Abstract

   Endometrial cancer (EC) is a prevalent gynecological malignancy
   worldwide, and 5-methylcytosine (m^5C) modification of mRNA is a
   crucial epigenetic modification associated with the development and
   occurrence of several cancers. However, the precise function of m^5C
   modification in EC remains elusive. This study aimed to investigate the
   expression and clinical significance of the primary m^5C modification
   writer, NSUN2, in EC. Our findings indicated that NSUN2 exhibited a
   substantial up-regulation in EC as a result of an epigenetic
   augmentation in H3K4me3 levels within the promoter region, which was
   triggered by the down-regulation of KDM5A. Moreover, gain- and
   loss-of-function experiments revealed the role of NSUN2 in enhancing
   m^5C modification of mRNA, thereby promoting EC cell proliferation. RNA
   bisulfite sequencing and transcriptomic sequencing were employed to
   elucidate the involvement of NSUN2 in the regulation of ferroptosis.
   Subsequent in vitro experiments confirmed that the knockdown of NSUN2
   significantly up-regulated the levels of lipid peroxides and lipid ROS
   in EC cells, thereby augmenting the susceptibility of EC to
   ferroptosis. Mechanistically, NSUN2 stimulated the m^5C modification of
   SLC7A11 mRNA, and the m^5C reader YBX1 exhibited direct recognition and
   binding to the m^5C sites on SLC7A11 mRNA via its internal cold shock
   domain (CSD), leading to an increase in SLC7A11 mRNA stability and
   elevated levels of SLC7A11. Additionally, rescue experiments showed
   that NSUN2 functioned as a suppressor of ferroptosis, which was
   dependent on SLC7A11. Overall, targeting the NSUN2/SLC7A11 axis
   inhibited tumor growth by increasing lipid peroxidation and ferroptosis
   of EC cells both in vitro and in vivo. Therefore, our study provides
   new insight into the role of NSUN2, suggesting that NSUN2 may serve as
   a prognostic biomarker and therapeutic target in patients with EC.

   Keywords: NSUN2, SLC7A11, m^5C, Endometrial cancer, Ferroptosis

1. Introduction

   Endometrial Cancer (EC) is a gynecological tumor originating from the
   endometrium, and its incidence rate worldwide is steadily increasing
   [[37]1]. Approximately 90 % of ECs are detected early and have an
   excellent 5-year survival rate because surgery alone or in combination
   with local therapy is generally curative [[38]2]. However, the
   prognosis remains poor for patients with recurrent EC, as the optimal
   adjuvant therapy is yet to be established [[39]3]. Considering the
   unsatisfactory treatment results in recurrent EC patients, there is a
   great need to further explore the mechanisms underlying EC initiation
   and progression and to develop innovative treatment modalities.

   More than 100 forms of RNA modifications have been identified,
   including N6-methyladenosine (m^6A), the most abundant internal mRNA
   modification that has been shown to play an essential role in cell
   activities and functions [[40]4]. There is also evidence that
   5-methylcytosine (m^5C) regulates RNA metabolism. m^5C is primarily
   found in tRNAs and rRNAs but has also been identified in mRNAs and
   other noncoding RNAs [[41]5,[42]6]. Similar to m^6A methylation, m^5C
   levels of RNAs are also regulated by a series of “writer”, “eraser”,
   and “reader” proteins. The m^5C methyltransferases consist mainly of
   NOL1/NOP2/sun (NSUN) family (NSUN1-7) and DNA methyltransferase 2
   (DNMT2), which act as “writers” to catalyze the methylation process
   [[43]7]. In comparison, alpha-ketoglutarate-dependent dioxygenase ABH1
   (ALKBH1) and ten-eleven translation family proteins (TET) act as
   demethylase (“Eraser”), which remove m^5C modifications from RNAs
   [[44]8,[45]9]. The ultimate fate of m^5C methylated RNAs depends on the
   “readers”, such as the Aly/REF export factor (ALYREF) and Y-box-binding
   protein 1 (YBX1). m^5C readers can recognize m^5C-modified sites, which
   may affect mRNA stability, translation, transcription, nuclear export,
   and cleavage of RNAs [[46]10,[47]11]. Recently, m^5C modification of
   RNA was found to play a significant role in tumourigenesis and cancer
   progression [[48][11], [49][12], [50][13]]. However, the biological
   significance of m^5C and its underlying regulatory mechanisms in EC
   remain elusive.

