treated with different doses of cisplatin for 24 h, followed by CCK8 assay. Dose-response curve of cisplatin
treatment in multiple OC line. (B) IC50 were measured after treating increasing dose of cisplatin for 24 h (log10 scaled). (C) Colony formation
treated with cisplatin 20 μM for 2 weeks. (D,E) Heatmap and volcano plot represent the differentially expressed mRNA in cisplatin resistant
SKOV3/DDP and counterpart SKOV3 cells. The red and green represent upregulated and down-regulated mRNA respectively. The arrow
indicates BIRC5. The mean ± SD of triplicate experiments was plotted (n = 3), *p < 0.05.

and the colony formation capacity was also hindered in reduced apoptotic rates, as assessed by flow cytometry
(Figure 2H). analyses alongside cleaved-PARP (Figure 3E,F). In sum-
Given that BIRC5 is an IAP, we posited that BIRC5 mary, these observations reinforced the notion that BIRC5
might attenuate cisplatin sensitivity by modulating tumor was pivotal for conferring cisplatin resistance in OC cells.
cell apoptosis. Following BIRC5 suppression, the DDP-
resistant OC cells were subjected to cisplatin treatment
at an optimal dose for 24 h, after which they underwent 3.3 | BIRC5 is regulated by
Annexin V-FITC/PI staining. Compared to control groups, METTL3-mediated m6A modification
the apoptotic rates in the BIRC5-deficient group showed
a significant increase (Figure 2I). Taken together, these Prior research has indicated that m6A modification plays
findings suggested that BIRC5 played a role in modulating an important role in drug resistance across various tu-
the sensitivity of OC cells to cisplatin. mors. To understand the molecular mechanism through
Simultaneously, we generated and transfected a BIRC5 which BIRC5 influences chemotherapy resistance in OC,
plasmid into SKOV3 and A2780 cells. The effectiveness we compared the m6A levels between parental SKOV3,
of this transfection was validated using both RT-qPCR A2780 cells, and their cisplatin-resistant counterparts:
and Western blotting analysis (Figure 3A). Enhanced SKOV3/DDP and A2780/DDP. Colorimetric analysis for
BIRC5 expression augmented cell viability and prolif- m6A RNA methylation quantification revealed that m6A
eration while diminishing cisplatin sensitivity, as evi- levels in mRNAs from SKOV3/DDP and A2780/DDP cells
denced by CCK8, IC50, and colony formation experiments were significantly elevated compared to those in SKOV3
(Figure 3B–D). Moreover, elevating BIRC5 levels resulted and A2780 cells (Figure 4A). Based on these findings, we
|

F I G U R E 2 Down-regulating BIRC5 reverses chemotherapy resistance of OC cells. (A) Relative expression of BIRC5 in OC tissues
(n = 30) and normal ovarian tissues (n = 30) was detected by RT-qPCR (p < 0.05). (B) The abundance of BIRC5 transcripts from the TCGA
database and GTEX database. (C) Expression of BIRC5 in cisplatin resistance cell and parental cells was detected by RT-qPCR and Western
blotting. (D) SKOV3/DDP and A2780/DDP were transfected with control shRNA and BIRC5 shRNA, and the effect of knockdown BIRC5
measured by Western blotting and RT-qPCR. (E,F) Then the cell viability monitored by CCK8, and IC50 after treating increasing dose of
cisplatin for 24 h. (G,H) EdU and colony formation assay were used to assess cell survival of knocked-down BIRC5 cisplatin resistance
cells after 20 μM cisplatin treatment. (I) Cell apoptosis was measured by flow cytometry in SKOV3/DDP and A2780/DDP with indicated
treatment, and right histograms represent apoptosis rate (early + late). The mean ± SD of triplicate experiments was plotted (n = 3), *p < 0.05.

hypothesized that m6A modification might play a part in BIRC5 in DDP-resistant OC cells (Figure 4B, Figures S1
regulating BIRC5 in cisplatin-resistant cells. and S2). Thus, we focused on the well-established m6A
To test this hypothesis, we assessed the correla- reader METTL3, which was also markedly upregulated in
tion between m6A methyltransferases and BIRC5. Data SKOV3/DDP and A2780/DDP cells in comparison to their
from RT- qPCR and Western blotting analysis showed respective control cells (Figure 4C).
that, upon overexpression of the methyltransferases, To investigate the role of METTL3 in modulating
only METTL3 consistently influenced the expression of BIRC5 in OC chemoresistance and malignant progression,
|

