【作者】 于志君；

【导师】 胡霞敏； 袁琼；

【作者基本信息】 武汉科技大学 ， 生物医药工程， 2021， 博士

【摘要】 背景缺血性脑卒中(ischemic stroke,IS),因发病率、死亡率及复发率较高,已成为严重威胁人类健康的重大疾病。目前临床用于治疗急性缺血性卒中的主要方法是溶栓药物(如rt-PA)治疗及机械取栓等。然而,由于溶栓治疗严格时间窗(一般起病后3-4.5h)及缺血性脑卒中本身复杂的病理机制,导致该疾病的治疗尚未取得突破性进展。大量研究发现,脑缺血再灌注(ischemic/reperfusion,I/R)引起的神经元凋亡或坏死是导致脑组织损伤的主要病理生理学机制之一。因此,从诱导神经元凋亡的发病机制出发,在基因调控网络中寻找关键分子,有望为急性缺血性脑卒中的预防及治疗提供新的研究方向。长链非编码RNA(long non-codingRNA,lncRNA)是在真核生物中发现的长度超过200nt的一类RNA,广泛存在于组织和细胞中,可通过在多个层面调控基因表达,如表观遗传、转录以及转录后等,参与重要的生物学进程。lncRNAs异常表达与脑I/R的发生密切相关。因此,寻找与脑I/R密切相关的lncRNAs并探讨其潜在作用及分子机制,为临床缺血性脑卒中的治疗提供新的治疗靶点具有极大的研究价值和意义。目的利用RNA测序(RNA-Sequencing,RNA-Seq)技术,获得缺血性脑卒中后发生显著改变的lncRNAs,并探讨其在脑I/R中的作用及潜在分子机制,为缺血性脑卒中的预防和治疗提理论依据。方法第一部分:小鼠脑I/R损伤相关lncRNA的差异表达及功能分析。成年健康雄性C57BL/6小鼠12只,随机分为假手术组(sham,n=6)及脑I/R模型组(I/R,n=6),线栓法制备右侧大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)模型。缺血1h/再灌注6h后提取患侧脑组织总RNA,质检合格后进行RNA-Seq。以|log2(Fold change)|>1,P<0.05为标准,筛选与脑I/R相关的lncRNAs,建立lncRNAs差异表达数据库。利用生物信息学方法对差异基因进行GO及KEGG分析。应用q RT-PCR方法对测序结果进行验证,获得上调表达明显的lncRNA PEG11as作为目标lncRNA进行功能学研究。第二部分:lncRNA PEG11as对脑I/R诱导神经元凋亡的影响。(1)lncRNA PEG11as对MCAO/R损伤小鼠脑神经元凋亡的影响:脑室注射lncRNA PEG11as干扰慢病毒(LV-ShRNA PEG11as)或阴性慢病毒(LV-ShRNA-NC),7天后构建小鼠MCAO/R模型,通过TTC染色、神经功能学评分、TUNEL染色、Western blot方法评估lncRNA PEG11as对脑I/R后对神经元损伤及凋亡的影响;(2)lncRNA PEG11as对OGD/R诱导N2a细胞凋亡的影响:体外培养N2a细胞构建氧葡萄糖剥夺(oxygen-glucose deprivation,OGD)/复氧复糖模型。N2a细胞转染lncRNA PEG11as干扰质粒(ShRNA-PEG11as)或阴性对照空载质粒(ShRNA-NC)后构建OGD3h/R24h模型,应用TUNEL染色及Western blot评估lncRNA PEG11as对OGD/R诱导N2a细胞凋亡的影响。第三部分lncRNA PEG11as对脑I/R诱导神经元凋亡的分子机制:应用RNA-FISH法确定lncRNA PEG11as的亚细胞定位;通过生物信息学方法,预测lncRNA PEG11as可作为竞争性内源RNA(competitive endogenousRNAs,ceRNA)在脑I/R损伤中发挥作用。RIP实验、免疫荧光、q RT-PCR及Western blot等方法检测lncRNA PEG11as/mi R-342-5p/Pfn1三者的ceRNA关系。TUNEL染色、Western blot、Hoechst 33258染色及Caspase 3酶活性检测等探讨mi R-342-5p及Pfn1在OGD/R诱导的N2a细胞凋亡中的作用。结果第一部分:lncRNA在小鼠脑I/R中表达:RNA-Seq测序结果显示,脑I/R组共254个lncRNA发生差异表达,其中上调249个,下调5个。验证结果发现,9个lncRNA表达趋势与测序结果一致,其中lncRNA PEG11as差异性倍数变化最为显著。lncRNA PEG11as在脑I/R体内、外模型中表达上调,具有时间依赖性。在I1h/R6h时上调倍数最大(P<0.01),随再灌注时间延长上调倍数逐渐降低,I1h/R48h时恢复至正常水平,与Sham或Control组比较无统计学差异(P>0.05)。GO及KEGG通路分析发现差异表达lncRNA主要富集细胞因子-细胞因子受体信号通路、NF-kappa B信号通路、MAPK信号通路及凋亡等。第二部分:lncRNA PEG11as对脑I/R神经元诱导凋亡的影响:体内敲减脑组织lncRNA PEG11as表达,可显著降低脑I/R后神经功能缺陷评分,缩小脑梗死体积,降低神经元凋亡率,抑制凋亡相关蛋白caspase 3活化,进而抑制神经元凋亡。体外敲低N2a细胞中lncRNA PEG11a表达,可显著降低OGD/R诱导的凋亡阳性细胞数及细胞凋亡率,减少cleaved caspase 3蛋白表达,进而抑制OGD/R诱导的细胞凋亡。第三部分:lncRNA PEG11as调控神经元凋亡与lncRNA PEG11as/mi R-342-5p/Pfn1通路相关:FISH亚细胞定位显示,lncRNA PEG11as主要定位于胞浆。生物信息技术预测,lncRNA PEG11as可作为mi R-342-5p内源性竞争RNA,通过影响Pfn1蛋白表达参与脑I/R损伤诱导的凋亡过程。q RT-PCR结果显示,脑I/R体内、外损伤模型中mi R-342-5p表达明显下调,敲低lncRNA PEG11as,可显著上调mi R-342-5p的表达,与lncRNA PEG11as表达呈负相关;RIP实验证实lncRNA PEG11as与mi R-342-5p可相互结合。过表达mi R-342-5p,可使OGD/R引起的细胞凋亡率降低,caspase 3蛋白活化减少,抑制细胞凋亡;而转染mi R-342-5p inhibitor则促进凋亡效应;共转染mi R-342-5p inhibitor及lncRNA PEG11as干扰质粒可使mi R-342-5p inhibitor促凋亡效应被部分逆转。生物信息学预测Pfn1是mi R-342-5p靶基因,在体内、外脑I/R中Pfn1蛋白表达显著上调,过表达/干扰mi R-342-5p,Pfn1蛋白表达降低/增加,与mi R-342-5p呈负相关。干扰lncRNA PEG11as,Pfn1表达水平下调,且这种效应被mi R-342-5p抑制剂部分中和,可见Pfn1与lncRNA PEG11as表达成正相关。干扰Pfn1表达,Hoechst凋亡阳性细胞数减少,caspase 3酶活性降低,细胞凋亡明显被抑制。结论脑I/R时,254个lncRNA表达发生差异性改变,其中lncRNA PEG11as上调显著;敲低lncRNA PEG11as的表达,可降低缺血再灌注诱导的神经元凋亡。其作用机制可能是,lncRNA PEG11as作为竞争性内源RNA(ceRNA),通过竞争性结合mi R-342-5p,上调mi R-342-5p靶基因Pfn1蛋白表达,促进神经元凋亡,参与脑I/R进程。本研究为lncRNA参与缺血性脑卒中的病理机制提供了新的理论依据,有望为缺血性脑卒中的临床防治提供新的干预靶点。更多还原