   Among all the m^5C regulatory proteins, the m^5C writer NSUN2 has been
   thoroughly investigated. As a major mRNA methyltransferase, NSUN2 is
   mainly located in the nucleus and catalyzes the formation of
   5-methylcytosine in RNA [[51]10]. Growing evidence has shown that NSUN2
   is highly expressed in multiple cancer types and is involved in various
   biological functions such as tumourigenesis [[52]14], cell
   proliferation [[53]12,[54]15], tumor invasion [[55]11,[56]16], and
   cellular differentiation [[57]13]. Sun et al. revealed through
   multi-omics analysis that NSUN2 is significantly upregulated in
   prostate cancer, indicating poor prognosis. Moreover, pathway
   enrichment suggests a relationship between NSUN2 and signaling pathways
   associated with endometrial cancer [[58]17]. Wang et al. discovered a
   significant upregulation of NSUN2 in cervical cancer, noting that
   silencing NSUN2 markedly inhibited the migration and invasion of
   cervical cancer cells. Additionally, they analyzed the expression
   levels of NSUN2 in early-stage endometrial cancer using expression
   profiling by array ([59]GSE17025) but did not observe any significant
   differences. However, this observation has not been validated in tissue
   samples [[60]18]. Furthermore, Yang et al. analyzed expression changes
   of RNA 5-Methylcytosine regulators in the TCGA database and identified
   NSUN2 as being significantly overexpressed in endometrial cancer (EC),
   suggesting its potential as a prognostic factor in EC [[61]19].
   However, a complete understanding of the involvement of NSUN2-mediated
   m^5C modification of mRNA in the pathogenesis of EC remains
   insufficient.

   This study aimed to investigate the mechanisms underlying the role of
   abnormal NSUN2-mediated mRNA m^5C modifications in promoting EC
   progression. We demonstrated for the first time that NSUN2 is
   overexpressed in EC and facilitates the malignant biological behavior
   of EC cells. Using mRNA bisulfite sequencing paired with transcriptome
   sequencing, we determined that NSUN2 is involved in the regulation of
   ferroptosis in EC. Further molecular biology experiments revealed a
   novel mechanism by which NSUN2 renders EC cells resistant to
   ferroptosis by regulating m^5C modification of SLC7A11 and enhancing
   its YBX1-dependent mRNA stability.

2. Materials and methods

2.1. Patients and sample collection

   All surgical specimens used in this study were obtained from the
   Department of Obstetrics and Gynecology at the Union Hospital of Tong
   Medical College (Wuhan, China) and were either immediately frozen in
   liquid nitrogen or fixed in poly-formalin after surgical excision. At
   least two experienced pathologists histopathologically confirmed the
   diagnosis of endometrial cancer. The institutional review board of
   Tongji Medical College at Huazhong University of Science and Technology
   granted ethical approval prior to the study implementation. All
   patients were informed, and written informed consent was obtained from
   the patients.

2.2. Cell culture and treatment

   The endometrial cancer cell lines used in this study, including HEC-1A,
   HEC-1B, KLE, Ishikawa, and RL95-2, were purchased from the American
   Type Culture Collection (ATCC) and cultured in media containing 10 %
   FBS according to the instructions of the manufacturer. The cells were
   cultured in a constant temperature incubator at 37 °C with 5 % CO2.
   Erastin (#S7242), Z-VAD-FMK (#S7023), 3-MA (#S2767), and Ferrostatin-1
   (Fer-1) (#S7243), were obtained from Selleck (Shanghai, China).

2.3. Vector construction and cell transfection

   To construct NSUN2, YBX1, SLC7A11, and KDM5A over-expression plasmids,
   human NSUN2 ([62]NM_017755.6), YBX1 ([63]NM_004559.5), SLC7A11
   ([64]NM_014331.4) and KDM5A ([65]NM_001042603.3) cDNAs were amplified
   by PCR and cloned into pcDNA 3.1 expression vectors (Shanghai, China).
   The small hairpin RNAs (shRNAs) of NSUN2 were synthesized and cloned
   into the PGMLVHU6-MCS-CMV-PGK-Puro vector by Genomeditech (Shanghai,
   China). Small interfering RNA (siRNA) targeting YBX1 and KDM5A genes
   were synthesized by Sangon Biotech (Shanghai, China). Transfections
   with siRNA (20 nM) were performed with RNATransMate (Sangon, #E607402)
   based on the manufacturer's instructions. Targeted sequences of shRNAs
   or siRNAs used in this study were listed in [66]Supplementary Table 1.

2.4. RNA extraction and RT-qPCR

   Total RNA from tissues and cell lines was extracted using TRIeasy Total
   RNA Extraction Reagent (Yeasen, #10606ES60) according to the
   manufacturer's protocol. Total RNA was reverse-transcribed into cDNA
   using ABScript III RT Master Mix for qPCR (Abclonal, #RK20428). RT-qPCR
   analysis was conducted on a CFX Connect Real-Time PCR Detection System
   (Bio-Rad, Hercules, CA, USA) using Universal SYBR Green Fast qPCR Mix
   (Abclonal, #RK21203). The housekeeping gene GAPDH was used as the
   internal reference gene for normalization. The primers used are listed
   in [67]Supplementary Table 2.

