LncRNA PEG11as Aggravates Cerebral Ischemia/Reperfusion Injury After Ischemic Stroke Through miR-342-5p /PFN1 Axis

LncRNAs are highly expressed in the CNS and regulate physiological and pathophysiological processes. However, the potential role of lncRNAs in cerebral ischemia/reperfusion (I/R) injury, a common pathological process after ischemic stroke (IS) remains widely unknown. From a RNA-Seq analysis, 254 lncRNAs were identi�ed to be differentially expressed in ischemic/reperfusion (I/R) mice brain tissue, including 234 upregulated and 5 downregulated lncRNAs. Among the 9 lncRNA used for veri�cation, lncRNA paternal expressed gene 11 antisense (PEG11as) was signi�cantly upregulated in the ischemic brain tissues of transient middle cerebral artery occlusion (MCAO) mice and N2a cells from oxygen-glucose deprivation/reoxygenation (OGD/R). Downregulate of PEG11as could markedly attenuate the brain infarct volume, alleviate neurological de�cit in vivo, and effectively promote neuron survival, attenuate neuronal apoptosis and caspase 3 activity both in vivo and in vitro. FISH assay discovered that PEG11as was mainly located in the cytoplasm, indicating that PEG11as could be used as a competitive endogenous RNA (ceRNA) to participate in post-transcriptional regulation. Furthermore, we demonstrate PEG11as was able to sponge miR-342-5p to inhibit miR-342-5p activity, resulting in pro�lin 1 (PFN1) overexpression and subsequently promoting apoptosis. Therefore, this study explored the role and underlying mechanisms of PEG11as in cerebral I/R-induced neuronal damage. It might contribute to the understand of the pathogenesis and provide a potential therapeutic option for the cerebral I/R.


Introduction
Ischemic stroke (IS) is an acute and severe neurological disorder that exists as one of the major causes of permanent morbidity and disability worldwide (Spescha et al. 2013; Knowland et al. 2014).Cerebral ischemia/reperfusion (I/R) injury is a common pathological process after IS.Previous studies have indicated that pathological mechanisms of cerebral I/R injury are complex, including energy failure, neurotoxicity of excitatory amino acid, in ammatory response, and depolarization of penumbra and apoptosis (Han et al. 2018).Apoptosis has been understood as an important mechanism mediating neurologic damages in IS.Therefore, a deeper understanding of the pathological processes and explorations, and development of new e cient therapeutic strategies to promote recovery in neurological functionality are urgently needed (Ikonomidou and Turski, 2002).
Long non-coding RNAs (lncRNAs) are a novel class of non-coding RNA that exceeds 200 bp in length, lacking or without an open reading frame (OFR).Based on present ndings, lncRNA plays an essential role in regulating gene expression and protein synthesis by interacting with DNA, RNA, and other proteins (Liu et al. 2019).The roles of lncRNAs in central nervous system (CNS) injuries were elucidated (Zhang and Wang 2019), notably in IS.Previous studies, however have described that the expression of lncRNAs in the adult rat MCAO/R model (Bridger and Baldwin 1990) and blood of IS patients have changed signi cantly (He et al. 2018).More recent studies have suggested that lncRNAs participate in several crucial biological processes after stroke.These include KCNQ1OT1 promotion by autophagy (Yu et  Through RNA-Sequencing (RNA-Seq), to compare the lncRNA pro ling of the ischemic tissue from middle cerebral artery occlusion (MCAO) mice model and sham mice, we established that lncRNA PEG11as was an upregulated lncRNA.Bioinformatic analysis revealed that PEG11as acted as a sponge for miR-342-5p.Furthermore, we discovered that pro lin 1 (PFN1), was predicted to be a target of miR-342-5p, and is upregulated in ischemic tissues of the MCAO/R model and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated N2a cells.Hence, we hypothesized that lncRNA PEG11as could regulate PFN1 levels via miR-342-5p, and eventually participate in the neuronal apoptosis in IS.

Animals
Adult male C57/BL6 mice, 22-25g, 8-9 weeks old, were purchased from the Experimental Animal Center of Hubei, China (license number: SCXK (Q) 2015-0018).The mice were housed under a standard environment of 24 ± 1 °C and 55% -70% humidity, with a 12-hour light/dark cycle.Food and water were available ad libitum.Animals experimental procedures involving animals were approved by the Experimental Animal Welfare and Ethics Committee of Wuhan University of Science and Technology.

