| Research background:Stroke is caused by cerebral ischemia and hypoxia,which can cause dysfunction of many distant organs and has a high morbidity rate.,morbidity and mortality[1-3],It has a great impact on people’s health and quality of life[4,5].At the moment,the most effective treatment for ischemic stroke is to achieve early reperfusion by performing thrombolysis and/or mechanical recanalization of occluded vessels within an effective time window.However,there are still a large number of patients who have not yet reached the hospital to complete this treatment within the effective time window,and no strong and effective drugs that can significantly improve the prognosis of stroke have been found in addition to the existing thrombolysis and thrombectomy.Therefore,there is an urgent need for Clinicians and researchers are developing more effective therapeutic approaches to overcome the limitations of current drug applications,reduce side effects,and expand the indications of new treatments.MicroRNA(miRNA)is an endogenous,small ribonucleic acid of about 19-25 nucleotides in length.The mechanism of action of miRNA is to bind to the 3’-UTR of mRNA and inhibit its protein translation.Each microRNA can regulate multiple target genes,It is hypothesized that MiRNAs regulate 1/3 of human genes.miRNAs are involved in a variety of biological processes such as proliferation,differentiation,metabolism and apoptosis[6-8].miR-224-5p is a member of the small non-coding RNA family and is located in the third intron of the GABAA receptor subunit(GABRE)gene on ChrXq28[9,10].Previous studies have shown that miR-224-5p may be co-transcribed with its host gene GABRE,which can be synergistically repressed by p53 and NF-κ Bp65 to promote granulosa cell proliferation[11,12].miR-224-5p is involved in a variety of life events,including tumour cell proliferation,metastasis,invasion and apoptosis[13].Its mechanism of action may be related to the activation of miR-224-5p/HOXD 10/p-PAK4/MMP-9 signaling pathway,AKT activation,and NF-κB inflammatory pathway[14-17].However,the molecular mechanisms underlying the effects of miR-224-5p on cortical neurons are not fully understood.Therefore,it becomes necessary to explore the molecular mechanism of miR-224-5p action on neuronal cells after ischemic-hypoxic injury.Research purposes:The etiology and pathological process of ischemic stroke are mediated by a cascade of various molecular mechanisms,and these molecular mechanisms are partly regulated by post-transcriptional activities.More and more evidences show that microRNA,as an important mediator of post-transcriptional gene silencing,plays an important role in both the physiology of brain development and the pathology of ischemic stroke.This study aims to study the molecular mechanism of stroke and explore the mechanism of miR-224-5p in ischemic and hypoxic cortical neuron damage.Research methods:Part One:The change trend of miR-224-5p in a rat model of focal cerebral ischemia-reperfusion injuryIn this experiment,clean-grade adult male Sprague-Dawley(SD)rats were used to establish the middle cerebral artery occlusion(MCAO)model of rats by the suture method.The four groups of experimental rats—the MCAO reperfusion 12 hours group,the sham operation group(Sham Group)(MCAO 12h group),MCAO reperfusion 24 hours group(MCAO 24h group),MCAO reperfusion 48 hours group(MCAO 48h group),the thread plug was removed 2 hours after the successful occlusion of the model,and ischemia Reperfusion.Rats in the sham operation group underwent the same surgical operation without inserting a thread plug.Refer to ZeaLonga’s 5-point system to evaluate neurological deficits through ZeaLonga score to determine whether the MCAO model is successfully established[24].At the same time,triphenyltetrazolium chloride(TTC)was used to observe the cerebral infarction lesions of MCAO rats,and again to evaluate whether the model was successfully established.Rats in each group were sacrificed at the corresponding time