2.5. Western blot

   Protein extracts containing protease inhibitors (Bimake, #[68]B14001)
   were prepared using RIPA buffer (Servicebio, #G2002). Protein
   concentrations were measured using the BCA kit (Biosharp, #BL521A).
   Equal amounts of total protein were separated by SDS-polyacrylamide gel
   electrophoresis and transferred onto a PVDF membrane (Merck Millipore,
   ISEQ00010). The membrane was then blocked for 1 h with 5 % non-fat dry
   milk in 0.1 % TBST. The blots were incubated with primary antibodies on
   a shaker at 4 °C overnight and then incubated with secondary antibodies
   for 1 h at room temperature. After 3 × 5 min washes in TBST,
   chemiluminescent signals were detected using ECL detection reagent
   (Biosharp, #BL520A). Antibodies against the interest proteins are shown
   in [69]Supplementary Table 3.

2.6. Chromatin immunoprecipitation assays (ChIP)

   The cells were subjected to appropriate disposal procedures prior to
   conducting the ChIP assay, which was performed using the ChIP Assay Kit
   (Beyotime, #P2078) in accordance with the manufacturer's instructions.
   Immunoprecipitation was carried out using anti-H3K4me3 and anti-KDM5A
   antibodies, while normal rabbit IgG was utilized as a negative control.
   The primer for ChIP-qPCR targeting NSUN2 promoter is provided in
   [70]Supplementary Table 2.

2.7. CUT&Tag

   CUT&Tag was performed using a commercial kit (Vazyme, China, TD903)
   according to the manufacturer's protocol. In brief, a total of
   50,000 cells were collected and subjected to a washing procedure, after
   which they were incubated with magnetic beads coated with Concanavalin
   A. Subsequently, the cells were exposed to an anti-H3K4me3 primary
   antibody (Abcam, #ab12209) for a duration of 2 h, followed by an
   additional hour of incubation with a secondary antibody at room
   temperature. To facilitate tagmentation, the hyperactive pG-Tn5
   transposonase was introduced. The reaction was then halted, and the
   resulting DNA fragments were extracted using PCI. Finally, PCR
   amplification was performed utilizing indexed P5 and P7 primers. The
   library products underwent enrichment, quantification, and subsequent
   sequencing using a Novaseq 6000 sequencer (Illumina) with the PE150
   model. The CUT&Tag experiment, as well as high-throughput sequencing
   and data analysis, were performed by Seqhealth Technology Co., LTD
   (Wuhan, China).

2.8. RNA immunoprecipitation (RIP) and m^5C-mRNA immunoprecipitation

   The RIP experiment was carried out with the Magna RIP RNA-Binding
   Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according
   to the manufacturer's instructions. Cell lysates were incubated with
   5 μg of primary antibodies against NSUN2, YBX1, and FLAG. The controls
   used were mouse or rabbit IgG. m^5C-mRNA immunoprecipitation was
   performed according to a previous description [[71]14]. Briefly, mRNA
   was purified from total RNA using a Hieff NGS mRNA Isolation Master Kit
   (Yeasen, #12603ES24). Anti-m^5C antibody was mixed with 50 μL protein G
   magnetic beads (Thermo Fisher, #10007D) and incubated for 30 min at
   room temperature with rotation. Subsequently, the purified mRNA was
   mixed with the antibody-bead mixture and incubated on a rotator
   overnight at 4 °C. The mRNA bound to the antibody was isolated using
   proteinase K (Beyotime, # ST532), followed by an additional round of
   purification with RNA Clean Magnetic Beads (Beyotime, #R0081), and the
   purified mRNA was then subjected to reverse transcription and
   quantitative PCR analysis.

2.9. Dot blot assays for m^5C

   The total RNA of EC cell lines was subjected to denaturation at 95 °C
   for 3 min, followed by cooling on ice. Equal amounts of RNA were
   spotted on a positively charged nylon membrane (Biosharp, #BS-NY-45),
   crosslinked with UV for 3 min at 120 mJ/cm2, and then washed with
   buffer to remove unbound RNA. After blocking with 5 % non-fat milk, the
   membrane was incubated with m^5C antibody overnight at 4 °C. The
   membrane was washed three times with buffer and incubated with a
   secondary antibody. Finally, chemiluminescence was used for detection,
   and methylene blue staining was used as a loading control.