Middle Cerebral Artery Occlusion/Reperfusion (MCAO) Model
The MCAO model was processed according to a previous study (Huang et al. 2021;Li et al. 2017).In brief, the mice were anesthetized with 10% chloral hydrate (350 mg/kg IP) to the right carotid arteries, external carotid artery (ECA), and internal carotid artery (ICA) were exposed through a longitudinal median neck incision.A 6-0 mono lament nylon suture with a silicone-coated (Ethicon, England) was inserted into the ECA incision and advanced towards the middle cerebral artery (MCA) origin through the ICA to obstruct the MCA.After 60 min of occlusion, the lament was withdrawn to allow blood reperfusion.Sham mice were subjected to the same operation, except the lament was not inserted.The surgery was done under a microscope and body temperature was maintained at 37 °C during surgery with a heating pad.

Neurological De cit Assessment
Behavioral assessments were performed 6 h, 12 h, 24 h, and 48 h after reperfusion.The mice were scored on a ve-point scale using the Longa method (Longa et al. 1989).The Longa scoring criteria are as follows: 0, no neurological de cit symptoms; 1, an incomplete extension of the left forelimb; 2, decreased resistance to lateral push, circle to the left side ; 3, inclination to the left side; and 4, no spontaneous locomotion and decreased level of consciousness.

2,3,5-triphenyl Tetrazolium Chloride (TTC) Staining
The mice were sacri ced by cervical dislocation after 24 hours of reperfusion.Their brains were carefully removed, cut into thick slices of 2 mm, and immersed in a 1% TTC (Biosharp Biotechnology, China) at 37 °C for 15 min in the dark then immersed in 4% paraformaldehyde solution overnight and photographed.
The infarct area in the brain slices was presented in white.The percentages of the infarct volume was measured using Image J software (ver.1.61).The percentage of the brain-infarct volume was calculated as infarct volume/total volume × 100%.

RNA-Seq Analysis
High-throughput sequencing was performed at RiboBio (Guangzhou, China).The total RNA samples were treated with Trizol (Life Technologies, USA) in accordance with the kit's instructions.The total RNA concentrations and quality were assessed by NanoDrop-1000 and Agilent 2200.Speci cally, OD260/OD280 remained between 1.8 and 2.2, and 28S/18S > 1.5, RIN >8, while the concentrations of total RNA were to be over 400 ng/μl.EpicentreRibo-Zero rRNA Removal Kit (Illumina, USA) was used to eliminate all rRNAs from the total RNA.Subsequently, puri ed RNA fragments were subjected to the rst strand and second-strand cDNA synthesis.Afterwards, adapter-ligated and enriched RNA-Seq libraries were prepared using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (NEB, USA).Finally, DNA was ampli ed.Meanwhile, the library's quality and concentration were validated by Agilent 2200 TapeStation (Agilent Technologies, USA).The obtained libraries were subjected to paired-end sequencing on an Illumina HiSeq2500 platform.The expression level of lncRNA was estimated by reads per kilobase of transcript per million mapped reads.A |log2FoldChange| >1 and q < 0.001 were used as thresholds for signi cantly altered lncRNAs.
The coordinates for the stereotaxic infusion were -0.5 mm posterior, -1.0 mm lateral, and -2.5 mm dorsal/ventral from the bregma (Zhang et al. 2019a) using a 10 μl syringe at a rate of 1 μl/min.After a period of seven days, the LV-shRNA-PEG11as and LV-shRNA-NC mice underwent MCAO surgery.
Oxygen and Glucose Deprivation/Reoxygenation ( (OGD/R) Model To mimic cerebral I/R in vitro, an OGD/R model was applied as aforementioned (Huang et al. 2021).In brief, N2a cells were inoculated in 6-well plates with a cell density of 3 × 105 per well.After 24 h, the normal medium was refreshed with the glucose-free DMEM (Gibco, USA) without serum.Afterwards, the plates were placed in a box containing a mixture of gas of 95% N2 and 5% CO2 at 37 °C for 3 h.Thereafter, the glucose-free medium was replaced by a complete DMEM.Conditioned medium was collected at 0 h, 6h, 12 h, 24 h, and 48 h after reoxygenation respectively for further studies.The control group was treated identically however they were not exposed to OGD.