points,and the materials were quickly collected and stored at-80℃.The change trend of miR-224-5p in rat brain tissue was analyzed by qRT-PCR.Part Two:The mechanism of miR-224-5p in hypoxic injury of cortical neurons1.In order to study the mechanism of miR-224-5p in ischemic and hypoxic neurons,we took newborn rats within 24 hours to isolate and culture primary cortical neurons,and established them in rat primary cortical neurons The oxygen-sugar deprivation(oxygenglucosedeprivationOGD)model was used,and the neuron morphology was observed under the bright field electron microscope 24 hours after the OGD model was made.Extract the neurons of the control group and the OGD group,and reverse transcription was performed.The change trend of miR-224-5p in primary rat cortical neurons was analyzed by qRT-PCR.2.Take newborn rats to isolate and culture primary cortical neurons,and divide the primary cultured cortical neurons into five groups:Control group(normally cultured primary cortical neurons),mimic-NC group(Transfection simulation-NC,nonsense miRNA),anti-NC group(transfection anti-NC,nonsense miRNA),miR-224-5p group(transfection miR-224-5p),anti-miR-224-5p group(Transfection anti-miR-224-5p group),culture for 7 days for the corresponding transfection operation,2-3 days after transfection,observe the red cy3-5’ fluorescence with a fluorescence microscope,and take pictures to calculate the transfection efficiency.Extract RNA for reverse transcription and qRT-PCR to observe the relative expression of miR-224-5p in each group3.Take newborn rats to isolate and culture primary cortical neurons,for 7 days and then transfect the primary cultured cortical neurons into Six groups:Control group(normally cultured primary cortical neurons),OGD group(only OGD modeling,no transfection),mimic-NC group(OGD+transfection simulation-NC,nonsense miRNA),anti-NC Group(OGD+transfection anti-NC,nonsense miRNA),miR-224-5p group(OGD+transfection miR-224-5p),anti-miR-224-5p group(OGD+transfection anti-miR-224-5p group).2-3 days after transfection,the OGD model was performed.24 hours after the model was successfully modeled,the morphological changes of the cells in each group were observed by a bright field microscope,and the number of neuronal cells was counted.4.Isolation and culture of primary cortical neurons,respectively inoculate cell slides in 12-well plates and 96-well plates,culture primary cortical neurons at 50-80%confluence for 7 days,and then transfect them.Subcultured cortical neurons are divided into four groups:Control group(normally cultured primary cortical neurons),OGD group(only OGD modeling),anti-NC group(OGD+transfection anti-NC,nonsense miRNA),anti-miR-224-5p group(OGD+transfection anti-miR-224-5p).2-3 days after transfection,perform OGD modeling,24 hours after successful modeling,perform Tuj 1+(neuronal labeling)staining and TUNEL staining(detecting apoptosis)to observe the changes in each group.At the same time,the 96-well plate was used to analyze the survival rate of each group of cells as a percentage of the control.Part Three:Mechanism of miR-224-5p/NR4A1 pathway in hypoxic injury of cortical neurons1.Use the bioinformatics database TargetScan,miRDB,miRmap and miRanda to predict the target genes of miR-224-5p and find the potential target genes.2.Isolation and culture of primary cortical neurons.After 10 days of culture,Divided into 2 groups by randomly method:Control group(normal culture of primary cortical neurons),OGD group(for OGD modeling),After24 hours qRT-PCR was used to observe whether NR4A1 could be down-regulated,and WB to observe whether NR4A1 protein expression decreased.3.A dual-luciferase reporter gene experiment was performed to verify whether the predicted target gene NR4A1 is the direct target gene of miR-224-5p.Construct an expression reporter vector and co-transfect 293T cells with miR-224-5p mimics,and verify the predicted target genes of miR-224-5p through the luciferase reporter system.The experiment is divided into four groups:NR4A1-WT-NC