2.10. RNA-bisulfite sequencing

   The total RNA of EC cell lines was extracted using Trizol, and the RNA
   samples were fragmented to lengths suitable for sequencing.
   Subsequently, EZ RNA Methylation Kit (Zymo Research, CA, USA) was used
   for Bisulfite treatment of RNA, followed by RNA library construction
   using KC UMI RNA Library Kit (Seqhealth, Wuhan, China), which was
   subjected to high-throughput sequencing. The raw FASTQ format
   sequencing data was quality controlled, the Clean Reads were aligned to
   the reference genome using Bismark (v0.22.3) software, and duplicate
   reads were removed. Bismark software was used to identify the
   methylation sites of cytosine (C). The detected methylation sites'
   corresponding methylation levels were calculated by mC/(mC + umC).
   Differentially methylated regions (DMRs) are regions where the
   methylation level between samples exhibits significant differences. The
   detection of DMRs between the two groups of samples was conducted using
   metilene software (v0.2-8) [[72]20], with filtering parameters of
   methylation difference greater than 0.1 and P-value less than 0.05. A
   complete list of differentially methylated genes was provided in
   [73]Supplementary Table 4.

2.11. RNA sequencing

   Total RNAs were extracted from EC cell lines using TRIzol Reagent
   (Invitrogen, c#15596026). RNA quality was evaluated by examining
   A260/A280 with Nanodrop OneCspectrophotometer. A total of 2 μg RNAs
   were used for stranded RNA sequencing library preparation using KCTM
   Stranded mRNA Library Prep Kit following the manufacturer's
   instructions. On the DNBSEQ-T7 sequencer (MGI Tech Co., Ltd. China),
   200–500 bps PCR products were enriched, quantified, and sequenced.
   Clean data were mapped to the hg38 human reference genome using STRA
   software (version 2.5.3a) with default parameters. Reads mapped to the
   exon regions of each gene were counted by featureCounts (Subread-1.5.1;
   Bioconductor), and then RPKMs were calculated. Differentially expressed
   genes between groups were identified using the edgeR package (version
   3.12.1). A p-value cutoff of 0.05 and a fold-change cutoff of 2 were
   used to judge the statistical significance of gene expression
   differences. A complete list of differentially expressed genes was
   provided in [74]Supplementary Table 5. RNA-Bisulfite sequencing and RNA
   sequencing experiments were conducted by Seqhealth Technology Co.
   (Wuhan, China).

2.12. RNA stability assay

   The EC cell lines were evenly seeded in a 6-well plate and treated with
   5 μg/mL actinomycin D (ActD) (Selleck, #S8964) for 0, 2, 4, 6, and 8 h,
   respectively. The collected cells were used for total RNA extraction
   and quantitative PCR detection of mRNA levels of target genes. The mRNA
   half-life was calculated based on the previously described method
   [[75]21].

2.13. RNA pull-down

   Biotin-labeled RNA fragments (50 pmol) containing SLC7A11 RNA sequences
   with (oligo-m^5C) or without m^5C modification at the m^5C site (chr4:
   138,242,036) were synthesized from Sangon (Shanghai, China;
   [76]Supplementary Table 2). RNA pull-down assays were performed using
   the Pierce™ Magnetic RNA-Protein Pull-Down Kit (20,164, Thermo
   Scientific), according to the manufacturer's instructions. The
   resulting RNA-protein complexes were further analyzed by immunoblotting
   or mass spectrometry.

2.14. Silver staining and mass spectrometry (MS) analysis

   After a duration of 75 min of electrophoresis at a voltage of 120 V,
   the gel underwent silver staining using the Fast Silver Stain Kit
   (P0017S, Beyotime) in accordance with the instructions provided by the
   manufacturer. The process of mass spectrometry (MS) was carried out by
   SpecAlly Life Technology Co., Ltd. (Wuhan, China). To summarize, the
   complexes bound to immobilized magnetic beads were subjected to
   cleaning and digestion using sequencing-grade modified trypsin.
   Following extraction and purification, the peptide samples were
   identified through mass spectrometry using the Q Exactive instrument
   manufactured by Thermo Finnigan in the United States. The acquired raw
   mass spectrometric data were analyzed using MaxQuant software
   (V1.6.2.10).

2.15. Transmission electron microscope (TEM)

   For the observation of the ultrastructure of cells, a TEM was used. The
   EC cells were collected and resuspended in TEM fixative solution at
   4 °C for 2–4 h. Then, the cells were pre-embedded in agarose, and 2 h
   of fixation was performed at room temperature using 1 % glutaraldehyde.
   After gradient alcohol dehydration, the samples were infiltrated and
   polymerized using a solution of propylene oxide and epoxy resin.
   Ultrathin sections were prepared and stained before observation using
   an HT7800 TEM (HITACHI). Images were collected for analysis.

