Research ArticleInfluence of miR-1 on Nerve Cell Apoptosis in Rats with CerebralStroke via Regulating ERK Signaling Pathway

Yuanding Jiang,1 Tao Wang,1 Jian He,1 Quan Liao,1 and Jingjing Wang 2

1Department of Neurosurgery, The Second Affiliated Hospital of South China University, Hengyang, 421001 Hunan Province, China2Department of Hemodialyses Room, The Second Affiliated Hospital of South China University, Hengyang,421001 Hunan Province, China

Correspondence should be addressed to Jingjing Wang; 364222074@qq.com

Received 25 March 2021; Accepted 1 August 2021; Published 20 August 2021

Academic Editor: Junyan Liu

Copyright © 2021 Yuanding Jiang et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

To explore the effect of miR-1 on neuronal apoptosis in rats with stroke through the ERK signaling pathway.Methods. Forty malerats (180-220 g) were selected and randomly divided into the sham, model, miR-1 inhibitor, and miR-1 mimic groups (10 rats pergroup) by average body weight. Cerebral ischemia/reperfusion (I/R) models were established using a modified middle cerebralartery wire thrombosis (MCAO) method in rats in the model group, miR-1 inhibitor group, and miR-1 mimic group. After thesuccessful model establishment, the miR-1inhibitor group and miR-1 mimic group were intravenously injected with miR-1inhibitor and miR-1 mimic, respectively, once a day for 3 days. The sham and model groups were given the same dose ofnormal saline. TTC staining was applied to detect the cerebral infarct size and calculate the infarct volume. Histopathologicalchanges in the hippocampus of rat brains were observed by HE staining. Flow cytometry was used to detect neuronal apoptosisin rat brains. The mRNA expressions of miR-1, ERK1/2, Bcl-2, and Bax in rat brain tissues were determined by QRT PCR, andthe protein levels of ERK1/2, Bcl-2, Bax, and caspase-3 were determined by Western blot analysis. Results. Compared with thesham group, the neurological impairment score, cerebral infarct size, and volume of rats in the model group were significantlyincreased (p < 0:05). Compared with the model group, the neurological impairment score, cerebral infarct size, and volume weresignificantly increased in the miR-1 mimic group and significantly decreased in the miR-1 inhibitor group (p < 0:05). In themodel group, the hippocampal tissue of rats had malaligned cells, neuron cell atrophy became smaller, the intercellular spacesbecame larger, and vacuoles appeared. Compared with the model group, the miR-1 inhibitor group could effectively alleviate thepathological changes in the hippocampus, and the miR-1 mimic group could significantly add to the pathological changes in therat hippocampus. Compared with the sham group, the mRNA expression of miR-1 and Bax in the brain of model rats increasedsignificantly (p < 0:05), and the mRNA expression of ERK1/2 decreased significantly; Compared with the model group, the miR-1 and Bax mRNA expressions in the brain tissues of rats in the miR-1 inhibitor group were significantly decreased, the ERK1/2and bcl-2 mRNA expressions were significantly increased, and the miR-1 and Bax mRNA expressions in the brain tissues of ratsin the miR-1 inhibitor group were significantly decreased, and the Bcl-2 mRNA expression was significantly increased (p < 0:05). Compared with the sham group, neuronal apoptosis was increased in the brain tissues of rats in the model group and miR-1mimic group. Compared with the model group, neuronal apoptosis was decreased in the brain tissues of rats in the miR-1inhibitor group. Compared with the sham group, the ERK1/2 proteins in the model group were significantly decreased, the Bcl-2, Bax, and caspase-3 proteins were significantly increased, and the ERK1/2, Bcl-2, Bax, and caspase-3 proteins in the miR-1inhibitor group and miR-1 mimic group were significantly increased. Compared with the model group, the protein levels ofERK1/2 and Bcl-2 in the miR-1 inhibitor group were significantly increased, the proteins of Bax and caspase-3 were significantlydecreased, and the protein levels of ERK1/2 and Bcl-2 in the miR-1 inhibitor group were significantly increased (p < 0:05).Conclusions. miR-1 can interfere with neuronal apoptosis in rats with stroke through the ERK signaling pathway.