group and NR4A1-WTmimc miR-224-5p group,NR4A1-Mut-NC group,NR4A1-Mut-mimic miR-224-5p group,use a microplate reader to determine the hRluc fluorescence value of each group according to the steps,and compare the fluorescence value of hRluc in each well with hluc The fluorescence value is compared,and the ratio is statistically analyzed with the ratio of the control well.4.Isolation and culture of primary cortical neurons.The cells were cultured at 50-80%confluence for 7 days and then transfected.The primary cultured cortical neurons were divided into five groups:Control group(normal Culture primary cortical neurons),OGD group(only OGD modeling,no transfection),anti-miR-224 group(OGD+transfection miR-224-5P inhibitor),anti-miR-224+si-NC group(OGD+transfection miR-224-5P inhibitor+nonsense chain of si-NR4A1),anti-miR-224+si-NR4A1 group(OGD+transfection miR-224-5P inhibitor+si-NR4A1 chain).2-3 days after transfection,perform OGD modeling,24 hours after successful modeling,perform Tuj1+(neuronal labeling)staining and TUNEL staining(detecting apoptosis)to observe the changes in each group.At the same time,the 96-well plate was used to analyze the survival rate of each group of cells as a percentage of the control.Results:Part One:Expression changes of miR-224-5p in a rat model of focal cerebral ischemia-reperfusion injuryWe successfully constructed MCAOmodel in SD rats.2 hours after MCAO,the wire plugs were removed for reperfusion,and 6 hours later the brains were removed and stained with TTC[18]to observe the infarcts,which showed white infarct areas in the MCAO group and red brain tissue with no obvious infarcts in the sham-operated group(Figure 1-2).This indicates that the middle cerebral artery was successfully blocked.Neural defects were assessed using the Longa score[19].There was a statistically significant difference in the scores at 12,24 and 48 hours post-MCAO compared to the sham-operated group(n=6,**P<0.01,Figure 1-1).This indicated that the MCAO model had been successfully established.We then found by qRT-PCR that the expression of miR-224-5p was significantly increased at 12,24 and 48 hours post-MCAO in rats(Figure 1-3).Part Two:The mechanism of miR-224-5p in hypoxic injury of cortical neurons1.In vitro,we modeled the OGD in primary cortical neurons.Bright-field images obtained by light microscopy are shown in Figure 2-1.After 2 h of oxygen glucose deprivation,the number of cortical neurons was significantly reduced within 24 h(Figure 2-1).We then found a significant increase in miR-224-5p expression after OGD(n=6,**P<0.01,Figure 2-2).2.To investigate the role of miR-224-5p,we transfected miR-224-5p mimics and inhibitors into primary cortical neurons.cy3 transfection is shown in Figure 2-3.Meanwhile,we found that mimics of miR-224-5p significantly increased the expression of miR-224-5p and inhibitors of miR-224-5p significantly decreased the expression of miR-224-5p(Figure 2-5).We then showed images of primary cortical neurons in the control,OGD,mock NC,anti-NC,miR-224-5p and anti-miR-224-5p groups(Figure 2-6).We found that the number of primary cortical neurons was significantly reduced after OGD compared to the control group(n=6,**P<0.01,Figure 2-7).Inhibition of miR-224-5p increased the number of neurons after OGD injury(**P<0.01 Figure 2-7).3.The number of Tuj1+cells was studied in the control,OGD,anti-NC and anti-miR-224-5p groups(Figure 2-8A).The results of the study showed that Tuj1+cells were reduced in the OGD group by the control group is significantly.However,the number of Tuj1+cells was significantly increased in the anti-miR-224-5p group compared to the anti-NC group(n=6,**P<0.01)(Figure 2-8B).In addition,Compared to the control group,the neuronal axon length in the OGD group there is a significant decrease.The mean axon length was significantly increased in the anti-miR-224-5p group compared to the anti-NC group(n=6,**P<0.01)(Figure 2-8C).TUNEL-positive cells were also observed in the control,OGD,anti-NC and