Real-time Reverse Transcription PCR (qRT-PCR)
Total RNA was extracted from the brain tissue and cells using Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA).The RNA concentrations were determined by OD260/OD280.First-strand cDNAs were synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, #K1622, MA, USA) following the manufacturer's protocols.The expression of PEG11as, miR-342-5p, β-actin, and U6 (Ribobio, Guangzhou, Guangdong, China) was analyzed using the CFX96 Real-Time PCR Detection System (BioRad, Richmond, CA, USA).Initially, denaturation was conducted at 95 °C for 2min, followed by 40 cycles, including denaturation at 95 °C for 10s, annealing at 60 °C for 20s, and elongation at 70 °C for 20s.Primers used in this study are listed in Table 1.Relative expression was determined by the 2−ΔΔCt method.The lncRNAs level was normalized to β-actin, and the miR-342-5p level was normalized to U6.

TUNEL Staining
Sections of the brain and cultured cells were labeled with the TUNEL Bright Red Apoptosis Detection Kit System (Vazyme Biotech, USA) in accordance with the instructions of the TUNEL kit.The sections were observed by Laser Scanning Confocal Microscope (CX31-32RFL, Olympus, Tokyo, Japan).The apoptotic cells were counted by Image J software (ver.1.61).The apoptotic ratio was calculated as apoptotic cells / total cells × 100%.

Western Blot
The proteins expression were detected by Western blotting analysis.Ischemic tissues and cells were kept in ice-cold RIPA buffer with 0.1 mM phenylmethylsulfonyl uoride (PMSF) (Sigma, Missouri, USA) to extract the total proteins.The proteins were separated by sodium dodecyl sulfate 12 % polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene uoride (PVDF) membrane under the condition of 300 mA for 1.5 h, and then incubated at room temperature for 2 hours in a TBST buffer (20 mM Tris-HCl, pH 7.6,150 mM NaCl, 0.1% Tween-20) containing 5% no-fat milk.Speci c primary antibodies were incubated at 4 °C overnight, including rabbit anti-caspase 3 (1:1000, CST, USA), rabbit anti-PFN1(1:500, Proteintech, China), and mouse anti-β-actin (1:1000, Santa,USA).All the antibodies were diluted in TBST buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.4).Furthermore, the PVDF membrane was incubated with corresponding HRP-conjugated secondary antibodies for 90 min at room temperature.Immunoblots were visualized by an enhanced chemiluminescent (ECL) reagent (Amersham Biosciences, Piscataway, USA) under a Bio-Rad ChemiDoc MP system (Bio-Rad, USA).The relative intensity of the bands was analyzed using Image J software.The results are presented as a ratio between the intensity of the target protein and β-actin.

Fluorescence in Situ Hybridization (FISH) Analysis
Cy3-labeled lncRNA PEG11as probes were designed and synthesized by BersinBio (Guangzhou, China).The probe signals were determined with the Fluorescent in Situ Hybridization Kit (RiboBio, Guangzhou, China) in accordance with the manufacturer's guidelines.In brief, the N2a cells grew to 60-70% con uency on the slides and were xed with 4% paraformaldehyde (PFA).Cells were pre-hybridized in 0.5% TritonX-100-PBS at room temperature.Later, the FISH probes were hybridized in a hybridization buffer in the dark at 37 °C overnight.After rinsing with the saline-sodium citrate (SSC) buffer, nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI).The images were acquired using a confocal laser microscope (CX31-32RFL, Olympus, Tokyo, Japan).

RNA Binding Protein Immunoprecipitation (RIP)
RIP was performed with a Magna RIP TM RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA), in accordance with the manufacturer's instructions.In summary, N2a cells lysates were incubated in RIP immunoprecipitation buffer with a magnetic bead-antibody complex, coupled with Argonaute 2 (AGO2) antibody (5μg, Proteintech, China) or IgG control antibody (5μg, Millipore, USA) at 4°C overnight.The immunoprecipitated RNA-protein complexes were collected, then the samples were subjected to proteinase K digestion and RNA extraction by Trizol(Life Technologies, USA).Co-precipitated lncRNA PEG11as and miR-342-5p levels were evaluated by qRT-PCR analysis.