2.16. Tumor xenograft model

   Four-week-old female nude mice were purchased from Vital River
   (Beijing, China) and raised in a separate ventilated SPF-level animal
   facility. Tumor cell suspension (1 × 10^6/point) was subcutaneously
   injected into the backs of the nude mice. Tumor size was measured every
   four days using a caliper. After four weeks, the mice were euthanized,
   and the xenograft tumors were harvested, weighed, and measured for
   volume. The xenograft tumors were fixed with formalin and embedded in
   paraffin for subsequent IHC staining.

2.17. Malondialdehyde (MDA) measurement

   The relative MDA content in the cell lysate was measured using a Lipid
   Peroxidation Assay Kit (Beyotime, #S0131S) according to the
   manufacturer's instructions. Briefly, EC cells were lysed using lysis
   buffer (Beyotime, #P0013) and centrifuged at 12,000 g for 10 min. A
   total of 100 μL supernatant was mixed with 200 μL MDA working solution
   and incubated at 100 °C for 15 min, followed by cooling to room
   temperature. The absorbance value at 532 nm was then measured.

2.18. Lipid reactive oxygen species (ROS) measurement

   EC cells were seeded in a six-well plate before treatment. After
   removing the culture medium, the cells were incubated with 5 μM
   C11-BODIPY (Thermo Fisher Scientific, #D3861) in HBSS at 37 °C for
   30 min. After washing the cells twice with PBS, trypsin was used to
   digest the cells, followed by centrifugation. The cells were
   resuspended in a serum-free medium for flow cytometric analysis
   (DxFLEX, Beckman Coulter, USA), and data were analyzed using FlowJo
   software (TreeStar).

2.19. Immunohistochemistry (IHC)

   The surgically resected tissues were immediately fixed with 4 %
   paraformaldehyde and embedded in paraffin. For immunohistochemical
   analysis, the tissues were dewaxed, dehydrated, and rehydrated,
   followed by overnight incubation with primary antibodies at 4 °C. The
   tissues were then incubated with secondary antibodies the next day, and
   DAB was used for chromogenic detection. Images were captured using a
   microscope equipped with a digital camera. The immunohistochemical
   score (IHC score) was defined as the product of staining intensity and
   staining area, where the staining intensity was scored as negative (0),
   weak (1), moderate (2), or strong (3), and staining area was scored as
   follows: 0 = no positive cells; 1 = <10 % of positive cells;
   2 = 10–50 % positive cells; 3 = 51–80 % positive cells; 4 = >80 %
   positive cells.

2.20. Cell proliferation and viability assays

   Cell proliferation assays were performed as previously described
   [[77]22]. Cell viability was evaluated using a CCK8 Cell Counting Kit
   (Topscience, Shanghai, China). In brief, 5 × 10^3 EC cells were seeded
   in a 96-well plate before treatment. After removing the culture medium,
   100 μL complete medium containing 10 μL of CCK8 reagent was added and
   incubated at 37 °C for 2 h in a CO2 incubator. The absorbance at 450 nm
   was measured, and the cell viability was calculated.

2.21. Statistical analysis

   Data are expressed as mean ± SD. The experiments were repeated at least
   three times with a minimum of three technical repeats. Statistical
   significance was determined with students' t-test. The Kaplan-Meier
   method was used to calculate OS. P-values less than 0.05 were
   considered statistically significant. Statistical analyses were
   performed using Prism 8.1.2 (GraphPad Software Inc.).

3. Results

3.1. Elevated NSUN2 expression correlates with poor prognosis in patients
with EC

   To investigate the potential role of m^5C modification in EC
   progression, we preliminarily analyzed the expression of critical m^5C
   regulatory genes in TCGA UCEC dataset. We found that NSUN2, which
   catalyzes mRNA m^5C, was significantly upregulated ([78]Fig. 1A,
   [79]Supplementary Figs. S1A and B). We also compared the mRNA levels of
   NSUN2 in paired EC tissues and adjacent normal tissues in the TCGA
   database, which suggested high NSUN2 expression in EC ([80]Fig. 1B).
   Moreover, based on public data from the CPTAC, we found higher protein
   expression of NSUN2 in EC than in normal endometrial tissues ([81]Fig.
   1C). RT-qPCR and western blotting were employed to assess the mRNA and
   protein levels of NSUN2 in surgically resected specimens, which
   confirmed the overexpression of NSUN2 in EC tissues compared to normal
   tissues ([82]Fig. 1D and E). Immunohistochemistry revealed that NSUN2
   was highly expressed in EC tissues and predominantly located in the
   nucleus ([83]Fig. 1F). We evaluated the prognostic significance of
   NSUN2 by analyzing clinicopathological factors and found that the
   expression levels of NSUN2 in EC were closely related to FIGO stage,
   histological grade, and pathological type ([84]Fig. 1G–I). Moreover,
   patients with high expression of NSUN2 showed a much worse prognosis
   ([85]Fig. 1J). Collectively, these findings demonstrated that NSUN2 is
   often upregulated in EC, making it a potential prognostic indicator.