HindawiBioMed Research InternationalVolume 2021, Article ID 9988534, 8 pageshttps://doi.org/10.1155/2021/9988534

1. Introduction

Cerebral stroke, also known as cerebralvascular accident, is akind of acute and sudden cerebrovascular disease. It is causedby obstruction and rupture of cerebral vessels, leading toblood stasis, preventing the blood from entering the brain,and triggering related diseases to hypoxic injury in the braintissues, including ischemic stroke and hemorrhagic stroke[1]. Clinically, ischemic stroke is a common type of cerebralstroke, which may be induced by carotid artery stenosis andvertebral artery occlusion, mostly in males aged over 40 yearsold, and can result in death in severe cases. The mortality rateof hemorrhagic stroke of the patients with cerebral stroke ismuch higher than that of ischemic stroke, according to stud-ies. Besides, cerebral stroke is the main reason of disabilityamong adults in China, seriously threatening the life andhealth of human [2–4]. However, there is no efficacious ther-apeutic measure in clinic at present.

Microribonucleic acids (miRNAs or miRs) are endoge-nous single-stranded noncoding small RNA molecules with22 nucleotides in length [5]. They regulate the expression oftheir complementary messenger RNAs (mRNAs) at the post-transcriptional level, thus affecting multiple physiologicaland pathological processes such as neurogenesis, neuronalmaturation, and development and regeneration of the ner-vous system [6]. Previous studies have shown that miR-1 isinvolved in the initiation and progression of several diseases,such as nerve injury [7–9], and the expression changes ofmiRNAs are time dependent after peripheral nerve injury.

Although cerebral stroke is correlated with various fac-tors in the body, studies related to the effect of miR-1 on neu-ronal apoptosis in rats with cerebral stroke are rarelyreported. Currently, the exact mechanism of cerebral strokein damaging the nerves remains unclear. Therefore, thisresearch is aimed at exploring the influence of the extracellu-lar signal-regulated kinase (ERK) signaling pathway on nervecell apoptosis in rats with cerebral stroke by intervening inmiR-1, providing certain experimental bases for the treat-ment of nerve cell apoptosis induced by cerebral stroke.

2. Materials and Methods

2.1. Laboratory Animals, Main Reagents, and Instruments. Atotal of 40 healthy male Sprague-Dawley rats aged 10-12weeks old and weighing 180-220 g were selected and thendivided into the sham-operation group (sham group, n = 10), model group (MCAO group, n = 10), miR-1 inhibitorgroup (n = 10), and miR-1 mimic group (n = 10) based onthe average body weight. The cerebral ischemia/reperfusion(I/R) injury model was established as per the modifiedMCAO method [10]. Specifically, the rats were anesthetizedby intraperitoneal injection of 20% urethane (5mL/kg), fixedon an operation table, and disinfected. Then, the skin was cutopen along the left neck, and the arteries were bluntly dis-sected. The external carotid artery was ligated, and the com-mon carotid artery was clipped using an artery clamp. Later,an oblique incision was made at the external carotid artery,from which a specialized nylon suture (0.26mm in diameter)for cerebral embolism was gently pushed in and stopped

when feeling resistance. After that, the suture was tightenedat the external carotid artery, and the incision was suturedand disinfected. 2 h later, the suture was pulled out. The bodytemperature of the rats was maintained at 37 ± 0:5°C duringthe whole surgical procedure.

In the sham group, all the operations were the sameexcept for the operation of nylon suture for cerebral embo-lism. Criteria of successful modeling are as follows: The ratswere scored using the EZ Longa method at 2 h after resusci-tation [6]. The rats with a score of 1-3 points were selectedas the objects of this research and randomly assigned intothe MCAO group, miR-1 inhibitor group, and miR-1 mimicgroup, with 10 rats in each group, after the model was pre-pared successfully. In addition, 10 rats were set as the shamgroup. Subsequently, the rats in the miR-1 inhibitor groupand miR-1 mimic group were intravenously injected withthe miR-1 inhibitor and miR-1 mimic, respectively, once aday for 3 days in total, while those in the sham group andMCAO group were given an equal dose of normal saline(Table 1).