Immuno uorescence Staining
Sections of the brain were xed with 4% PFA for 15 min, permeabilized with 0.1% 0.1%Triton®X-100 for 15 min, and after blocked with 10% donkey serum in tris-buffered saline (TBS) for 30 min at RT.The sections were incubated overnight at 4°C with the rabbit anti-PFN1 primary antibody (1:200, Proteintech, China).After rinsing with the TBS, the sections were incubated with the secondary antibody Alexa Fluor®488 donkey anti-rabbit lgG(H+L) for 45 min.Sections were then washed in TBST and counterstained with DAPI for 10 minutes.Finally, the sections were washed with TBST, and 4, 6-diamino-2phenylindole (DAPI, Roche, USA) was used for nuclear staining.The stained tissue sections were then examined with a uorescence microscope (Olympus, Tokyo, Japan), and images were taken at × 200 magni cation.

Hoechst Staining
Apoptosis in cells transfected with PFN1 siRNA was examined using Hoechst Staining Kit System (Beyotime, China) following the manufacturer's manual.In brief, the N2a cells were cultured in 24-well plates with 5 × 104 cells per well, then transfected with PFN1 siRNA when the cells were grown to 60-70% con uency.Forty-eight hours after transfection, the cells were treated with OGD 3h / R 24h, and then xed with 4% paraformaldehyde at 4°C for 10 min.Following, the cells were washed with PBS and stained with Hoechst 33258 solution in the dark for 10min.The N2a cells were observed using a Laser Scanning Confocal Microscope (CX31-32RFL, Olympus, Tokyo, Japan).The apoptotic ratio was then, calculated as apoptotic cells / total cells × 100%.

Caspase 3 Activity Assay
Our experiment performed the Caspase 3 enzyme activity to detect apoptosis in the OGD/R after transfer of the PFN1 siRNA in N2a cells.The N2a cells were cultured in the 6-well plates, then transfected with PFN1 siRNA when the cells were grown to 60-70% con uency.After forty-eight hours of transfection, the cells were treated with OGD 3h/R24h, and then extracted of the total proteins.The protein concentration was detected using Bradford Protein Assay Kit (Beyotime, China) in accordance with the manufacturer's manual.The enzyme reaction was performed for 60 min at 37°C in a reaction mixture containing 100μl cell lysates and an equal volume of reagent based on the protocols of the Caspase 3 enzyme activity unit detection kit (Beyotime, China).The absorbance of the color was measured at 405 nm.

Statistics
The results are expressed as the mean ± SEM after repetition of at least three times.The data was analyzed using a t-test, one-way ANOVA.The t-test was used to compare the differences between the two groups, whereas the one-way ANOVA was used to compare the differences among multiple groups.Statistical analysis was performed by GraphPad Prism (ver.8.0) and values of P < 0.05 were considered to be statistically signi cant.

Quality Assessment of MCAO/R Model
To ensure the successful construction of the MCAO/R model, neurological de cit scores and TTC staining were analyzed.The results showed all animals exhibited signi cant neurological de cit (2.14 ± 0.69) except for the sham group (Supplemental Fig. 1a).The infarct volume was signi cantly increased in the I/R group (27.06 ± 8.78%) compared with the sham group (Supplemental Fig. 1b,c) .The results imply that the model was successful.

LncRNA Expression Pro les Signi cantly Alters after Cerebral I/R
To investigate the expression of lncRNAs in ischemic stroke, was performed to determine the lncRNA expression pro ling between the MCAO/R group and sham group through an Illumina HiSeqTM 2500.
Three samples from the same group constituted of independent biological replicates.After RNA-Seq and normalization, 254 lncRNAs were identi ed to be differentially expressed (fold change≥2, P < 0.05) between the MCAO/R group and sham group, of which 249 lncRNAs were upregulated and 5 lncRNAs were downregulated.As shown in Fig. 1a, the top 50 upregulated or downregulated lncRNAs were converted into a heatmap to show the distinguishable lncRNA expression pro ling between the MCAO/R group and the sham group.The volcano plots, GO (Supplemental Fig. 2a) and KEGG (Supplemental Fig. 2b) are demonstrated.
To verify the results of RNA-Seq, 9 differentially expressed lncRNAs were selected with the screening criteria (fold change≥2, P < 0.05, treat >1), including 8 upregulated lncRNAs and 1 downregulated lncRNA.The qRT-PCRs were performed to validate the expression of these selected lncRNAs.The expression of these lncRNAs was consistent with the data of RNA-Seq (Fig. 1b).We also found that PEG11as was the most signi cantly upregulated lncRNA in cerebral I/R.Subsequently, we detected the expression level of PEG11as at different time-points after reperfusion in vivo and in vitro, respectively.
The qTR-PCR data revealed that PEG11as were signi cantly upregulated at 0h, 6h, 12h, and 24h after reperfusion in vivo, reaching a peak at 6 h, and recovering at 48 h (Fig. 1c).Similarly, the results were found in vitro(Fig.1d), except the PEG11as expression did not change signi cantly in OGD/R 0h.All of the above results suggests that lncRNA PEG11as can play an important role in cerebral I/R.PEG11as, also known as 6430411K18Rik, accession: NR002848, a 1315 bp transcript with 1 exon and localizes in chromosome 12qF1.Hence, PEG11as was selected for further in-depth research in subsequent experiments.