F I G U R E 3 Upregulation of BIRC5 promotes cisplatin resistance of OC cells. (A) SKOV3 and A2780 cells transfected with
overexpressed-BIRC5 vector, and the effect were tested by RT-qPCR and Western blotting. (B–D) CCK8 and colony formation assay revealed
the cell viability of overexpression BIRC5 cells treated with 20 μM cisplatin, and the sensitivity to cisplatin of overexpressed BIRC5 cells
measured by IC50. (E,F) Flow cytometry assays were performed to observe the change of percentage of apoptosis cells (early + late) after
upregulating BIRC5, and the expression level of apoptosis biomarker PARP was detected by Western blotting. The mean ± SD of triplicate
experiments was plotted (n = 3), *p < 0.05.

we suppressed METTL3 expression using shRNA common motif of these peaks were identified as DRACH
(shMETTL3) in SKOV3/DDP and A2780/DDP cells by the RMBase website (Figure 4H). This finding suggested
(Figure 4D). Both RT-qPCR and Western blotting anal- that METTL3 might modulate target gene expression via
ysis revealed that METTL3 positively influenced the ex- methylation modification in OC. This mechanism could
pression of BIRC5 in DDP-resistant OC cells (Figure 4E). be a primary factor for the elevated BIRC5 expression in
Additionally, the depletion of METTL3 notably reduced chemotherapy-resistant OC cells relative to control cells.
m6A modification levels (Figure 4F).
To further confirm the regulatory role of METTL3 in
OC chemoresistance, we used the SRAMP tool (http:// 3.4 | IGF2BP1 promotes OC patients'
www.cuilab.cn/sramp/) to predict m6A modification sites chemotherapy resistance
on BIRC5. The tool highlighted that BIRC5 possessed mul-
tiple m6A sites (Figure S3). MeRIP-seq analysis of m6A According to recent research, the IGF2BP protein family
modifications in control and METTL3-depleted SKOV3/ has the ability to bind m6A-modified mRNA, which plays
DDP cells showed that m6A peaks were predominantly a pivotal role in RNA stability. We utilized m6A2Target
situated near the 3′-UTR (Figure 4G). Furthermore, the (http://m6a2target.canceromics.org/) to predict potential
|

F I G U R E 4 BIRC5 is regulated by METTL3-mediated m6A modification. (A) RNA extracted in SKOV3, SKOV3/DDP, A2780 and
A2780/DDP, and m6A status was examined by 450 nm absorbance according to m6A methylation assay kit manuscript (Abcam, USA).
(B) Overexpression of predicted writers and measure the expression of BIRC5 in mRNA and protein level. (C) The mRNA and protein
expression level of METTL3 in OC cells. (D–F) The effect of silenced METTL3, with the decreased BIRC5 expression level and m6A level. (G)
MeRIP-seq confirmed the m6A peak location. (H)Predicted binding motif of METTL3 on 3′UTR of BIRC5 mRNA from RMBase website. The
mean ± SD of triplicate experiments was plotted (n = 3), *p < 0.05.

targets, as illustrated in Figure S4. To validate the regu- in SKOV3/DDP and A2780/DDP cells compared to con-
latory relationship with BIRC5, we overexpressed reader trol cells (Figure 5B).
proteins in both SKOV3/DDP and A2780/DDP cells. Our We conducted an RNA immunoprecipitation (RIP)
findings demonstrated that upregulating IGF2BP1 sig- assay in DDP- resistant OC cells to confirm the in-
nificantly augmented the expression of BIRC5 at both teraction between IGF2BP1 and BIRC5 mRNA. Our
the mRNA and protein levels (Figure 5A; Figure S5). results indicated that BIRC5 mRNA was distinctly en-
Moreover, there was a notable upregulation of IGF2BP1 riched when using the anti- IGF2BP1 antibody. This
|

F I G U R E 5 IGF2BP1 promotes OC patients' chemotherapy resistance. (A) BIRC5 expression level in mRNA and protein after
overexpressed “readers”. (B) The expression of IGF2BP1 in cisplatin-resistant cells and counterpart cells. (C–D) RIP assay was used to
detect the ability of IGF2BP1 binding to BIRC5. (E,F) Point mutation in m6A modification motif with lower luciferase reporter activities,
and mutated BIRC5 with silenced IGF2BP1 was represented to explore the m6A roles in BIRC5 expression. The mean ± SD of triplicate
experiments was plotted (n = 3), *p < 0.05, ns was not statistically significant.

