Abstract
Cerebral ischemia-reperfusion injury (CIRI) is common in ischemic
stroke and seriously affects the prognosis of patients. At present,
N^6-methyladenosine (m^6A) modification of lncRNAs and mRNAs has been
reported in other diseases, such as cancer, but its role in CIRI has
not been clarified. In this study, we aimed to investigate the m^6A
lncRNA and m^6A mRNA modification profiles in CIRI. First, we detected
the total level of m^6A and the changes in related m^6A
methyltransferases and demethylases in the brain tissue of rats with
CIRI and then identified differentially modified lncRNAs and mRNAs in
CIRI by lncRNA and mRNA epigenetic transcriptomic microarray. In
addition, bioinformatics analysis was used to predict the underlying
functions and related pathways of related lncRNAs and mRNAs. We found
that the total m^6A methylation level was significantly increased, and
the expression of fat mass and obesity-associated protein (FTO) was
downregulated after CIRI. In addition, a large number of m^6A-modified
lncRNAs and mRNAs appeared after CIRI, and these genes were mainly
enriched for the Toll-like receptor signaling pathway, peroxisome
proliferator-activated receptor (PPAR) signaling pathway, and
mitogen-activated protein kinase (MAPK) signaling pathway. Our findings
provide the basis and insights for further studies on m^6A modification
in CIRI.
Keywords: cerebral ischemia-reperfusion injury, N^6-methyladenosine,
lncRNA, mRNA, FTO
Introduction
The primary treatment principle for ischemic stroke is to recanalize
the blood flow in the ischemic area and restore the blood oxygen supply
to the infarcted brain tissue as soon as possible, but it may cause
cerebral ischemia-reperfusion injury (CIRI) and secondary injury to the
brain tissue ([38]van der Steen et al., 2022). The mechanism underlying
CIRI is very complex and may be related to oxidative stress, calcium
overload, inflammation, etc. ([39]Stegner et al., 2019), which
eventually lead to nerve cell damage, apoptosis, or necrosis. Further
exploration of the pathogenesis and prognostic biomarkers of CIRI or
identification of therapeutic targets may be of great significance.
Long noncoding RNAs (lncRNAs) are a class of RNAs that cannot encode
translational proteins but can actively participate in many important
biological processes by regulating gene expression at the
transcriptional and posttranscriptional levels. In recent years,
studies have shown that lncRNAs are involved in pathophysiological
responses such as inflammation, oxidative stress, angiogenesis, and
nerve regeneration after cerebral ischemia ([40]Akella et al., 2019).
Currently, the number of studies involving related lncRNAs involved in
CIRI is increasing, and a recent study reported lncRNA expression
profiles after CIRI ([41]Yang et al., 2022). However, the roles of
N^6-methyladenosine (m^6A)-related lncRNA posttranscriptional
modifications in CIRI remain unknown.
M^6A is the most extensive modification of mRNA and ncRNA observed in
eukaryotes, accounting for 80% of RNA methylation modifications, which
can functionally regulate the transcriptome of eukaryotes, thereby
affecting RNA splicing, nucleation, localization, translation, and
stabilization ([42]Meyer et al., 2012; [43]Frye et al., 2018). M^6A RNA
methylation occurs in a variety of important cellular life processes,
such as stem cell differentiation and the production of biological
rhythms, and is also involved in the occurrence of various diseases
([44]Dominissini et al., 2012). The process of methylation is
reversible, and the level of RNA m^6A methylation modification is
regulated by methyltransferases and demethylases ([45]Roundtree et al.,
2017). Several current studies suggest that lncRNAs may regulate tumor
growth through m^6A modification in cancer ([46]Steponaitis et al.,
2022). Unfortunately, the role of m^6A-related lncRNAs and mRNAs in
CIRI has not been elucidated.
In the present study, we identified the m^6A lncRNA and mRNA
modification profiles for the first time in CIRI. Bioinformatics
analysis was used to predict the potential functions and related
pathways of lncRNAs and mRNAs dysregulated by m^6A modification in
CIRI. Moreover, further analysis indicated that m^6A modification of
lncRNAs may exert biological functions through the lncRNA-miRNA-mRNA
transcriptional network, as shown in [47]Figure 1A.