Downregulation of PEG11as Improved the Neurological Function after Cerebral I/R
To investigate the effect of PEG11as after cerebral I/R in vivo, we performed knockdown PEG11as using intraventricular injection (ICV) of LV-shRNAs-PEG11as 7 days prior to cerebral I/R.After reperfusion 24h, PEG11as expression was detected using qRT-PCR and veri ed that PEG11as was downregulated successfully (Fig. 2a).Furthermore, we evaluated the effect by neurological de cit score and TTC straining.As shown in Fig. 2b, in I 1h /R 24h the neurological de cits score was notably higher than the Sham.Downregulated PEG11as signi cantly improved neurological function (1.67 ± 0.82) as opposed to the LV-shRNA-NC group (2.83 ± 0.41) ( P < 0.01).The results of the TTC staining was consistent with that of the behavioral assessments.The infarct volume of the I 1h/R 24h signi cantly increased (31.33 ± 5.10%) compared with that of the Sham, whereas the LV-shRNA-PEG11as group signi cantly decreased (15.77 ± 6.19%) compared with that of the LV-shRNA-NC group (31.31 ± 9.511%) ( P < 0.01) (Fig. 2c, d).These observations demonstrated that knocking down PEG11as improved neurological function and reduced I/R-induced brain damage.Downregulation of PEG11as Attenuated Cerebral I/R-induced Apoptosis Both in Vivo and in Vitro Cerebral neuronal apoptosis is an important characteristic of I/R-induced brain injury.To determine the effect of PEG11as on cerebral I/R-induced neurons apoptosis, TUNEL staining and cleaved caspase 3 protein expression were detected in vivo.Compared with the Sham group(4.48 ± 1.37; 1.00 ± 0.35 , the ratio of apoptosis of brain neurons (55.87 ± 5.58%) and cleaved caspase 3 protein expression (3.45 ± 0.34) were signi cantly increased in the I 1h/R 24h group.PEG11as knockdown inhibited the ratio of TUNEL-positive cells (31.49 ± 3.20%) (Fig. 3a, b) and the protein expression of cleaved caspase 3 (1.92 ± 0.49) (Fig. 3c) in the brain cortex neurons related to LV-shRNA-NC group (54.41 ± 2.14%; 3.70 ± 0,32).According to the above results, we concluded that PEG11as may be a promising gene for cell apoptosis, downregulation of its expression could decrease apoptosis in vivo induced by I/R.

miR-342-5p is A Target of PEG11as
Several studies have indicated that the subcellular localization of lncRNAs is related to their biological functions.Thus, we explored the cellular localization of PEG11as using RNA FISH assay.As shown in Fig. 4a, PEG11as is mostly distributed in the cytoplasm.We speculated that lncRNA PEG11as participates in the pathological process through ceRNA.After, we performed the bioinformatics analysis to predict the possible target miRNA by miRanda Miranda (http://www.miRanda.org), Pita (http://genie.weizmann.ac.il/pubs/miR07/miR07_data.html), and RNAhybrid (http://bibiserv.techfak.unibielefeld.de/rnahybrid)databases.We discovered that 71 miRNAs were predicted in all three databases (Fig. 4b).Among the given data, miR-342-5p was a human and mouse homologous gene (Supplemental Fig. 3a).Bioinformatic predictions presented that miR-342-5p has 6 binding sequences on PEG11as (Supplemental Fig. 3b).To investigate the interaction between PEG11as and miR-342-5p, we detected the expression levels of miR-342-5p in the I/R mice and OGD/R cells.The results showed that miR-342-5p was downregulated, while its expression was signi cantly upregulated when PEG11as was knockdown in vivo (Fig. 4c) and in vitro (Fig. 4d).Subsequently, to assess the direct interaction between PEG11as and miR-342-5p, AGO2-RIP assays were performed.As shown in Fig. 4e, f, both PEG11as and miR-342-5p were signi cantly enriched in AGO2 compared with IgG and the input.These results indicated that PEG11as functions as a miR-342-5p sponge to suppress its expression.
Together, this data supports that PEG11as serves as a ceRNA for miR-342-5p to regulate PFN1 expression and affect the process of apoptosis in cerebral I/R.