The miR-1 inhibitor and miR-1 mimic were synthesizedby Guangzhou Ribio Biotech Co., Ltd., and eosin stainingsolution was provided by Shenyang Chemical Reagent No.5 Factory. Fluorescence quantitative polymerase chain reac-tion (qPCR) reagent and consumables were purchased fromTaKaRa (Dalian, China), and goat anti-rabbit IgG primaryantibodies (Abcam, Germany) and rabbit anti-rat horserad-ish peroxidase- (HRP-) labeled secondary antibodies (TIAN-GEN Biotech, China) were used for Western blotting. Inaddition, miRNeasy Mini kit (TIANGEN Biotech, China),PCR instrument (Thermo, USA), Gaoke TD-5A table-toplow-speed centrifuge (Jintan Changzhou Instrument Factory,China), ultra-micro spectrophotometer (Thermo, USA),TGL-16G centrifuge (Shanghai Anting Scientific InstrumentFactory, China), PT-3502B microplate reader (Thermo,USA), ECL instrument (Thermo, USA), and HH-4 digitalthermostatic water bath (Changzhou City Jintan HuateExperimental Instrument Co., Ltd., China) were applied.

2.2. Methods

2.2.1. 2,3,5-Triphenyltetrazolium Chloride (TTC) StainingMethods. The brain tissues of the rats were taken after the lastdrug administration, which were frozen at -20°C for 25minand sliced to prepare frozen sections. Then, the sections wereplaced in 1% TTC solution, incubated at 37°C for 15min, and

Table 1: EZ Longa scoring criteria.

No. Symptom Score

1 There is not any symptom of neurological deficit. 0

2 The right forepaw cannot straighten completely. 1

3The rat tilts to the right involuntarily or circles

to the right when walking.2

4The rat tumbles to the right or has paralysisof the right side of the body when walking.

3

5 The rat has coma and cannot walk autonomously. 4

6 The rat dies. 5

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fixed with 10% formaldehyde solution. Finally, the infarctionarea in the sections was measured using Image-Pro Plus soft-ware, so as to calculate the cerebral infarction volume. Thered parts stand for noninfarcted tissues and the white partsfor infarcted tissues.

2.2.2. Fluorescence qPCR. The brain tissue samples stored inliquid nitrogen were taken out to extract the total RNA usinga TRIzol reagent method, which was reversely transcribedaccording to the instructions of reverse transcription (RT)kit. Then, target genes were amplified as per the instructionsof the qRT-PCR kit, with the RT product (complementarydeoxyribonucleic acid (cDNA)) as the template. FluorescenceqPCR system: 10μL of qPCR Master Mix (2×), 1μL of PCRreverse primer, 2μL of cDNA, and 11μL of ddH2O. Finally,the results were calculated via 2-△△Ct method (Table 2).

2.2.3. Hematoxylin-Eosin (HE) Staining. The brain tissuesfixed for 24 h were taken out, washed in clear water, anddehydrated with ethanol, followed by embedding in paraffinand slicing into sections. Then, the sections were stained withhematoxylin at room temperature for 10min, washed withclear water for 30min, and soaked in 1% hydrochloric acidfor 30 s. After washing in clear water again for 30 s, the sec-tions were stained with eosin at room temperature for5min, dehydrated with ethanol, and then sealed in neutralgel in ventilation environment. Finally, the changes in thebrain tissues were observed under a microscope.

2.2.4. Detection of Cell Apoptosis via Flow Cytometry. Freshbrain tissues in each group were put in plates, added withnormal saline, and cut into pieces using scissors, followedby homogenization in a tissue homogenizer. Then, thehomogenate was aspirated and filtered into 10mL centrifugetubes by 100-mesh nylon nets, followed by centrifugation at1000 rpm for 5min and washing with normal saline for 3times. Next, the cells were resuspended in 5mL of PBS, fil-tered by 300-mesh nylon nets, and detected using a flowcytometer.