FIGURE 1.
[48]FIGURE 1
[49]Open in a new tab
Establishment of the CIRI model (A) Experimental design (B) TTC
detection (TTC staining displayed the infarcted areas in white and the
normal areas in red) (C) Nissl staining (D) Neurobehavioral score.
Materials and methods
Animals
Specific pathogen-free (SPF) male Sprague Dawley (SD) rats that were
8 weeks old and weighed 250–260 g were purchased from Hunan Silaike
Jingda Co., LTD (Changsha, China) using production license number SCXK
(Xiang) 2019-0004. The animals were housed in the SPF animal room of
the First Affiliated Hospital of Hunan University of Chinese Medicine
at a temperature of 24–26°C, a relative humidity of 40%–60%, a
day-night cycle of 12 h, and free access to food and water, and
adaptive rearing was performed for 1 week before the experiment. This
animal experiment was approved by the Experimental Animal Ethics
Committee of the First Affiliated Hospital of Hunan University of
Chinese Medicine (ZYFY20201215-1).
Drugs and reagents
2,3,5-Triphenyl tetrazolium chloride (TTC) dye (Chengdu clone Chemicals
Co., Ltd., CAS 298-96-4); arterial embolus (Beijing Cinontech CO., Ltd,
2636A2); RNA wait tissue preservation solution (Dalian Meilunbio Co.,
Ltd, MA0208); m^6A RNA methylation assay kit (Abcam, ab185912); TRIzol
reagent (Life Technologies, T9424); affinity-purified anti-m^6A rabbit
polyclonal antibody (Synaptic Systems, 202003); sheep anti-Rabbit IgG
(Invitrogen, 11203D); and a lncRNA&mRNA epigenetic transcriptomic
microarray (8 × 60 k) customized by Arraystar were used.
Instruments
A centrifuge 5418 (Eppendorf, Germany); ultramicro spectrophotometer
(Nanodrop, United States); bioanalyzer 2100 (Agilent, United States);
G2505C microarray scanner (Agilent, United States); Vectra3 intelligent
tissue slice imaging system (Perkin Elmer, United States); and Gene Amp
PCR System 9700 (Applied Biosystems, United States) were used.
Model preparation and grouping
Consistent with the method described previously, rats were randomly
selected to establish the CIRI model ([50]She et al., 2019): after the
rats were anesthetized, an incision was made in the middle of the neck,
the left internal and external carotid arteries and the common carotid
arteries were exposed; the common carotid arteries were clipped to one
small orifice, a wire plug was inserted until reaching the internal
carotid artery at a depth of 2 mm from the bifurcation of the artery,
and the internal carotid artery was ligated. Reperfusion was performed
after the insertion of wire plugs for 2 h, and blood could re-enter the
middle cerebral artery by the circle of Willis to achieve cerebral
vascular reperfusion. The rats in the control group were operated on in
the same way as the model group but were not given a plug line to
occlude the middle cerebral artery. Finally, the wound was sutured, and
the state of the rats was observed. The degree of neurological deficit
in the rats was assessed by reference to the Zea-Longa scoring method
([51]Longa et al., 1989), and a score of one–three points indicated
successful modeling.
Neurobehavioral score
Recent studies have indicated that the seventh day after cerebral
ischemia is the optimal time for brain tissue recovery ([52]Dos Santos
et al., 2021). Therefore, in this study, the Longa 5-point scale was
used to score neurobehavioral scores on the seventh day after CIRI in
rats. The scoring criteria were as follows: 0 points, normal, no
neurological signs; one point, the animal could not fully extend the
left forelimb; two points, the animal’s left limb was paralyzed, the
animal turned around to the left when walking, and tail-collision
occurred; three points, the animal walked to the left and fell
sideways, or the animal was unable to stand or roll; four points, no
spontaneous movement, impaired consciousness.
