乙酰胆碱受体抗体阳性重症肌无力外周血单个核细胞miRNA表达谱

目的 本研究对比乙酰胆碱受体抗体阳性重症肌无力患者（AchR-MG）和正常对照组外周血单个核细胞miRNA,预测对AchR-MG发病可能产生影响的通路,为进一步探讨发病机制打下基础。方法 采用病例对照研究方法,基于高通量测序,筛选了AchR-MG特异性表达的miRNA。利用TargetScan、miRanda进行靶基因交叉预测,利用基因条目（GO）和京都基因与基因组百科全书（KEGG）进行富集分析。结果 共筛选出差异性miRNA 28种,其中上调17种,下调11种。差异最显著的前5个为：mmu-miR-3968、miR-4785、miR-210-3p、miR-664a-3p、miR-2277-5p。miR-4785预测到METTL22、TMEM38A、ZNF324、ITGB4、CDC34等395种靶基因。最终识别了319条GO term（P<0.01）,获得了119个的风险通路（P<0.05）。结论 AchR-MG特异性表达miR-4785、miR-210-3p、miR-664a-3p、miR-2277-5p等28种miRNA。以Wnt信号通路为代表的多种通路可能参与AchR-MG的发病。     

重型颅脑损伤病人外周血miRNA生物信息学分析

目的 通过生物信息学方法分析重型颅脑损伤（sTBI）病人外周血微小RNA（miRNA）的靶基因及功能。方法 从GEO数据库中检索获取sTBI病人和对照组外周血的基因芯片数据,应用生物信息学方法筛选差异表达的miRNA,并进行靶基因预测和生物学功能及信号通路分析,构建miRNA及靶基因的调控网络。结果 检索得到芯片GSE21854,筛选得到145个差异表达的miRNA,预测得到靶基因共580个。这些靶基因的功能主要为细胞增殖负性调控、转换生长因子β受体信号通路负性调控等,主要分布在Ras信号通路、转换生长因子β信号通路等。miRNA及靶基因的调控网络图显示hsa-miR-125a-5p、hsa-miR-760、hsa-miR-217、hsa-miR-199a-3p、hsa-miR-543是调控核心。结论 sTBI病人外周血存在差异性表达的miRNA,hsa-miR-125a-5p、hsa-miR-760、hsa-miR-217、hsa-miR-199a-3p、hsa-miR-543与sTBI的进展密切相关。     

无症状性脑动脉粥样硬化与大动脉粥样硬化性卒中患者血浆微小RNA表达分析

目的 建立无症状性脑动脉粥样硬化（ath e ro s cl e ro s i s,AS）及大动脉粥样硬化（l arge arte ry atherosclerotic,LAA）性卒中患者血浆微小核糖核苷酸（microribonucleic acid,miRNA）差异表达谱。 方法 收集2013年1月～2013年2月于青岛大学医学院附属医院神经内科就诊的8名AS患者（AS组）、 LAA性卒中患者（LAA组）和对照者（control组）的空腹前臂静脉血血浆标本,应用Solexa高通量测序 技术检测每组血浆标本miRNA表达谱,筛选出差异表达的miRNA,并进行靶基因预测和功能分析,采 用实时荧光定量聚合酶链反应（polymerase chain reaction,PCR）技术对高通量检测结果进行验证。 结果 3组间比较,表达差异均具有显著性的miRNA有miR-let-7a-5p、miR-146b-5p、miR-26a-5p、 miR-23a-3p等26个;在AS组与LAA组表达一致,较对照组表达差异具有显著性的miRNA有miR-10b-5p、 miR-126-5p、miR-143-3p、miR-192-5p等41个。本研究生物信息学分析发现,差异miRNAs调控的靶基 因主要与细胞黏附、分化、增殖等生物学过程相关。实时荧光定量PCR检测miR-146b-5p、miR-23a-3p、 miR-10b-5p,结果与高通量检测结果一致。 结论 AS患者及LAA性卒中患者血浆miRNAs表达谱既有差别又有相同之处。     