【Abstract】 BackgroundIschemic stroke has the characteristics of high morbidity,high disability,high mortality and high recurrence rate,and it has become a major disease that seriously threatens human health.At present,recombinant tissue plasminogen activator(rt-PA)and mechanical clot removal have been used clinically to treat acute ischemic stroke.However,due to the short therapeutic window(normally 3-4.5h after onset)and the complicated pathological mechanism,the clinical treatment of ischemic stroke has not yet made breakthrough progress.A large number of studies have reported that neuronal apoptosis and necrosis induced by ischemic/reperfusion(I/R)have been considered as one of the main pathophysiological mechanisms leading to brain damage.Therefore,focusing on the pathogenesis of inducing neuronal apoptosis,we might find the key molecules in the gene regulatory network,which is expected to provide new research approch for the prevention and treatment of acute ischemic stroke.Long non-codingRNAs(lncRNAs)is a type ofRNA with a length of more than200 nucleotides in eukaryotes.LncRNAs widely exist in tissues and cells,which regulate gene expression at the level of epigenetic,transcription,post-transcriptional and play an important role in many biology process.The abnormal expression of lncRNAs are closely related to the occurrence of cerebral ischemia reperfusion injury.Therefore,searching for lncRNAs,which are related to cerebral ischemia-reperfusion injury,is the important approch that explores their potential molecular mechanism,leading to the new targets for the treatment of ischemic stroke.ObjectiveTo obtain lncRNAs that is related to the pathological process of cerebral ischemia-reperfusion injury byRNA sequencing(RNA-Seq)technology,and further to explore the role and its molecular mechanism of the target lncRNA in cerebral ischemia-reperfusion,which might provide a theoretical basis for the prevention and treatment of ischemic stroke.MethodsPart 1.RNA-Seq technology was used to detect lncRNA expression profile in mouse cerebral I/R injury.Twelve C57BL/6 mice were randomly divided into the sham and cerebral I/R groups(n=6).The suture method was used to construct the model of middle cerebral artery occlusion(MCAO)in mice.After ischemic 1h and reperfusion 6h(I1h/R6h),brain tissue samples were collected to extract totalRNA,the expression prifile was detected byRNA-Seq sequencing.We screened the lncRNAs related to cerebral I/R and established the lncRNA differential expression database according