Discussion
LncRNAs modulate various biological functions at epigenetic, transcriptional, and post-transcriptional levels, and are widely involved in various physiological and pathological processes (Huang 2018).They act predominantly as biomarkers (Li et  In this study, we employed RNA-Seq approaches to evaluate the differential expression pro les.Further, we explored the function and mechanism of lncRNA PEG11as in IS.The expression of PEG11as signi cantly increased in I/R mice and in OGD/R-treatment cells, however knockdown of PEG11as decreased the brain infarct volume and alleviated the neurological de ciencies.In addition, we found that the silencing of lncRNA PEG11as inhibited neuronal apoptosis and caspase 3 activity.Mechanistic experiments demonstrated that PEG11as functioned as a ceRNA through harboring miR-342-5p to counteract its suppressive effect on the target gene PFN1 in cerebral I/R. Recent studies have shown that IS signi cantly alters the expression of lncRNAs, where the number of lncRNAs induced is the maximum at 6 h, and only a handful of lncRNAs were newly expressed beyond 6 h (Bhattarai et al. 2017).Therefore, in this study, we employed RNA-Seq approaches to evaluate the differential expression pro les of lncRNA in the mouse MCAO1h/R6h model.The results indicated that a total of 254 lncRNAs (249 up-regulated and 5 down-regulated) were differentially expressed.LncRNA PEG11as is present in a conserved imprinted gene cluster, which is located on the human chromosome 14 and mouse chromosome 12 (Bidwell et al. 2004), with a total length of 1315bp.LncRNA PEG11as originated from paternally expressed gene11 (PEG11 also known as Rtl1) opposite strand, similar to GTL2 (also referred to as MEG3) and MEG8, belonging to a maternally expressed non-coding gene (Byrne et al. 2010).LncRNA PEG11as was signi cantly up-regulated in the infarcted cortex and OGD/R-treated N2a cells.To this date, there have been no additional studies on the function of PEG11as in IS.  (Fuchs and Steller 2015).Our previous research has shown that neuron apoptosis characterized by progressive cell death has mainly pathological changes that occur in the peri-infarct zone after transient global ischemia, causing I/R damage (Li et al. 2017;Zhang et al. 2019a) In the present study, we investigated the potential role of lncRNA PEG11as on neuronal apoptosis in vivo and in vitro.We veri ed that downregulation of PEG11as signi cantly attenuated neurological de cits, decreased infarct areas in the MCAO/R mouse model, and knockdown PEG11as signi cantly inhibited apoptosis in vivo and vitro.These results strongly suggest that the suppression of PEG11as expression can effectively promote neuron survival, attenuate I/R or OGD/R-induced apoptosis, and downregulation of lncRNA PEG11as plays a protective role in ischemic stroke.
The localization of lncRNAs within the cell is the primary determinant of their molecular functions (Carlevaro-Fita and Johnson 2019).To further disclose the underlying molecular mechanism of lncRNA PEG11as, the subcellular localization analysis of lncRNA PEG11as by FISH was utilized.The current study has revealed that lncRNA PEG11as was mainly located in the cytoplasm.Hence, we speculated that lncRNA PEG11as acted as a competing endogenous RNA (ceRNA) that participates in I/R-induced apoptosis.CeRNA, a regulatory mechanism has been identi ed in which crosstalk between lncRNAs and mRNAs occurs by competing for shared microRNA response elements(MREs).In this scenario, the lncRNAs sponge of miRNAs modulate the derepression of the miRNA targets, imposing an additional level of post-transcriptional regulation, and play an important role in these pathological conditions (Mao et al. 2019;Liang et al. 2018;Cao et al. 2020;Zhang et al. 2019e).For instance, in an ischemic stroke, the lncRNA small nucleolar RNA host gene 6 (SNHG6) functions as a ceRNA for miR-181c-5p, which in turn represses its downstream target of Bcl-2 interacting mediator of cell death (BIM) inhibiting cell apoptosis (Zhang et al. 2019c).MEG3 functions as a ceRNAs and competes with the programmed cell death 4 (PDCD4) mRNA for direct binding to miR-21, whereas the knockdown of MEG3 protects against ischemic damage and improves overall neurological functions (Yan et al. 2017).Subsequently, in support of the hypothesis, the bioinformatic analysis revealed lncRNAs PEG11as potential interactions with miR-342-5p to affect the expression of PFN1.