生长激素腺瘤中差异表达的微小RNAs及其靶基因的相关生物信息学分析

目的探讨垂体生长激素(GH)细胞腺瘤中差异表达的微小RNAs(miRNAs)及其靶基因的生物学功能和两者的调控关系。方法利用miRDB、miRwalk、Targetscan7.2及starbase数据库对差异表达的miRNAs进行靶基因预测并对靶基因进行GO、KEGG和蛋白-蛋白相互作用网(PPI)分析,随后筛选出有意义的核心靶基因,取核心靶基因和数据库的mRNAs测序结果进行对比,构建可视化的miRNAs-靶基因调控关系网。结果以蛋白相互作用程度≥20为标准,本研究得到113个上调的miRNAs核心靶基因和128个下调的miRNAs核心靶基因,进一步取交集得到13个目的核心靶基因,这些基因主要涉及的通路为泛素介导蛋白水解通路,其相对应的调控miRNAs为miR-15b、miR-365、miR-32-3p、miR-486-5p。结论本研究应用生物信息学分析工具构建了与GH细胞腺瘤密切相关的miRNAs-核心靶基因相互调控网,发掘了一些可能与GH细胞腺瘤发病机制和病理过程有关的蛋白和通路。     

帕金森病患者血清胞外囊泡中miRNAs表达水平变化及其对疾病进展的影响

目的 探讨帕金森病患者血清胞外囊泡中微小RNAs(miRNAs)表达水平变化及其对疾病进展的影响。方法 采用回顾性研究,收集本院85例帕金森病患者的临床资料,根据发病年龄段的不同分为早发组(早发性帕金森病,发病年龄不超过50岁)37例和晚发组(晚发性帕金森病,发病年龄超过50岁)48例,另以本院30例体检健康人员为对照组; 采集3组血清标本,并提取胞外囊泡,采用miRNA表达谱芯片和实时荧光定量聚合酶链反应对miRNAs表达水平进行检测; 通过Cytoscape分析miRNAs共同调控的靶基因,并通过String数据库探讨靶基因的相互作用和功能; 采用实时荧光定量聚合酶链反应检测早发组与晚发组血清胞外囊泡中靶基因的相对表达水平。结果 晚发组血清胞外囊泡中miR-500a-5p和miR-451表达水平较对照组均显著降低,而miR-1180-3p和miR-143-3p表达水平较对照组显著升高(P<0.01)。与早发组比较,晚发组血清胞外囊泡中miR-143-3p表达水平明显降低(P<0.01); 2组血清胞外囊泡中miR-451,miR-500a-5p及miR-1180-3p表达水平比较均无明显差异(P>0.05)。经Cytoscape分析发现,Akt1和Bcl-2是miR-143-3p的关键靶基因。与早发组比较,晚发组血清胞外囊泡中Bcl-2表达水平明显升高(P<0.01); 2组血清胞外囊泡中Akt1表达水平比较并无明显差异(P>0.05)。结论 miR-143-3p与其靶基因Bcl-2均异常表达于帕金森病患者,其中miR-143-3p在早发性帕金森病患者血清胞外囊泡中的表达水平明显提高,Bcl-2的表达水平明显降低,提示二者表达水平的异常改变可能与帕金森病的疾病进展密切相关。     

胶质母细胞瘤患者外周血中miRNA特异性表达研究

目的检测GBM患者和健康人外周血中miRNA的异常表达。方法利用基因芯片分析GBM患者和正常对照者血清中miRNA的表达量,然后进一步进行靶基因的生物信息学分析。结果miRNA芯片表明在血清中15例GBM组和15例正常对照组的外周血miRNA表达有明显差异。752种miRNA中,GBM组有95种miRNA表达上调,22种表达下调。(倍数≥2.0,P0.01)。通过深入分析,我们发现GBM患者miR-576-5p、miR-340和miR-626过表达,但miR-320、let-7g-5p、miR-7-5p低表达。进一步相关生物信息学分析,我们发现,他们在脑胶质瘤信号通路的调控中发挥重要的作用。结论与正常人比较,以上6种miRNA在GBM患者外周血中有显著差异。外周血中的miRNA表达谱中一些特异性表达的miRNA可能作为高特异性和灵敏度诊断胶质瘤的新靶向标志物。     