2.2.5. Western Blot

(1) Western Blotting. The brain tissues of the rats taken outfrom liquid nitrogen were added with RIPA lysis buffer con-taining PMSF and homogenized in a glass homogenizer for4min. Later, the homogenate was collected and centrifugedat 12,000 rpm for 10min. The supernatant was taken, mixedwith 5× protein loading buffer and boiled for 3min. AfterSDS-PAGE, the samples were transferred onto a polyvinyli-dene fluoride (PVDF) membrane. Subsequently, the PVDFmembrane was sealed in 5% skim milk powder solution atroom temperature for 1 h, incubated with correspondinglydiluted antibodies (1 : 1000) at 4°C overnight, and washedwith TBST for 5 min 3 times. Next, the membrane was sealedwith relevant HRP-labeled antibodies (diluted at 1 : 5000) at37°C for 1 h, followed by color development with developerand quantitative analysis using Quantity One software.

2.3. Data Analysis. The experimental data were analyzed viaSPSS 19.0 software. The mean ± standard deviation (�χ ± s)was adopted for intragroup comparison, one-way analysisof variance was utilized for intergroup comparison, and p <0:05 suggested statistical differences.

3. Results

3.1. Comparison of Neurological Deficit Score among Groups.As shown in Table 3, compared with the sham group, theneurological impairment scores of rats in the model groupwere significantly increased (p < 0:05). Compared with themodel group, the neurological impairment scores of themiR-1 mimic group were significantly increased, as werethe scores of the rats in the miR-1 inhibitor group (p < 0:05).

3.2. Comparison of Infarction Volume in Brain Tissues amongGroups. As shown in Figure 1 and Table 4, compared withthe sham group, the cerebral infarct size and volume of themodel rats were significantly increased (p < 0:05).Comparedwith the model group, the cerebral infarct size and volumewere significantly increased in the miR-1 mimic group, andthe neurologic impairment score and the cerebral infarct sizeand volume were significantly decreased in the miR-1 inhib-itor group (p < 0:05).

3.3. Comparison of Pathological Changes in HippocampalTissues among Groups. In contrast with those in the shamgroup, the cells in the hippocampal tissues of the rats werearranged irregularly, the neuronal cells became atrophicand small, the intercellular space was increased, and vacuoles

Table 2: Primer sequences for qRT-PCR.

Primer Sequence

miR-15′-CGTCCTTCAAGTAATCCAGGA-3′5′-GCAGGGTCCGAGGTATTC-3′

ERK1/25′-GAACTCCAAGGGCTATACCAAGT-3′5′-GGAGGGCAGAGACTGTAGGTAGT-3′

B-cell lymphoma-2(Bcl-2)

5′-CCGGGAGATCGTGATGAAGT-3′5′-ATCCCAGCCTCCGTTATCCT-3′

Bcl-2-associatedX protein (Bax)

5′-GTGGTTGCCCTCTTCTACTTTG-3′5′-CACAAAGATGGTCACTGTCTGC-3′

GAPDH5′-AGGTCGGTGTGAACGGATTTG-3′

5′-TGTAGACCATGTAGTTGAGGTCA-3′

Table 3: Comparison of neurological deficit after MCAO amonggroups (points, �χ ± s).

Group n Neurological deficit score

Sham group 10 0:31 ± 0:07MCAO group 10 2:49 ± 0:55a

miR-1 inhibitor group 10 0:79 ± 0:27b

miR-1 mimic group 10 3:65 ± 0:52abap < 0:05 vs. the Sham group, bp < 0:05 vs. the MCAO group.

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occurred in MCAO group (Figure 2). The pathologicalchanges in the hippocampal tissues of the rats were relievedeffectively in the miR-1 inhibitor group but aggravated dis-tinctly in the miR-1 mimic group.

3.4. Changes in mRNA Expressions of miR-1, ERK1/2, Bcl-2,and Bax in Brain Tissues in each Group of Rats. Accordingto Figure 3, compared with the sham group, the mRNAexpression of miR-1 and Bax in the brain of model ratsincreased significantly (p < 0:05), and the mRNA expressionof ERK1/2 decreased significantly. Compared with the modelgroup, the mRNA expression of miR-1 and Bax in the braintissues of rats in the miR-1 inhibitor group was significantlydecreased, and the mRNA expression of ERK1/2 and Bcl-2was significantly increased. The mRNA expression of miR-1 and Bax was significantly decreased, and the mRNAexpression of Bcl-2 was significantly increased (p < 0:05) inthe brain of rats in the miR-1 mimic group.