to the standard of |log2(Fold change)|>1,P<0.05,and then the analysis of Gene Ontology(GO)and the Kyoto Encyclopedia of Genes and Genomes(KEGG)for the differentially expressed genes using bioinformatics methods.Quantitative real time polymerase chain reaction(q RT-PCR)was used to validate the genes selected fromRNA-Seq.Among them,the up-regulated lncRNA PEG11 as was identified as the target lncRNA involved in cerebral I/R.Part 2.The effect of lncRNA PEG11 as on cerebral I/R-induced neuronal apoptosis.(1)The effect of lncRNA PEG11 as on cerebral I/R-induced neuronal apoptosis in mice: C57BL6 mice were intraventricular injection with the interfering lentivirus of lncRNA PEG11as(LV-ShRNAPEG11as)or its negative lentivirus(LV-ShRNA NC)for7days,and then subjected to MCAO/R.After 24 h reperfusion,TTC staining,neurological score,TUNEL staining and western blot were used to evaluate the effect of lncRNA PEG11 as on infarct volume,neurological deficit,neuronal apoptosis and the expression of apoptotic related genes in brain tissue.(2)The effect of lncRNA PEG11 as on OGD/R-induced apoptosis in N2 a cells: N2 a cells were transfected with lncRNA PEG11 as interfering plasmid(ShRNA-PEG11as)or its negative plasmid(ShRNA-NC)for 48 h,and then exposed to OGD 3h/R 24 h.TUNEL staining and western blot were used to evaluate neuronal apoptosis and the level of the apoptotic related genes.Part 3.The mechanism of lncRNA PEG11 as on regulating neuronal apoptosis in cerebral I/R injury:RNA-FISH was used to determine the subcellular location of lncRNA PEG11as;furthermore,the bioinformatics analysis was used to predict that lncRNA PEG11 as could be used as a competitive endogenousRNA(ceRNA)in cerebral I/R damage.The analysis ofRNA-binding protein immunoprecipitation(RIP),immunofluorescence,q RT-PCR and western blot was used to detect ceRNA interaction of lncRNA PEG11as/mi R-342-5p/Pfn1.TUNEL staining,western blot,hoechst 33258 staining and the activity of caspase 3 enzyme were used to investigate the role of mi R-342-5p and Pfn1 in OGD/R-induced N2 a cell apoptosis.ResultsPart 1.The differential expression of lncRNA involved in mice cerebral I/R.RNA-Seq sequencing results showed that compared with the sham group,a total of 254 lncRNAs were differentially expressed in the cerebral I/R group.Among them,249 lncRNAs were up-regulated and 5 lncRNAs were down-regulated.The q RT-PCR was used to verified the expression of 9 lncRNAs predicted byRNA-Seq,and lncRNA PEG11 as was identified to be the most significantly expression lncRNA.LncRNA PEG11 as expression was up-regulated,and associated with the time-dependent changes in I/R and OGD/R.At I1h/R6 h,the expression of lncRNA PEG11 as was the highest(P<0.01),and then decreased with the extension of reperfusion time.At I1h/R48 h,the expression of lncRNA PEG11 as was returned to normal,and there was no statistically significant difference compared with