血浆及外周血单个核细胞微小RNA在精神分裂症诊断中的研究

目的:探索血浆及外周血单个核细胞(PBMC)中微小RNA (miRNA)在精神分裂症(SZ)诊断中的价值。方法:根据文献报道筛选出与SZ相关的7种miRNA (has-miR-449a,has-miR-34a-5p,has-miR-652-3p,has-miR-564,has-miR-432-5p,has-miR-548d-5p和has-miR-572);提取35例初诊的SZ患者(患者组)及15名健康体检者(对照组)的抗凝全血的血浆及PBMC中的RNA,采用实时逆转录聚合酶链式扩增反应技术测定血浆及PBMC中这7种miRNA水平,计算其相对表达量。结果:患者组血浆及PBMC中4种miRNA(miR-652-3p,miR-564,miR-432-5p和miR-548d-5p)相对表达量较对照组明显增加(P均0.05);患者组7种miRNA的相对表达量血浆明显高于PBMC(P均0.05);多变量ROC曲线显示,患者组血浆miRNA(has-miR-652-3p,has-miR-564,has-miR-548d-5p)ROC曲线下面积为98%,灵敏度为90.9%,特异度为95.4%;PBMC miRNA(has-miR-652-3p,has-miR-564,has-miR-432-5p以及has-miR-548d-5p)ROC曲线下面积为86%;灵敏度为88.2%,特异度为78.3%。结论:SZ患者血浆及PBMC中的miRNA(miR-652-3p,miR-564,miR-432-5p和miR-548d-5p)相对表达量明显增高;血浆miRNA更有潜力成为SZ的分子诊断标志物。     

微小核糖核酸miR-146a-5p、miR-23a-3p在儿童耐药性癫痫血清中的表达

目的 探讨miR-146a-5p、miR-23a-3p在耐药性癫痫患儿血清中的表达水平及其临床意义.方法 收集68例耐药性癫痫患儿、64例非耐药性癫痫患儿及55例体检健康者静脉血标本,用Hiseq测序法对血清微小核糖核酸(microRNA,miRNA)测序并测定表达量,用RT-qPCR验证耐药性癫痫患儿血清差异性表达的miR-146a-5p、miR-23a-3p.结果 与非耐药性癫痫比较,Hiseq 测序显示耐药性癫痫患儿血清中,78种miRNA 表达量上升,22种miRNA表达量下降,miR-146a-5p、miR-23a-3p的fold-change分别为1.241和1.302.RT-qPCR结果表明,与非耐药性癫痫miR-146a-5p(1.183±0.848)、miR-23a-3p(1.633±0.970)表达水平比较,耐药性癫痫患儿分别为(1.563±1.831)和(2.119±1.543),表达水平均升高,差异有统计学意义(t 分别为2.142 和2.150,P 均<0.05),而且与非耐药性癫痫用药前比较,二者在耐药性癫痫用药前血清中表达已经上升,差异有统计学意义(t分别为2.381和2.345,P 均<0.05).结论 耐药性癫痫患儿血清miR-146a-5p、miR-23a-3p表达升高,有望成为早期诊断及评估预后的分子标记物.     

血清microRNA在烟雾病发病机制中的潜在作用

目的 烟雾病发病机理尚有诸多未知,烟雾病血清微小RNA(microRNA,miRNA)表达谱可能会为该疾病诊断和预后判断提供新的生物学标记物.本研究拟寻找在烟雾病发病机制中扮演重要角色的血清miRNAs.方法 10例烟雾病患者和10例正常对照血清使用基因芯片筛选差异表达的miRNAs.通过实时聚合酶链反应(RT-PCR)进行结果验证.通过基因本体论(GO)分析诠释关键信号通路和参与烟雾病发病机制的相关miRNAs.结果 基因组miRNA序列显示烟雾病患者血清中94个miRNAs差异表达,其中上调50个,下调44个.RT-PCR验证了miRNA106b,miRNA130a和miRNA126显著上调,而miRNA125a-3p则显著下调.通过目标预测软件检测并定义潜在功能目标后鉴定了1989个潜在功能目标.GO分析表明差异表达的miRNAs在代谢过程、转录和信号转导中富集.结论 本研究通过基因芯片检测烟雾病miRNA标志物并揭示环指蛋白213 (RNF213)和乳腺癌易感基因复合物3(BRCC3)的蛋白表达在烟雾病发病机制中可能发生的作用.     

维吾尔族烟雾病病人血清miRNA的异常表达及其基因分析

目的 探讨我国新疆地区维吾尔族（简称维族）烟雾病病人血清异常表达的miRNA。方法 收集2017年2月至2018年8月新疆莎车县人民医院救治的8例维族烟雾病与8例维族健康者（对照组）的血样,运用高通量全基因芯片技术检测血清miRNA,筛选差异表达水平大于1.5倍的miRNA,然后利用targetscan软件进行靶基因的预测和分析。结果 发现54个差异表达的miRNA,选择表达水平变化倍数R在0.67~1.5的差异表达miRNA共4个,其中显著上调3个（miR-188-5p、miR-4284及miR-5100）,显著下调1个（miR-6277-3p）。靶基因生物学分析结果显示维吾尔族烟雾病可能与某类锌指蛋白有关。结论 维族烟雾病病人血清miRNA有异常表达,通过生物信息学分析,其致病基因可能某类锌指蛋白有关。     