TTC detection
Consistent with the method described previously ([53]Ni et al., 2022),
after neurological evaluation, the rats were anesthetized; the whole
brain was quickly removed; and the olfactory bulb, cerebellum, and
lower brainstem were removed. The brains in each group were removed
after being frozen in a freezer at -20°C for 15 min; placed on ice
disks from the frontal pole to the occipital pole; centered at the
level of the optic chiasm; and subjected to coronal, equal thickness
sectioning with a slice thickness of 2 mm. The brain slices were
immersed in 2% TTC staining solution and incubated at 37°C at constant
temperature in the dark for 15 min. The stained hindbrain slices were
placed in 10% formalin after fixation, removed, and blotted with filter
paper to dry the surface moisture. The brain slices were arranged
neatly in the anterior-posterior order of the brain, and a digital
camera was used to obtain images.
Nissl staining
After anesthetizing the rats, the whole brains were removed, fixed with
4% paraformaldehyde, and embedded in paraffin. The processed brain
tissue was sectioned coronally and morphologically evaluated by Nissl
staining. The sections were observed and photographed under a
microscope.
Quantification of total m^6A levels in brain tissues
Total m^6A levels were detected using a commercial m^6A RNA methylation
Quantification Kit. In brief, total RNA was first extracted using the
TRIzol method, 200 ng of total RNA was added to each well, and reagents
were added in steps according to the manufacturer’s instructions. Then,
m^6A levels were measured colorimetrically by reading the absorbance of
each well at a wavelength of 450 nm.
RT‒qPCR validation
The relative gene expression of m^6A methylase was verified by RT‒qPCR.
These genes included methyltransferases (writers), including
methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14)
and Wilms tumor one associated protein (WTAP), and demethylases
(erasers), including fat mass and obesity-associated protein (FTO) and
alkylation repair homolog 5 (ALKBH5). The experimental process strictly
followed the steps of the kit. The internal reference gene was β-actin,
and the 2^−ΔΔCt method was used to calculate the relative expression of
the gene. The sequences of each primer are shown in [54]Supplementary
Table S1.
Epigenetic transcriptomic microarray assays
The brains were decapitated directly after the rats were anesthetized,
and the cortical tissue on the ischemic side was rapidly separated on
ice, placed into a cryovial prefilled with RNA wait solution, stored
strictly according to the operation procedure, and sent to Aksomics
Biotechnology (Shanghai, China) for microarray assays. In brief, total
RNA was first extracted using the TRIzol method, then the total RNA was
immunoprecipitated with anti-m^6A antibodies. The “IP” grade fraction
of the immunoprecipitation was highly enriched for m^6A methylated RNA,
and the supernatant “Sup” grade contained unmodified RNA. The above two
RNA types were amplified as cRNAs and mixed after labeling with Cy5 and
Cy3. The samples were hybridized to the microarray for 17 h at 65°C in
an Agilent Hybridization Oven. Slides were scanned with an Agilent
G2505C microarray scanner. Data were extracted using Agilent feature
extraction software. The generated raw data obtained from the files
were normalized by GeneSpring software for subsequent data analysis.
Gene ontology (GO) functional analysis and kyoto encyclopedia of genes and
genomes (KEGG) pathway enrichment analysis
GO function and KEGG pathway enrichment analyses were performed on the
associated mRNAs ([55]Chen et al., 2022).
MeRIP-PCR validation
Three lncRNAs (LOC100912312, uc.440- and uc.77-) and three mRNAs
(protein phosphatase 1F (PPM1F), o-linked N-acetylglucosamine
transferase (OGT) and schlafen family member 13 (SLFN13)) were randomly
selected for validation. Total RNA was first immunoprecipitated with
anti-m^6A antibodies. The immunoprecipitated “IP” fraction contained
enriched m^6A methylated RNA, and the supernatant “Supernatant”
fraction contained unmodified RNA. The above two kinds of RNA were
converted into cDNA, amplified, and subsequently subjected to RT‒qPCR
using gene-specific primers. In addition, MeRIP-PCR assays were
performed in three replicates in each group (n = 3). The primer
sequences are shown in [56]Supplementary Table S2, and the proportion
of m^6A methylation modification of each gene was calculated according
to the following formula.