enrichment was markedly reduced upon METTL3 de- Mut construct exhibited a pronounced decrease in lucifer-
pletion (Figure 5C,D). ase reporter activity. This finding suggested that IGF2BP1
To decipher the role of IGF2BP1 in cisplatin resistance, played a crucial role in stabilizing mRNAs with WT se-
we carried out luciferase assays. The luciferase activity of quences. Furthermore, when comparing the Mut to the
wild-type (WT) or mutant (Mut) 3′UTR sequences was an- WT, the luciferase activity in the control group showed
alyzed in SKOV3/DDP and A2780/DDP cells. Compared a decrease. However, upon silencing IGF2BP1, there was
to cells with the WT construct, those transfected with the no significant change in luciferase activity, indicating that
|

the primary binding sites were site 1 and site 2 (Figure 5E; A2780/DDP cells with sh- IGF2BP1. The silencing of
Figure S6). IGF2BP1 led to a marked reduction in both mRNA and
protein expression levels of BIRC5 (Figure 6A). Through
CCK8 and colony formation assays, we found that the
3.5 | METTL3/IGF2BP1/BIRC5 axis is downregulation of IGF2BP1 significantly increased
essential for cisplatin resistance of OC the sensitivity of the cells to cisplatin and reduced the
IC50 value in both SKOV3/DDP and A2780/DDP cells
To further investigate the role of IGF2BP1 in OC chem- (Figure 6B–D). To corroborate these findings, we con-
otherapy resistance, we transfected SKOV3/DDP and ducted an RNA decay assay. SKOV3/DDP and A2780/