Circulating Plasma Micro RNAs in Patients with Major Depressive Disorder Treated with Antidepressants: A Pilot Study

ObjectiveSignificant progress was made in the understanding etiopathogenic factors related to MDD, including through research on the role of micro RNAs (miRs). We investigated plasma miRs as potential markers for MDD in patients treated with antidepressants.MethodsAt the initiation and at the end of twelve weeks of treatment, blood samples were collected and a structured diagnostic interview and a standardized depression rating scale for the presence and severity of major depression were done. The average decrease in HAMD score was 76.89%. Plasma miR expression profiling was performed by real time PCR. The lists of up-regulated (cut-off=2) and down-regulated miRs were imported into the miRWalk2.0 algorithm and used for target predictions. KEGG database pathways analysis was used to retrieve the pathways significantly targeted by at least two of the miRs.ResultsOf the 222 miRs detected in plasma samples of MDD patients, 40 were differentially expressed after treatment. Twenty-three miRs were significantly overexpressed with fold changes between 1.85 and 25.42, and 17 miRs were significantly downregulated with fold changes from 0.28 to 0.68. Pathway analysis revealed a list of 29 pathways for up-regulated miRs, and 20 pathways for down-regulated miRs. Six dysregulated miRs are common to all the top five pathways (Wnt signaling, Cancer, Endocytosis, Axon guidance, MAPK signaling): miR-146a-5p, miR-146b-5p, miR-221-3p, miR-24-3p, miR-26a-5p.ConclusionOverall, our miRWalk analysis of changes in plasma microRNAs after treatment of patients with major depression might open a new avenue for the understanding of Escitalopram mode of action and for its side effects.     

MicroRNA abundance is altered in synaptoneurosomes during prion disease

Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimer's disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p).     

Bioinformatics and microarray analysis of microRNA expression profiles of murine embryonic stem cells,neural stem cells induced from ESCs and isolated from E8·5 mouse neural tube

Abstract

To better understand whether microRNAs (miRNAs) are involved in the self-renewal of stem cells and fate determination of neural stem cells and to identify the miRNA expression patterns of different neural stem cells (NSC) in vitro and in vivo, we examined miRNA expression profiles of murine embryonic stem cells (ESC), NSC induced from ESC and isolated from E8·5 mouse neural tube (E8·5-NSC) using microarray technique. It was found that a total of 40 miRNAs had similar expression level in all the three cells [false discovery rate (FDR)=0, fold change <3·0]. Moreover, q-PCR showed that some members of miR-106b and miR-17–92 families were expressed in the ESC, NSC induced from ESC (ESC-NSC) and hematopoietic stem cells (HSC). Bioinformatical analysis showed that 'stemness genes' (p21/CDKN1A, p57/CDKN1C and PTEN) were putative targets of miR-106b and miR-17–92 families. A total of 95 miRNAs were found to experience significant change (FDR=0, fold change >5·0) when the ESC differentiated into NSC. On the basis of miRNA, mRNA expression variance and predicted target genes of miRNA, we formulated a bioinformatical model for miRNA control of ESC-NSC differentiation. Then, the miRNA expression pattern was compared between NSC obtained in vitro and in vivo, and it was found that only 8% of miRNAs were different between the two NSCs. This study suggested that miR-106b and miR-17–92 families may promote the renewal of stem cells by targeting PTEN, p21/CDKN1A and p57/CDKN1C. Some miRNAs may play a key role in gene re-programming during ESC-NSC differentiation, and a substantial homogeneity exists between NSCs derived in vitro and those in vivo.     

MiR-29a-3p Enhances the Viability of Rat Neuronal Cells that Injured by Oxygen-Glucose Deprivation/Reoxygenation Treatment Through Targeting TNFRSF1A and Regulating NF-κB Signaling Pathway