3.5. Comparison of Cranial Nerve Cell Apoptosis amongGroups. The MCAO group and miR-1 mimic group hadenhanced nerve cell apoptosis in the brain tissues comparedwith the sham group, but the miR-1 inhibitor group exhib-ited no apparent changes in nerve cell apoptosis in the braintissues (Figure 4). Moreover, in comparison with the MCAOgroup, the miR-1 inhibitor group displayed weakened nervecell apoptosis in the brain tissues, while the miR-1 mimicgroup showed no obvious changes in nerve cell apoptosis.

3.6. Protein Expressions of ERK, Bcl-2, Bax, and Caspase-3 inBrain Tissues in each Group of Rats. As shown in Figure 5,compared with the sham group, the protein of ERK1/2 inthe model group decreased significantly, the protein ofBcl-2, Bax, and caspase-3 increased significantly, and theprotein of ERK1/2, Bcl-2, Bax, and caspase-3 increased sig-nificantly in the miR-1 inhibitor group and the miR-1mimic group; Compared with the model group, the proteinlevels of ERK1/2 and Bcl-2 in the miR-1 inhibitor group weresignificantly increased, the proteins of Bax and caspase-3were significantly decreased, and the protein levels ofERK1/2 and Bcl-2 in the miR-1 inhibitor group were signifi-cantly increased (p < 0:05). Figure 5 shows the ERK, Bcl-2,Bax, and caspase-3 protein expressions in the brain of ratsin each group.

4. Discussion

In China, cerebral stroke is the leading cause of disability inadults, and it is the second major cause of death in peopleaged over 60 years old [11]. In addition, cerebral stroke is

(a) (b) (c) (d)

Figure 1: TTC staining results of brain tissues in each group. Note: (a) sham-operation group, (b) MCAO: model group, (c) miR-1 inhibitorgroup, and (d) miR-1 mimic group.

Table 4: Comparison of cerebral infarction volume after MCAOamong groups (�χ ± s).

Group n Cerebral infarction volume (%)

Sham-operation group 10 1:08 ± 0:05MCAO group 10 42:48 ± 8:42a

miR-1 inhibitor group 10 16:18 ± 3:45b

miR-1 mimic group 10 50:44 ± 5:58aap < 0:05 vs. the Sham group, bp < 0:05 vs. the MCAO group.

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the fifth most important cause of death in population aged15-59 years old in the world. About 2.4 million cases of cere-bral stroke events have been recorded since 2013; 75% ofwhich suffer from different degrees of disability [12]. It hasbeen revealed in studies that cell apoptosis is a crucial reasonof nerve cell death triggered by cerebral I/R injury [13–15].The mitogen-activated protein kinase (MAPK)/ERK signal-ing pathway participates in the regulation of such biologicalprocesses as cell proliferation, differentiation, and apoptosisthrough modulating the activity of transcription factors.Moreover, the abnormal activation of the MAPK/ERK sig-naling pathway can induce cell apoptosis [16].

As a category of conserved small noncoding RNAs, miR-NAs can repress protein translation at the posttranscrip-tional level via complementary base pairing with thespecific 3′-UTR of mRNAs, so the abnormal expression ofmiRNAs is closely associated with various diseases [17].Studies have demonstrated that aberrant miR-1 expression

will affect the occurrence and development of diverse dis-eases. It is also investigated that miRNAs are capable ofdegrading the mRNAs of target genes and inhibiting theirtranslation, thus playing vital roles in regulating pathophysi-ological processes such as cell proliferation, differentiation,and apoptosis [18–20]. miR-1 is currently known to bemainly expressed in the skeletal muscle and myocardium,and it is involved in the modulation of heart development,arrhythmia, myocardial ischemia, and other pathologicalprocesses according to studies [21]. It can be seen that miR-1 has close correlations with multiple diseases.