anti-miR-224-5p groups(Figure 2-9A).Apoptosis of primary cortical neurons was increased in the OGD group compared to the control group(n=6,**P<0.01)(Figure 2-9B).The number of apoptotic neuronal cells was reduced in the anti-miR-224-5p group compared to the anti-NC group,and induction of miR-224-5p inhibition reduced apoptosis in hypoxic-ischemic neurons.Also,neuronal survival was significantly increased in the anti-miR-224-5p group compared to the OGD group(n=6,**P<0.01)(Figure 2-9C).Part Three:Mechanism of miR-224-5p/NR4A1 pathway in hypoxic injury of cortical neurons1.Potential target genes of miR-224-5p may be nuclear receptor subfamily 4 group A member 1(NR4A1),interleukin 1 receptor antagonist(IL1RN)and ring finger protein 38(RNF38)(Figure 3-1).2.qRT-PCR analysis showed reduced mRNA expression of NR4A1 but not IL1RN and RNF38 in hypoxic-ischaemic neurons(Figure 3-2).3.The 3’-UTR of NR4A1 was cloned into the pmi-R-RB-REPORT plasmid and the predicted binding site for miR-224-5p is shown in Figure 3-5.Mutant constructs were generated using targeted mutagenesis of the reporter gene containing the 3’-UTR of the NR4A1 gene(Figure 3-5).Treatment with miR-224-5p significantly reduced relative luciferase activity,while inhibition of miR-224-5p was eliminated in the plasmid containing the NR4A1 mutant 3’-UTR(Figure 3-6).4.Inhibition of miR-224-5p improved neuronal morphology through the target gene NR4A1.A reduction in the number of Tuj1+cells was observed in the control,OGD,anti-miR-224-5p,anti-miR-224-5p+si-NC,and anti-miR-224-5p+si-groups compared to the anti-miR-224-5p+si-NC groups(**P<0.01)(Fig.3-7B).Neuronal axon length was quantified to be reduced in the anti-miR-224-5p+si-NR4A1 group compared to the anti-miR-224-5p+si-NC group(**P<0.01)(Figure 3-7C).TUNEL staining revealed that inhibition of MiR-224-5p reduced neuronal apoptosis via NR4A1(Figure 3-8A).Apoptosis of neurons(red)was observed in the control group,OGD group,anti-miR-224-5p group,anti-miR-224-5p+si-NC group,and anti-miR-224-5p+.After quantifying the rate of neuronal apoptosis,the rate of neuronal apoptosis was significantly increased in the anti-miR-224-5p+ si-NR4A1 group compared to the anti-miR-224-5p+si-NC group(**P<0.01)(Figure 3-8B).Neuronal viability was quantified by MTT and was significantly reduced in the anti-miR-224-5p+si-NR4A1 group compared to the anti-miR-224-5p +si-NC group(**P<0.01)(Figure 3-8C).Conclusions:1.The SD rat MCAO model was successfully constructed.The expression of miR-224-5p was found to be significantly increased by qRT-PCR at 12 h,24 h and 48 h post-MCAO.2.OGD model was successfully constructed.qRT-PCR verified that miR-224-5p was also upregulated after OGD.After transfection with miR-224-5p inhibitor,the number of neurons was significantly increased in the miR-224-5p inhibitor group compared with the OGD group,and the survival rate of neurons in the anti-MIR-224-5P group was significantly increased compared with the control group,and the apoptosis rate was significantly decreased.3.The potential target genes predicted for miR-224-5p may be NR4A1,IL1RN and RNF38.qRT-PCR results confirmed that NR4A1 was significantly reduced after hypoxic-ischemic injury,and luciferase reporter gene assays verified that NR4A1 was the direct target of miR-224-5p direct target of action.4.Inhibition of miR-224-5p improved neuronal morphology through the target gene NR4A1.Inhibition of miR-224-5p reduced neuronal apoptosis through the action of NR4A1.miR-224-5p plays a key role in hypoxic-ischemic neuronal injury through NR4A1,which may be an important regulatory mechanism of hypoxic-ischemic brain injury,and this may be an important indicator of the impact of hypoxic-ischemic damage in the brain.Functional reversion experiments showed that the protective effect of low expression of miR-224-5p in hypoxic neurons was achieved through NR4A1.miR224-5p/NR4A1 pathway plays an important role in hypoxic injury of cortical neurons. |
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