Previous studies have demonstrated that miR-342-5p is extensively expressed in the brain and regulates the neural stem cell proliferation and differentiation (Gao et al. 2017).A recent study reported that miR-342-5p elicits anti-apoptotic effects via the inhibition of Caspase 9 and Jnk2 in ischemic/reperfused hearts (Hou et al. 2019).Throughout the course of this study, we employed qRT-PCR, RIP to prove the binding ability of miR-342-5p and PEG11as.As per our expectations, we discovered that miR-342-5p was remarkably downregulated in I/R mice and OGD/R cells, and similarly by downregulating PEG11as signi cantly up-regulated the expression of miR-342-5p.The results suggest that miR-342-5p expression has an inverse correlation with lncRNA PEG11as.In addition, PEG11as miR-342-5p coimmunoprecipitation with anti-AGO2 demonstrated a physical interaction in the N2a cells, providing further support for lncRNA PEG11as's miRNA sequestering activity.Furthermore, we discovered that the miR-342-5p mimic inhibited the OGD/R-induced apoptosis in the N2a cells, whilst the miR-342-5p inhibitor treatment showed a pro-apoptotic effect.However, the knockdown of lncRNA PEG11as overturned the pro-apoptotic effect of the miR-342-5p inhibitor.These results demonstrate that lncRNA PEG11as serves as a sponge for miR-342-5p, inhibiting both miR-342-5p expression and function in IS.
Bioinformatics revealed that PFN1 was one of the potential targets of miR-342-5p, which was a widely known conserved actin-binding protein that plays an essential role in tumor (Zhang et al. 2021), cardiomyocytes hypertrophy (Yang et al. 2017), and amyotrophic lateral sclerosis (ALS) (Yang et al. 2016).Existing reports have mentioned of PFN1's importance as an apoptosis regulator in several cancers, and that the overexpression of PFN1 sensitizes cancer cells to apoptosis through the typical intrinsic apoptotic pathway (Chen et al. 2013).Additionally, the PFN1 expression had signi cantly increased in AGEs-induced cardiomyocytes hypertrophy.Downregulation of PFN l expression can decrease cleaved caspase 3 expression and Hoechst 33258 staining, followed by attenuation through AGEs-induced H9c2 cells apoptosis (Yang et al. 2017).Meanwhile, knockdown of PFN1 was involved in M2 microglial polarization to prevent cerebral ischemia/reperfusion injury in ischemic stroke (Lu et al. 2020).In our study, we discovered that PFN1 was elevated in the brain I/R model and OGD/R treatment N2a cells, and knockdown of the expression of PFN1 inhibited cell apoptosis.Our results further indicate that PEG11as knockdown can inhibit PFN1 expression both in vivo and in vitro.In vitro functional assays, miR-342-5p could negatively regulate the expression of PFN1, whilst miR-342-5p inhibitor reversed the effect of PEG11as downtown on PFN1 expression.These results suggest that PFN1 can mediate the effect of PEG11as on the apoptosis of cerebral neurons through response of miR-342-5p.However, the present study also has some limitations in that in our experiment, we did not detect the promoter activation between lncRNA PEG11as, miR-342-5p, and PFN1 through a luciferase reporter assay.

Declarations Figures
al. 2019), lncRNA MEG3 mediation of neuronal apoptosis (Xiang et al. 2020), lncRNA H19 regulation of oxidative stress (Zeng et al. 2019) and, lncRNA 1810034E14Rik reduction in microglial activation (Zhang et al. 2019b) et al. in cerebral IS.However, the mechanisms of IS are far more complicated, consisting of multiple new lncRNA to be involved in the different stages.Meanwhile, the expression and function of lncRNA in IS are still uncertain.
al. 2018), potential therapeutic targets (Wang et al. 2019b), or indicators of prognosis (Zhao et al. 2018a) having received widespread attention.With the development of sequencing technology, a bulk of aberrantly expressed lncRNAs have been screened out in ischemic stroke patients (He et al. 2018) or ischemia insulted animals (Bhattarai et al. 2017).However, only a few cerebral I/R-related lncRNAs functions and mechanisms have been proven.