Fig. 1.

   [86]Fig. 1
   [87]Open in a new tab

   NSUN2 overexpression predicts poor prognosis in endometrial cancer. (A)
   The mRNA levels of NSUN2 were compared between EC and normal tissues in
   TCGA UCEC dataset. (B) Different mRNA expression of NSUN2 between EC
   and the paired normal tissues. (C) The protein expression levels of
   NSUN2 based on the CPTAC database. (D) RT-qPCR analysis displaying the
   mRNA levels of NSUN2 in surgical EC and normal samples. (E) Western
   blot was used to detect the protein levels of NSUN2 in EC and paired
   normal tissues. (F) Representative images of immunohistochemical
   staining for NSUN2 and the comparison of the ISH scores in EC and
   normal tissues. The expression levels of NSUN2 were presented in
   different FIGO stages (G), histological grade (H), and pathological
   type (I). (J) Kaplan-Meier (KM) curve for OS of patients with UCEC in
   TCGA and patients were stratified into low and high expression based on
   the median NSUN2 expression. *P < 0.05; **P < 0.01; ***P < 0.001.

3.2. NSUN2 upregulation is induced by the epigenetic alteration of H3K4me3

   Post-translational modifications (PTMs) of histones play essential
   roles in the regulation of gene expression in cancer cells [[88]23].
   Consequently, we used the WashU Epigenome Browser and the Cistrome Data
   Browser to explore whether NSUN2 is regulated by epigenetic mechanisms
   [[89]24,[90]25], which discovered significant enrichment of H3K4me3 in
   the promoter region of NSUN2 ([91]Fig. 2A). Gene promoters enriched for
   H3K4me3 are generally associated with transcriptional activation
   [[92]26], suggesting that elevated NSUN2 might be regulated by histone
   methylation. Moreover, chromatin immunoprecipitation (ChIP) assays were
   performed to further determine histone methylation levels in EC cells,
   and the ChIP-PCR results indicated that histone H3 lysine 4 (H3K4)
   trimethylation (H3K4me3) was significantly enriched in the promoter
   region of NSUN2 ([93]Fig. 2B). To yield more insight into the
   underlying mechanisms, we analyzed the expression of lysine methylases
   and demethylases of H3K4me3 in TCGA UCEC dataset. The findings indicate
   that the decreased expression of KDM5A may be the primary factor
   contributing to the elevated levels of H3K4me3 at the NSUN2 promoter
   region ([94]Supplementary Fig. S2A). We then used siRNA technology to
   knockdown endogenous KDM5A expression in EC cells and found that the
   mRNA level of NSUN2 increased significantly ([95]Fig. 2C). Furthermore,
   Western blot results also indicated that KDM5A knockdown led to an
   increase in NSUN2 protein levels and H3K4me3 levels, whereas
   over-expression of KDM5A produced the opposite result ([96]Fig. 2D,
   [97]Supplementary Figs. S2B and C). Consistently, ChIP-PCR was
   performed in KDM5A-overexpressed EC cells, and the results suggested
   that the overexpression of KDM5A resulted in a significant decrease of
   H3K4me3 enrichment in the NSUN2 promoter region ([98]Fig. 2E).
   Similarly, we also found the enrichment of KDM5A in the NSUN2 promoter
   region in the Cistrome Data Browser, and ChIP-PCR assay using KDM5A
   antibody also confirmed that KDM5A bound the NSUN2 promoter in EC
   ([99]Fig. 2F and G). Moreover, we proceeded to suppress KDM5A in EC
   cell lines and conducted CUT & Tag analysis. The findings indicated
   that diminishing KDM5A levels can augment the accumulation of H3K4me3
   in the promoter region ([100]Fig. 2H). Additionally, the IGV map
   reveals an enhanced signal of H3K4me3 enrichment in the NSUN2 promoter
   region subsequent to KDM5A knockdown([101]Fig. 2I). Taken together, the
   above results further revealed that NSUN2 upregulation occurs because
   of an increase in H3K4me3 mediated by a decrease in KDM5A.

Fig. 2.