[MATH: %Input=2−
Ct MeRIP2−Ct MeRIP+2−Ct Supernatant×1
00% :MATH]
(1)
Competing endogenous RNA (ceRNA) network establishment
We selected the above three validated lncRNAs for ceRNA network
establishment. Similar to the results of a previous study ([57]Chen et
al., 2022), differentially methylated mRNAs with levels that were
significantly positively correlated with lncRNA levels were first
screened based on a Pearson coefficient >0.8. Then, miRBase and
TargetScan were used to predict lncRNA‒miRNA relationship pairs.
mRNA‒miRNA relationship pairs were predicted by the miRDB and miRWalk.
Finally, a lncRNA‒miRNA-mRNA transcription network was established with
miRNA as a bridge.
Statistical analysis
Differentially expressed m^6A methylation genes were screened by fold
changes (FCs) of ≥1.5 and p values of <0.05. Measurement data are
expressed as the mean ± standard deviation. If the data in each group
conformed to a normal distribution, a one-way ANOVA was performed, and
results with p values of <0.05 were considered statistically
significant. Analysis was performed using GraphPad Prism 8 graphing
software.
Results
Establishment of the CIRI model
TTC staining showed that the brain tissue in the control group was dark
red, and no obvious pale cerebral infarction was found, while the model
group showed obvious pale infarction, as shown in [58]Figure 1B. Nissl
staining showed a clear and complete neuronal cytoarchitecture in the
cortical region of control rats, with uniform cytoplasmic and nuclear
staining showing a pale blue color, normal intercellular spaces, and
abundant numbers of Nissl bodies showing a dark blue color. Unlike in
the control group, in the ischemic side of the cortex in the model
group, neuronal cell body shrinkage was severe, vacuolar degeneration
in the cytoplasm was obvious, cell arrangement lost regularity, and the
number of Nissl bodies was reduced, as shown in [59]Figure 1C. The
neurobehavioral score of the rats in the model group was significantly
higher than that observed in the control group (p < 0.01), as shown in
[60]Figure 1D. In conclusion, it was suggested that the rat model of
CIRI was successfully established, and pathological damage was observed
on the ischemic side of the rat model of cerebral ischemia.
Detection of total m^6A levels after CIRI
The results showed that the total m^6A methylation level in the cortex
tissue of the ischemic side was significantly higher in the model group
than in the control group (p < 0.01), as shown in [61]Figure 2A. This
result suggested that CIRI resulted in the abnormal methylation of
cortical tissue on the ischemic side.
FIGURE 2.
[62]FIGURE 2
[63]Open in a new tab
M^6A modification and methyltransferases and demethylases validation
(A) The total level of m^6A modification measured in cortical tissue on
the ischemic (B) Expression analysis of m^6A methyltransferases and
demethylases by RT-qPCR. ^** p < 0.01 vs Control group.
Verification of m^6A methyltransferases and demethylases
We studied the expression levels of five m^6A methyltransferases and
demethylases and found that the expression of FTO was lower (p < 0.01)
in the model group than in the control group, while no significant
changes were observed in the expression of methyltransferases,
including METTL3, WTAP, and METTL14. In addition, we also found no
significant changes in the expression levels of ALKBH5, as shown in
[64]Figure 2B. These results suggest that the increase in total m^6A
levels in CIRI may be caused by the imbalance in the expression of FTO.
M^6A modification profiles of lncRNAs and mRNAs
Samples from the model and control groups were analyzed using m^6A
lncRNA and mRNA epigenetic transcriptomic microarrays. The results
showed that a total of 108 lncRNAs exhibited differences in m^6A
modification between the model group and the control group, of which 54
were hypermethylated and 54 were hypomethylated, as shown in [65]Figure
3A. In addition, 590 mRNAs harbored differential m^6A modifications, of
which 375 were hypermethylated and 215 were hypomethylated, as shown in
[66]Figure 3B. The raw data supporting this result have been uploaded
to the GEO database ([67]GSE201258).
FIGURE 3.
[68]FIGURE 3
[69]Open in a new tab
M^6A modification profiles (A) Hierarchical clustering analysis of the
differentially methylated lncRNAs (B) Hierarchical clustering analysis
of the differentially methylated mRNAs (C) GO functional analysis of
the hypermethylated mRNAs (D) KEGG pathway enrichment analysis of the
hypermethylated mRNAs (E) GO functional analysis of the hypomethylated
mRNAs (F) KEGG pathway enrichment analysis of the hypomethylated mRNAs.