In this research, modified MCAO method was applied toestablish the rat model of cerebral I/R injury, and miR-1mimic and miR-1 inhibitor were utilized to investigate theimpacts of ERK on the nerve cell apoptosis in rats with cere-bral stroke. Then, the rats were administered with miR-1inhibitor and miR-1 mimic for 3 consecutive days after suc-cessful modeling, and the cerebral infarction area and volume

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Figure 2: Pathological changes in hippocampal tissues in each group of rats (HE staining, ×200). Note: (a) sham-operation group, (b) MCAO:model group, (c) miR-1 inhibitor group, and (d) miR-1 mimic group.

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Figure 3: mRNA expression of miR-1, ERK1/2, Bcl-2, and Bax in the brain of rats from each group. Note: sham: sham-operation group;MCAO: model group; miR-1 inhibitor: miR-1 inhibitor group; miR-1 mimic: miR-1 mimic group, ∗p < 0:05 and ∗∗p < 0:01 vs. the shamgroup.

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in the brain tissues of the rats were measured via TTC stain-ing. It was found that there were fairly apparent infarcted tis-sues in the MCAO group, while the infarction area andvolume were decreased remarkably after inhibiting miR-1,suggesting that the suppression of miR-1 can ameliorate theinfarction caused by I/R injury in the rats. Later, the HE

staining was performed to observe the hippocampal tissuesin the rat brain, and it was discovered that the infarction inthe rats was relieved after inhibiting miR-1. According tostudies, miR-1 can affect cardiomyocyte apoptosis byinfluencing the ERK signaling pathway. Therefore, themRNA expression of ERK1/2 in the brain tissues of rats

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Figure 4: Influence on cranial nerve cell apoptosis in each group of rats. Note: (a) sham-operation group, (b) MCAO: model group, (c) miR-1inhibitor group, and (d) miR-1 mimic group.

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Figure 5: Protein expressions of ERK, Bcl-2, Bax, and caspase-3 in brain tissues in each group of rats. (a) Western blot result. (b)Quantification analysis. ap < 0:05 vs. the Sham group, bp < 0:05 vs. the MCAO group. Note: sham: sham-operation group; MCAO: modelgroup; miR-1 inhibitor: miR-1 inhibitor group; miR-1 mimic: miR-1 mimic group.

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was detected via qRT-PCR, and the results manifested thatmiR-1 was able to influence the ERK expression in the braintissues of rats. Subsequently, the nerve cell apoptosis in thebrain tissues of rats was determined by means of flow cytom-etry, and it was shown that repressed miR-1 could improvethe nerve cell apoptosis in the brain tissues of rats with I/Rinjury. In contrast, miR-1 mimic could enhance the nerve cellapoptosis in rats, illustrating that miR-1 can intervene in thenerve cell apoptosis in rats through ERK. Finally, the aboveconclusions were verified using Western blotting.

Nevertheless, the precise mechanism of miR-1 in affect-ing the nerve cell apoptosis in rats with cerebral stroke mayalso be related to other target genes of miR-1. A latest studyindicated that the level of miR-1 rises in cardiomyocytes,and the overexpression of miR-1 attenuates the phosphoryla-tion of MAPK and the activation of PI3K p85α and regulatesthe energy metabolism of cardiomyocytes by virtue of IRS2,Akt, and ERK. It has been revealed that miR-1 regulatesdiversified target genes, including MTSS1, HOXA10,SMAD5, and JAK2. In this research, however, the impactson the ERK signaling pathway were merely testified, whilethe impacts on other signaling pathways were not elaborated,which will lay an experimental foundation for subsequentstudies on the pathogenesis of nerve cell apoptosis in ratswith cerebral stroke.

Data Availability

The datasets used and/or analyzed during the current studyare available from the corresponding author on reasonablerequest.

Ethical Approval

The study was approved by the ethics committee of The Sec-ond Affiliated Hospital of South China University.

Conflicts of Interest

The authors declare that they have no competing interests.

Acknowledgments

This study was supported by the Hengyang science and tech-nology project (No. 2020jh042777).

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