   [102]Fig. 2
   [103]Open in a new tab

   NSUN2 upregulation is regulated by the epigenetic alteration of
   H3K4me3. (A) ChIP-seq data for mapping H3K4me3 modifications near the
   NSUN2 promoter in the different cell lines were obtained from the
   Cistrome Data in the WashU Epigenome Browser. (B) ChIP-PCR analysis of
   H3K4me3 modification in the promoter of NSUN2. (C) The mRNA levels of
   NSUN2 were detected by qPCR after the knockdown of KDM5A. (D) Protein
   expression in HEC-1B or Ishikawa cells with overexpression or silencing
   of KDM5A was assessed using Western blot; GAPDH and H3 were used as the
   loading control. (E) ChIP-PCR analysis of H3K4me3 modification of the
   NSUN2 promoter in KDM5A overexpression EC cells. (F) KDM5A ChIP-seq of
   different cell lines at NSUN2 promoter is displayed using the WashU
   Epigenome Browser. (G) ChIP-PCR analysis of KDM5A in the promoter of
   NSUN2. (H) Heatmaps illustrating H3K4me3 levels around gene body
   regions. (I) IGV tracks presenting the enrichments of H3K4me3 by
   CUT&Tag. ***P < 0.001.

3.3. NSUN2 deposits m^5C on mRNA and promotes EC proliferation in vitro

   To further demonstrate the regulatory effects of NSUN2 on tumor
   progression in vivo, we evaluated the mRNA and protein expression of
   NSUN2 in different EC cell lines and in normal endometrium (NE).
   Increased NSUN2 levels were observed in cancer cell lines in contrast
   to primary normal cells ([104]Fig. 3A and B), and HEC-1B, KLE, and
   Ishikawa cell lines were selected for subsequent studies. We silenced
   NSUN2 in HEC-1B and KLE cells using shRNA knockdown and overexpressed
   NSUN2 in Ishikawa cells using NSUN2 expression vectors
   ([105]Supplementary Figs. S3A–C). We purified mRNA from all three EC
   cell lines transfected with the indicated vectors and performed dot
   blot analysis with an antibody recognizing m^5C, which demonstrated
   that NSUN2 knockdown resulted in reduced m^5C levels, whereas NSUN2
   overexpression resulted in an increased level of m^5C mRNA ([106]Fig.
   3C). Subsequently, a series of experiments were conducted to assess the
   effect of NSUN2 on EC cell proliferation, including CCK-8, clone
   formation, and EDU assays. As shown in [107]Fig. 3D–F and
   [108]Supplementary Figs. S3D and E, NSUN2-knockdown robustly inhibited
   the proliferation of HEC-1B and KLE cells, whereas overexpression of
   NSUN2 promoted the proliferation of Ishikawa cells.

Fig. 3.

   [109]Fig. 3
   [110]Open in a new tab

   NSUN2 is involved in the regulation of m^5C levels and the
   proliferation ability of EC cells. (A, B) The mRNA and protein levels
   of NSUN2 in normal endometrium and five different EC cell lines. (C)
   The m^5C levels of total RNA in NSUN2 knockdown HEC-1B and KLE cells or
   NSUN2 overexpression Ishikawa cells were indicated by m^5C dot blot.
   (D–F) CCK-8, colony formation, and EdU assays were carried out to
   detect the proliferation ability of EC cells with NSUN2 knockdown or
   overexpression. *P < 0.05; **P < 0.01; ***P < 0.001.

   As the primary mRNA m^5C methyltransferase, it has been confirmed that
   mutations in the releasing (cysteine 271) and catalytic (cysteine 321)
   sites of NSUN2 completely abrogated its regulatory role on mRNA m^5C
   levels [[111]10]. Thus, we constructed wild-type and double-mutant
   NSUN2 overexpression plasmids and transfected them into EC cells with
   stable knockdown of NSUN2. In comparison with wild-type NSUN2, the
   mutant NSUN2 failed to restore the proliferation ability of EC cells
   ([112]Supplementary Figs. S4A–C). Collectively, these results indicate
   that NSUN2 facilitates EC proliferation in vitro via m^5C modification.