GO function and KEGG pathway enrichment analysis of differentially methylated
mRNAs
First, we performed an enrichment analysis of the hypermethylated mRNAs
and found that the biological processes involved were mainly protein
binding, lipid binding, and glutathione peroxidase activity, and the
cellular functions involved were mainly cell activation and immune
response. The results of the KEGG pathway enrichment analysis mainly
identified the chemokine signaling pathway and toll-like receptor
signaling pathway, as shown in [70]Figures 3C, D.
Moreover, we performed an enrichment analysis of the hypomethylated
mRNAs and found that the biological processes involved were mainly ion
binding and oxidoreductase activity, and the cellular components
involved were mainly cell junctions, organelles, and synapses. The
molecular functions involved mainly included various types of
metabolism and biosynthesis. The results of the KEGG pathway enrichment
analysis were mainly for steroid biosynthesis and the peroxisome
proliferator-activated receptor (PPAR) signaling pathway, as shown in
[71]Figures 3E, F.
Validation of differentially methylated genes
Validation of the microarray results was performed by MeRIP-PCR. Three
differentially methylated lncRNAs and three mRNAs were randomly picked.
The results showed that in the model group, the m^6A methylation ratios
of LOC100912312, uc.440-, OGT and SLFN13 were significantly increased
(p < 0.05 or 0.01), and the m^6A methylation ratios of uc.77- and PPM1F
were significantly decreased (p < 0.01), consistent with the trends of
change observed in the microarray results, as shown in [72]Figure 4.
FIGURE 4.
FIGURE 4
[73]Open in a new tab
M^6A analysis of differentially methylated genes by MeRIP-PCR. ^* p <
0.05, ^** p < 0.01 vs Control group.
CeRNA analysis of lncRNAs
To clarify the biological function of related lncRNAs, we performed
ceRNA network analysis on the validated LOC100912312, uc.440- and
uc.77- lncRNAs based on the ceRNA hypothesis. A ceRNA network
consisting of three lncRNAs, 17 miRNAs, and 16 mRNAs was established,
as shown in [74]Figure 5A. The enrichment analysis of mRNAs in this
network showed that the biological processes were mainly involved in
the regulation of voltage-gated calcium channel activity and
neuromuscular process controlling posture, the cellular components were
mainly involved in the costamere and sarcolemma, and the molecular
functions were mainly involved in transforming growth factor
beta-activated receptor activity ([75]Figure 5B). The results of the
KEGG pathway enrichment analysis mainly identified the
mitogen-activated protein kinase (MAPK) signaling pathway, as shown in
[76]Figure 5C.
FIGURE 5.
[77]FIGURE 5
[78]Open in a new tab
CeRNA analysis of lncRNAs (A) CeRNA network for LOC100912312, uc.440-
and uc.77- (B) GO functional analysis involved in the ceRNA network (C)
KEGG pathway enrichment analysis involved in the ceRNA network.
Discussion
M^6A is one of the most prevalent modifications present in the RNA of
higher eukaryotes, and increasing amounts of evidence suggest that m^6A
modification of RNA plays important biological roles in physiological
and pathological processes in the central nervous system (CNS)
([79]Zhang et al., 2022). This finding was confirmed in cerebral
ischemic disease, in which lncRNAs were identified as important
biomarkers ([80]Chokkalla et al., 2019; [81]Xu et al., 2020a).
Unfortunately, to date, reports on the dysregulation of lncRNA m^6A
modification in cerebral ischemia-reperfusion injury and studies on the
biological functions of related lncRNAs have not been reported.