ObjectiveWe attempt to investigate the role of TNFRSF1A and its underlying mechanism in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in rat pheochromocytoma PC12 cells.MethodsPublic datasets GSE61616 and GSE106680 were downloaded from GEO database. PC12 cells were used to construct OGD/R models. QRT-PCR and western blot were implemented to test the relative mRNA and protein levels, respectively. The miRNA online prediction website TargetScan was used to predict TNFRSF1A upstream regulated miRNAs, which were then confirmed by luciferase reporter assay. The changes in cell viability and apoptosis were evaluated using cell counting kit 8 (CCK-8), lactose dehydrogenase (LDH), and flow cytometry assays.ResultsBioinformatics analysis demonstrated that the expression of TNFRSF1A was upregulated in CI/RI and middle cerebral artery occlusion models compared with control, respectively. And a significant upregulation was also observed in OGD/R-damaged PC12 cells. Depletion of TNFRSF1A can notably enhance the cells proliferation after OGD/R treatment, while enlargement of TNFRSF1A presented the opposite outcomes. Moreover, miR-29a-3p was shown to be the upstream regulatory miRNA of TNFRSF1A. The levels of TNFRSF1A were inversely mediated by miR-29a-3p. Overexpression of miR-29a-3p can raise the cell viability, decrease the LDH activity, and reduce the apoptotic ratio in OGD/R-treated cells. Besides, TNFRSF1A can attenuate the protective effect of miR-29a-3p on OGD/R-treated cells. Furthermore, miR-29a-3p mimic inhibited, while overexpression of TNFRSF1A promoted the activation of NF-κB signaling pathway, and TNFRSF1A can attenuate the suppressive effect of miR-29a-3p on the NF-κB pathway.ConclusionOur research illustrated that the potential regulatory role of miR-29a-3p/TNFRSF1A axis in neurons cells suffered from OGD/R, and their effects on NF-κB signaling pathway, providing a possible bio-target for protecting cells from OGD/R damage .     

Epigenetics Modifications in Large-Artery Atherosclerosis: A Systematic Review

ObjectivesIn recent years, the evidence of the relationship between epigenetics and acute ischemic stroke (AIS) were accumulating, however, the epigenetic characteristics that directs specifically towards the aetiology of large-artery atherosclerosis (LAA) remain ambiguous. The aim of this study was to highlight the overall evidence concerning the epigenetic mechanisms associated with the occurrence of LAA.Materials and methodsStudies that involve investigations related to epigenetic markers (DNA methylation and RNA modifications) and LAA were retrieved from eleven scientific publication databases. The studies were screened through the pre-set inclusion and exclusion criteria prior to the NOS evaluation.ResultsEligible studies (n=25) were evaluated. Of which, six reported on DNA methylation and 19 studies assessed RNA modifications (16 on miRNAs, two on lncRNAs, and one study on circRNA). Hypomethylation of MTRNR2L8 and ERα promoters; microRNAs (miR-7-2-3p, miR-16, miR-34a-5p, miR-126, miR-143, miR-200b, miR-223, miR-503, miR-1908, miR-146a rs2910164 C/G, miR-149 rs2292832 T/C, miR-200b rs7549819 T/C, miR-34a rs2666433); lncRNA of ZFAS1; and circRNA of hsa_circRNA_102488 were associated with LAA significantly.ConclusionCurrent systematic review highlighted hypomethylation of miRNAs and lncRNA might be the potential biomarkers for LAA.     

Neuroprotective effect of ischemic preconditioning via modulating the expression of cerebral miRNAs against transient cerebral ischemia in diabetic rats

Objectives: In this study, we aimed to evaluate the effect of the Ischemic preconditioning (IPreC) on the expression profile of cerebral miRNAs against stroke by induced transient middle cerebral artery occlusion (MCAo) in diabetic rats.

Methods: Eighty male Spraque Dawley rats were allocated to eight groups. In order to evaluate the expression profile of miRNAs, we induced transient MCAo seven days after STZ-induced diabetes (DM). Also we performed IPreC 72 h before transient MCAo to assess whether IPreC could have a neuroprotective effect against ischemia-reperfusion injury.

Results: The general characteristics of STZ-treated rats included reduced body weight and elevated blood glucose levels compared to non-diabetic ones. We demonstrated that miRNA expression profiles, which are determined for biological functions such as aquaporin 4 formation (miR-29b-2, miR-124a-3p, miR-130a, miR-223 and miR-320a), glutamate toxicity (miR107, miR-145, miR-223), salvageable ischemic area (miR-9a, miR-19b, miR-29b-2, miR-341, miR-339–5p, miR-15–5p, miR-99b-5p), and neoangiogenesis (let-7f-5p, miR-126a and miR-322–3p), were regulated following IPreC. Ischemic preconditioning before cerebral ischemia significantly reduced infarction size compared with the other groups [IPreC + MCAo (27 ± 11 mm³) vs. MCAo (109 ± 15 mm³) p < 0.001; DM + IPreC + MCAo (38 ± 9 mm³) vs. DM + MCAo (165 ± 41 mm³) p < 0.001, respectively].

Discussion: The study results revealed the neuroprotective effects of ischemic preconditioning, supported with the upregulated pro-survival miRNAs in MCA infarcts.