3.4. NSUN2 stimulates m^5C hypermethylation in EC

   As NSUN2 is a major mRNA methyltransferase, we performed bisulfite
   sequencing (BS-seq) of mRNA derived from HEC-1B cells transfected with
   control or sh-NSUN2 vectors ([113]Fig. 4A). As shown in [114]Fig. 4B,
   NSUN2 knockdown resulted in a considerable reduction in m^5C
   modification sites. A higher frequency of C[A/C]GGGG downstream of the
   m^5C site was observed in the probability sequence context generated
   using WebLogo ([115]Fig. 4C). The distribution density map of m^5C
   sites on the mRNA of HEC-1B was drawn, and it indicated that m^5C sites
   are located in the coding DNA sequences (CDS): 5′UTR and 3′UTR
   ([116]Fig. 4D). Differentially Methylated Regions (DMRs) are regions
   with significant differences in methylation levels between samples that
   may play an essential role in the regulation of gene expression. Using
   Metilene software, we identified differentially methylation-modified
   genes between the control and NSUN2 knockdown groups. The cut-off for
   significant DMR was |methylation changes|> 0.1 and p < 0.05 ([117]Fig.
   4E). Among these differentially methylated genes, there were more genes
   whose methylation levels were decreased by more than one after NSUN2
   knockdown ([118]Fig. 4F). Differentially methylated genes were also
   subjected to pathway enrichment analysis using KOBAS3.0, and
   significant enrichment for the oxidative stress response, ferroptosis,
   and iron uptake was observed ([119]Fig. 4G).

Fig. 4.

   [120]Fig. 4
   [121]Open in a new tab

   Identification of NSUN2-mediated regulation of m^5C-modified mRNAs. (A)
   Flow chart of the bisulfite sequencing. (B) The number of m^5C modified
   sites on different chromosomes in control or NSUN2 knockdown HEC-1B
   cells. (C) The sequence context of 10 bases upstream and downstream of
   NSUN2-m^5C sites is depicted in the probability pattern. (D)
   Distribution and methylation level of all m^5C sites in different
   segments of mRNAs. (E) The heatmap showed differentially methylated
   genes in HEC-1B cells with NSUN2 knockdown. The screening criteria were
   as follows: methylation difference greater than 0.1, P-value less than
   0.05. (F) The number distribution histogram of differentially
   methylated mRNAs. (G) Pathway enrichment analysis of differentially
   methylated genes.

3.5. The downregulation of NSUN2 renders EC susceptible to ferroptosis

   Considering that differentially methylated genes were enriched in
   ferroptosis-related pathways, we speculated that NSUN2 may participate
   in the regulation of ferroptosis in EC. Ferroptosis is characterized by
   iron-dependent cell death induced by cysteine depletion and massive
   lipid peroxidation [[122]27]. In previous studies, we found that
   significant ferroptosis resistance exists in EC cells and that
   targeting ferroptosis is a potential treatment for EC [[123]28].
   Functionally, NSUN2 knockdown significantly promoted erastin-induced
   cell death in a concentration-dependent manner ([124]Fig. 5A and B).
   Notably, Fer-1, an inhibitor of ferroptosis, rather than inhibitors of
   other forms of cell death such as apoptosis (Z-VAD-FMK) and autophagy
   (3-methylademine, 3-MA), suppressed the erastin-induced increase in
   cell death in HEC-1B and KLE cells with NSUN2 knockdown ([125]Fig. 5C
   and D). This indicated that NSUN2 is involved in the regulation of
   ferroptosis in EC cells. Similarly, EC cells were treated with erastin
   or DMSO for 12h and subjected to live or dead staining, which suggested
   that NSUN2 knockdown significantly increased erastin-induced cell death
   ([126]Fig. 5E). Furthermore, the relative intracellular MDA
   concentration and lipid ROS levels suggested that NSUN2 knockdown
   caused a robust increase in the extent of lipid peroxidation and lipid
   ROS ([127]Fig. 5F and G). Transmission electron microscope (TEM)
   analysis further revealed that NSUN2 knockdown EC cells exhibited
   typical morphological features of ferroptosis, including shrunken
   mitochondria with elevated membrane density ([128]Fig. 5H). Taken
   together, these observations support the conclusion that the
   downregulation of NSUN2 renders EC susceptible to ferroptosis.

Fig. 5.

   [129]Fig. 5
   [130]Open in a new tab

   NSUN2 knockdown promotes ferroptosis in EC. (A, B) CCK-8 assay was
   performed to determine the cell viability after treatment with
   different concentrations of erastin in HEC-1B or KLE cells. (C, D)
   Evaluation of cell viability after treatment with DMSO, erastin,
   erastin and Fer-1, erastin and Z-VAD, erastin and 3-MA in the indicated
   cells. (E) Calcein-AM (green)/PI (red) staining was performed to
   visualize living and dead cells, respectively. (F) Intracellular lipid
   peroxides MDA levels were measured with an MDA assay kit. (G) Lipid
   peroxidation was measured by flow cytometry after C11-BODIPY staining
   in the indicated cells. (H) Representative TEM images of mitochondria
   in control or NSUN2 knockdown EC cells. ***P < 0.001; ns, not
   statistically significant. (For interpretation of the references to