In this study, we first detected significantly elevated m^6A levels in
ischemic lateral brain tissue, which is consistent with the results of
previous studies ([82]Xu et al., 2020b). M^6A modifications are known
to be dynamic and reversible, installed by “writers” and removed by
“erasers” ([83]Shi et al., 2019). Therefore, we further investigated
the expression levels of five common m^6A methyltransferases and
demethylases and found that FTO expression was downregulated after
CIRI, while no significant changes were observed in the expression of
methyltransferases, including METTL3, WTAP, and METTL14. The high
expression of METTL3, WTAP, and METTL14 may lead to an increase in the
m^6A methylation level ([84]Zhang et al., 2022), but at present, there
are few reports related to cerebral ischemia, which need to be further
studied. FTO was initially considered related to obesity ([85]Jia et
al., 2011). With further research, FTO has been confirmed to be an
important regulator of m^6A methylation ([86]Wei et al., 2018; [87]Wei
et al., 2022). FTO is an m^6A demethylase with abundant expression in
the brain ([88]Li et al., 2018). Studies have shown that FTO expression
is specifically downregulated in cerebral ischemic cortical neurons
([89]Yi et al., 2021). In addition, the expression of FTO is
downregulated in myocardial infarction ([90]Mathiyalagan et al., 2019),
suggesting that the expression of FTO is generally low in ischemic
injury. Conversely, overexpression of FTO was shown to reverse m^6A
methylation and reduce the levels of neuronal apoptosis caused by
cerebral ischemia ([91]Xu et al., 2020b). Moreover, FTO has also been
found to affect neurogenesis, memory formation, regulation of
neuropsychiatric disorders, etc. ([92]Li et al., 2017; [93]Walters et
al., 2017; [94]Wang et al., 2022), suggesting that it may be closely
related to the CNS. Moreover, we also found no significant changes in
the expression levels of ALKBH5. Although ALKBH5 has been reported to
selectively demethylate BCL2 transcripts after cerebral ischemia, which
prevents degradation of B-cell lymphoma-2 (BCL2) mRNA, enhances
expression of anti-apoptotic BCL2 protein, and inhibits neuronal
apoptosis, the role of ALKBH5 is still less understood ([95]Xu et al.,
2020b).
Microarray analysis showed that 590 mRNAs exhibited differences in m^6A
modification in CIRI, of which 375 were hypermethylated and 215 were
hypomethylated. Several studies have reported on the roles of related
mRNAs. For instance, apolipoprotein E (ApoE) was recognized as a
hypomethylated RNA in this study. In the CNS, ApoE is synthesized and
secreted by astrocytes and is involved in maintaining the homeostasis
of cholesterol and phospholipids, regulating the mobilization and
redistribution of cholesterol and phospholipids during neural membrane
remodeling and thus regulating the maintenance of synaptic plasticity
as well as repair when neuronal cells are damaged
([96]Cantuti-Castelvetri et al., 2018). In addition, vascular
endothelial growth factor A (VEGFA) is recognized as a hypermethylated
RNA; VEGFA is a factor that is closely related to angiogenesis after
cerebral ischemia, and hypermethylated VEGFA can promote angiogenesis
through multiple pathways ([97]Xin et al., 2022). GO function and KEGG
pathway enrichment analyses showed that these m^6A-modified mRNAs were
mainly involved in lipid binding, immune reactions, and oxidoreductase
activity, and the signaling pathways involved were the Toll-like
receptor signaling pathway and PPAR signaling pathway. The Toll-like
receptor signaling pathway is a bridge between innate immunity and
acquired immunity and has been confirmed to be closely related to the
inflammatory cascade observed after cerebral ischemia ([98]Eltzschig
and Eckle, 2011). Proliferator-activated receptors (PARs) are
ligand-activated receptors in the nuclear hormone receptor family that
control metabolic processes in many cells. Studies have shown that
ligand-activated PPAR can inhibit the expression of a variety of
related inflammatory factors, thus inhibiting the inflammatory response
and playing a protective role in CIRI ([99]Wu et al., 2018). In
addition, studies have supported the idea that PPAR activation has a
direct transcriptional regulatory effect on the expression of several
key endogenous antioxidants. For example, the activation of PPAR can
activate the expression of antioxidant enzymes in female rats and
protect them from CIRI ([100]Mohagheghi et al., 2013).