F I G U R E 6 METTL3/IGF2BP1/BIRC5 axis is essential for cisplatin resistance of OC. (A) The expression level of BIRC5 in silenced
IGF2BP1 OC cells. (B–D) Cell viability detected by CCK8, the sensitivity of cisplatin detected by IC50, and the clonogenic ability in silenced
IGF2BP1 OC cells (left panel), quantification of the colony formation assay results (right panel). (E) The mRNA expression of BIRC5 was
measured by RT-qPCR in SKOV3/DDP and A2780/DDP, who overexpressed or silenced IGF2BP1 upon actinomycin D (ActD) treatment for
8 h. (F–I) Nude mice bearing SKOV3/DDP cells treated with shBIRC5, or shBIRC5 combined with pcDNA-IGF2BP1 plasmid (IGF2BP1) or
corresponding negative control, and after 7 days the mice treated with cisplatin (10 mg/kg). Tumor volume were measured and record 4 days
after injection. Tumor were removed after 28 days, and their weights had significantly differences. The protein expression of PARP was
measured by Western blotting. The mean ± SD of triplicate experiments were plotted (n = 3), *p < 0.05,**p < 0.01.
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DDP cells were treated with actinomycin D, an RNA mice, corroborated these findings. Notably, we observed
transcription inhibitor, at different intervals. Results distinct differences in tumor weight and volume among
indicated that BIRC5 mRNA remained highly stable the control group, the BIRC5 knockdown group, and the
upon IGF2BP1 overexpression. Conversely, a decrease group with BIRC5 knockdown combined with IGF2BP1
was observed in the cells where IGF2BP1 was depleted overexpression.
(Figure 6E). BIRC5, also known as survivin, belongs to the IAP
To further explore the roles of the METTL3/IGF2BP1/ family. It encodes proteins that act as negative regula-
BIRC5 axis in OC in vivo, we subcutaneously implanted tors, inhibiting cell death. Aberrant expression of BIRC5
BALB/c nude mice with SKOV3/DDP cells. These cells has been documented in various malignancies. This ex-
were stably transduced with either a lentiviral vector con- pression has been directly linked to aspects of cancer
taining a negative control sequence (LV-vector), a shBIRC5 progression such as tumor growth, invasion, metastasis,
sequence (LV-shBIRC5), or a combination of lentivirus poor prognosis, immune infiltration, and resistance to
containing IGF2BP1 and shBIRC5 (LV- shBIRC5 + LV- chemotherapy.22,23 BIRC5 is central to several signaling
IGF2BP1). After 7 days incubation, three groups of mice pathways. Consequently, understanding the upstream
received 10 mg/kg cisplatin respectively, and we measured molecules that regulate its expression and function has
the xenograft volumes in mice every 4 days. Our observa- become a focal point in cancer therapy research. These
tions indicated that the depletion of BIRC5 substantially regulatory molecules encompass a wide range, includ-
reduced the tumor volume. However, this effect could ing miRNAs, transcription factors, binding proteins, and
be offset by the co-treatment involving IGF2BP1 over- protein regulators. For instance, there is emerging evi-
expression (Figure 6F,G). After sacrificing the mice, we dence suggesting that USP1 enhances BIRC5 stabilization
assessed the tumor weights, which aligned with our vol- through ubiquitin, which significantly prevents apopto-
ume findings (Figure 6H). Furthermore, tumors treated sis induced by ML323 and TRAIL.24 Suppression of both
with LV- shBIRC5 exhibited elevated PARP protein ex- Mcl-1 and BIRC5 has been shown to efficiently curtail cell
pression—a well-known marker for apoptosis. This effect growth and increase drug sensitivity.25 In the context of
was mitigated upon co-treatment with LV-IGF2BP1 over- OC, studies have indicated that miR-203 targets BIRC5,
expression (Figure 6I). Collectively, these results under- suppressing OC metastasis.26 Furthermore, low-dose ra-
scored that IGF2BP1 contributed to cisplatin resistance diation has been found to reverse cisplatin resistance by
by modulating BIRC5 mRNA stability, emphasizing the down-regulating BIRC5.27 Although BIRC5 plays an in-
importance of the METTL3/IGF2BP1/BIRC5 axis in OC strumental role in chemoresistance, its precise underlying
cisplatin resistance. mechanism remains elusive.
Cisplatin, known for its potent antitumor efficacy, is
a primary chemotherapeutic drug for OC. However, the
4 | DI S C USSION onset of chemoresistance poses a significant challenge for
OC treatment. Cisplatin binds to the DNA of cancer cells,
We uncovered the potential role and function of BIRC5 facilitating both intra-chain and inter-chain cross-linking.
in cisplatin resistance in OC through an m6A-dependent When damage from this process is irreparable, it induces
mechanism. The resistance to chemotherapy remains apoptosis. Chemoresistance mechanisms vary based on
a significant hurdle in OC treatment, making it crucial their sites and timeframes and can be categorized as pre-
to comprehend its underlying mechanisms. While prior target, target, post-target, and off-target drug resistance.
research has highlighted the upregulation of BIRC5 in Among these, epigenetic modifications form part of
various tumors,19–21 our study delved deeper to show that the off-target drug resistance mechanisms. These mod-
BIRC5 regulation was associated with RNA methylation ifications span DNA, RNA, and protein adjustments.
involving specific methyltransferases. Depleting BIRC5 Notably, RNA methylation has emerged as a focal point
not only heightens sensitivity to cisplatin but also esca- in recent research. m6A, known for its role in post-
lates the rate of cisplatin-induced apoptosis. transcriptional regulation, has diverse functions in
Bioinformatics analyses indicated that BIRC5 had cancer progression.28,29 Many studies have extensively
abundant m6A binding sites, pinpointing METTL3 and explored METTL3 across various cancer types. METTL3,
IGF2BP1 as potential regulators. Utilizing methods like an m6A “writer”, can independently or collaboratively
meRIP-seq, RIP assays, and luciferase assays, we vali- regulate aspects of cancer, such as growth, invasion,
dated that METTL3 and IGF2BP1 could indeed modulate metastasis, and drug resistance.30–33 For instance, it's
the expression level of BIRC5 and its RNA stability in an been reported that METTL3 partners with YTHDF1 to
m6A-dependent manner. Moreover, in vivo experiments, boost hepatocellular carcinoma progression,34 triggers
wherein tumors were transplanted into BALB/c nude the PI3K/AKT signaling pathway to enhance prostate
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cancer growth,35 and plays roles in chemoresistance ETHICS STATEMENT

across various cancers like oral cancer,36 acute myeloid Written informed consent was obtained from all the par-
leukemia,37 and more. ticipants prior to the enrollment of this study. And all ex-
In this study, using MeRIP-seq and RIP assays, we periments were conducted in accordance with the ethical
determined that METTL3 activated BIRC5, leading to standards of the ethics committee of The Second Affiliated
increased expression levels and decreased cisplatin sensi- Hospital, Hainan Medical University.
tivity. The further analysis predicted potential RBPs bind-
ing to BIRC5. Subsequent tests, including RIP and dual ORCID
luciferase reporter assays, identified IGF2BP1 as a stabi- Yadan Fan https://orcid.org/0000-0002-5592-904X
lizer of BIRC5 mRNA. Recognized as a “reader” of RNA- Haohao Huang https://orcid.org/0000-0003-3787-4302
binding proteins, our research substantiated IGF2BP1's
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