sham or control group(P >0.05).GO and KEGG pathway analyses identified significantly enriched pathways in cytokines-cytokine receptor signaling pathway,NF-kappa B signaling pathway,MAPK signaling pathway and apoptosis signalling pathway.Part 2.The effect of lncRNA PEG11 as on cerebral I/R-induced neuron apoptosis.Knockdown of lncRNA PEG11 as reduced the neurological deficit score,decreased cerebral infarct volume,suppressed the expression of apoptosis-related protein cleaved caspase 3 and inhibited neuronal apoptosis in mice cerebral I/R.Consistent with the in vivo findings,lncRNA PEG11 as inhibition also significantly reduced the number of apoptotic positive cells and attenuated the rate of apoptosis,decreased the protein expression of cleaved caspase3,and thereby inhibit OGD/R-induced apoptosis in N2 a cells.Part 3.lncRNA PEG11 as regulates neuronal apoptosis associated with the lncRNA PEG11as/mi R-342-5p/Pfn1 pathway.FISH subcellular localization revealed that lncRNA PEG11 as was mainly located in the cytoplasm.Bioinformatics predicted that lncRNA PEG11 as could act as a ceRNA for mi R-342-5p to affecting Pfn1 protein expression and participate in the apoptotic process in cerebral I/R-induced.The q RT-PCR results showed mi R-342-5p was downregulated in cerebral I/R and OGD/R.Knockdown of lncRNA PEG11 as significantly upregulated mi R-342-5p expression,which was negatively negatively regulated by lncRNA PEG11 as.RIP assays revealed that lncRNA PEG11 as bound to mi R-342-5p directlly.mi R-342-5p mimic decreased the rate of apoptosis of N2 a cells,surpressed the level of cleaved caspase 3 protein and inhibited OGD/R-induced apoptosis,while effects were reversed after mi R-342-5p inhibitor treatment.Co-transfection of mi R-342-5p inhibitor and shRNA-lncRNA PEG11 as attenuated the pro-apoptotic effect of mi R-342-5p inhibitor.Bioinformatics predicts that Pfn1 was a target gene of mi R-342-5p.Pfn1 protein expression is significantly upregulated in cerebral I/R and OGD/R.Further investigations demonstrated that Pfn1 expression level was negatively correlated with mi R-342-5p and positively correlated with lncRNA PEG11 as.Silencing Pfn1 led to the attenuation in the number of hoechst apoptosis-positive cells and decrease in caspase 3 enzyme activity,ultimately,apoptosis was inhibited.ConclusionIn total,254 lncRNAs were identified in cerebral I/R,of which lncRNA PEG11 as was significantly up-regulated.LncRNA PEG11 as knockdown could reduce cerebral I/R-induced neuronal apoptosis.Mechanistic study further revealed that lncRNA PEG11 as functions as a ceRNA for mi R-342-5p and regulates the Pfn1 expression thus promoting cerebral I/R-induced apoptosis.This study provides a new theoretical basis for lncRNA to participate in the pathological processes of ischemic stroke,and provides a novel intervention target for the clinical prevention and treatment of ischemic stroke.更多还原