In recent years, m^6A-modified lncRNAs have received extensive
attention. For example, m^6A modification can drive the interaction
between related lncRNAs and downstream genes, which indicates that the
m^6A modification status in lncRNAs may control the biological
functions of lncRNAs ([101]Lan et al., 2021). In this study, we
identified 108 lncRNAs with differential m^6A modification, of which 54
were hypermethylated and 54 were hypomethylated. Studies have found
that although lncRNA cannot directly encode translational proteins, it
can act as a “miRNA sponge”, indirectly reducing the binding between
miRNA and downstream mRNA targets by absorbing the miRNA into this
sponge, thus affecting the expression of target genes. This is
essentially the mechanism underlying ceRNA regulation ([102]Salmena et
al., 2011). Subsequently, we established a potential ceRNA network of
validated lncRNAs LOC100912312, uc.440-, and uc.77- to clarify the
biological functions of relevant lncRNAs. Enrichment analysis indicated
that this network was mainly closely related to the MAPK signaling
pathway. MAPK signaling is an important regulatory pathway after
cerebral ischemia and hypoxia and is closely related to pathological
processes such as oxidative stress, the inflammatory response,
apoptosis, and autophagy ([103]Zhen et al., 2016). Experiments have
confirmed that the activated MAPK pathway after cerebral ischemia can
activate the NF-E2–related factor 2(Nrf2) pathway, and inhibiting the
phosphorylation of the MAPK pathway can reverse the nuclear
translocation of Nrf2 and reduce oxidative stress ([104]Meng et al.,
2018); other studies have indicated that the MAPK signaling pathway is
associated with inflammatory responses. It can activate the downstream
nuclear factor kappa-B (NF-κB) pathway after cerebral ischemia and
promote the inflammatory response ([105]Wang et al., 2014) while
inhibiting the activation of the MAPK pathway can reduce the activation
of the inflammatory response and have neuroprotective effects ([106]Guo
et al., 2012).
Undeniably, this study has several limitations. First, the sample size
of this study was relatively small, and the above results may need to
be validated in a large sample study in the future. In addition,
targeting FTO in CIRI requires further exploration. Despite these
limitations, we present the first systematic analysis of m^6A lncRNA
and m^6A mRNA modification profiles in CIRI and constructed a related
lncRNA‒miRNA‒mRNA network, laying a foundation for further revealing
the pathogenesis of CIRI.
Conclusion
In summary, we identified for the first time that m^6A lncRNA and m^6A
mRNA were differentially modified in CIRI, and total m^6A levels were
increased in CIRI, which might be caused by the downregulation of FTO
expression. In addition, bioinformatics analysis was used to predict
the potential functions of differentially m^6A-modified lncRNAs and
m^6A mRNAs, which could provide a reference for further revealing the
mechanism of CIRI.
Data availability statement
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and accession
number(s) can be found below: [107]https://www.ncbi.nlm.nih.gov;
[108]GSE201258.
Ethics statement
The animal study was reviewed and approved by This animal experiment
was approved by the Experimental Animal Ethics Committee of the First
Affiliated Hospital of Hunan University of Chinese Medicine
(ZYFY20201215-1).
Author contributions
BL designed the experiments, analyzed the data, and prepared the
manuscript. LS and BC performed the experiments, analyzed the data, and
prepared the manuscript. QW optimized the language of the manuscript.
YX and JY performed the experiments. The manuscript was revised by QW
and ZG. All authors confirmed the final manuscript.
Funding
This work was supported by grants from the National Natural Science
Foundation of China (82074251, 82004346), Natural Science Foundation of
Hunan Province (2022JJ30357, 2021JJ40425), Outstanding Youth Fund of
Hunan Education Department (20B433), the Science and Technology
Development Fund, Macau SAR (0098/2021/A2), Project of Hunan Provincial
Health Commission (202103071507), Hunan University of Chinese Medicine
First-class Subject Open Fund Project (2021ZYX02, 2021ZYX38). Hunan
University of Chinese Medicine Postgraduate Innovation Project
(2021CX20).
Conflict of interest
The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or claim
that may be made by its manufacturer, is not guaranteed or endorsed by
the publisher.
Supplementary material
The Supplementary Material for this article can be found online at:
[109]https://www.frontiersin.org/articles/10.3389/fgene.2022.973979/ful
l#supplementary-material.
[110]Click here for additional data file.^ (12.7KB, XLSX)
[111]Click here for additional data file.^ (12.7KB, XLSX)
References