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.     

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。     

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。     

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。     

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。     

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(P0.05)。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。     

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。     

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。     

miRNA调控SCN2A参与癫痫的分子机制研究

目的在体内和体外癫痫模型中,探讨miRNA-132在癫痫中的作用及其机制。方法利用氯化锂-匹罗卡品构建大鼠颞叶癫痫模型及对照,通过real-time PCR检测miRNA的表达;利用Retinoic acid(RA)构建体外癫痫细胞模型,分别用Real-time PCR和Western blot检测miR-132-3p及SCN2A的miRNA及蛋白表达水平;在体外癫痫细胞模型中加入miR-132-3p的抑制剂,检测miR-132-3p及SCN2A的表达水平;在体内大鼠癫痫模型中,分析二者表达的相关性。结果在体外细胞癫痫模型中,miR-132-3p的miRNA和蛋白表达显著增加,而SCN2A的miRNA和蛋白表达显著下降;在体外细胞模型中证实miR-132-3p与SCN2A之间存在靶向抑制关系;在大鼠癫痫模型中证明miR-132-3p与SCN2A的表达呈现负相关关系。结论在体内和体外癫痫模型中,miR-132的表达升高,而其靶基因SCN2A的表达降低,且两者呈现负相关关系,表明miR-132通过其靶基因SCN2A参与癫痫的发生发展。     

miR-221在脑胶质瘤中的表达及调控关系

目的探讨miR-221在脑胶质瘤组织中的表达变化及其对胶质瘤细胞系细胞增殖的影响。方法收集2014年1月至2015年8月手术切除的胶质瘤标本123例及颅脑损伤行内减压的正常脑组织36例,荧光定量PCR法检测miR-221表达水平。按照miR-221的表达水平将123例胶质瘤病人分为高表达组(miR-221/U6的相对表达量≥0.6,72例)和低表达组(miR-221/U6的相对表达量0.6,51例),采用Kplan-Meier法分析miR-221表达水平与胶质瘤生存时间的关系。MTT法检测miR-221对胶质瘤细胞系U251、U87细胞增殖的影响,双荧光试验和蛋白印迹法、PCR方法筛选和验证miR-221的靶基因。结果胶质瘤组织miR-221表达水平明显高于正常脑组织(P0.01);高级别胶质瘤miR-221表达水平明显低于低级别胶质瘤(P0.01)。miR-221低表达胶质瘤病人生存时间较高表达病人明显延长(P0.05)。敲除miR-221基因显著抑制U251、U87细胞增殖(P0.01);而且,U251、U87细胞敲除miR-221基因后,正性调节区锌指蛋白2(PRDM2)mRNA和蛋白表达水平明显增高(P0.01)。结论miR-221在胶质瘤中异常高表达,可作为胶质瘤预后判断的生物标志物;PRDM2可能是miR-221的靶基因。     

Patterns of microRNA expression in normal and early Alzheimer’s disease human temporal cortex: white matter versus gray matter

MicroRNA (miRNA) expression was assessed in human cerebral cortical gray matter (GM) and white matter (WM) in order to provide the first insights into the difference between GM and WM miRNA repertoires across a range of Alzheimer's disease (AD) pathology. RNA was isolated separately from GM and WM portions of superior and middle temporal cerebral cortex (N = 10 elderly females, postmortem interval < 4 h). miRNA profiling experiments were performed using state-of-the-art Exiqon^© LNA-microarrays. A subset of miRNAs that appeared to be strongly expressed according to the microarrays did not appear to be conventional miRNAs according to Northern blot analyses. Some well-characterized miRNAs were substantially enriched in WM as expected. However, most of the miRNA expression variability that correlated with the presence of early AD-related pathology was seen in GM. We confirm that downregulation of a set of miRNAs in GM (including several miR-15/107 genes and miR-29 paralogs) correlated strongly with the density of diffuse amyloid plaques detected in adjacent tissue. A few miRNAs were differentially expressed in WM, including miR-212 that is downregulated in AD and miR-424 which is upregulated in AD. The expression of certain miRNAs correlates with other miRNAs across different cases, and particular subsets of miRNAs are coordinately expressed in relation to AD-related pathology. These data support the hypothesis that patterns of miRNA expression in cortical GM may contribute to AD pathogenetically, because the aggregate change in miRNA expression observed early in the disease would be predicted to cause profound changes in gene expression.     

Transcriptome analysis reveals disparate expression of inflammation-related miRNAs and their gene targets in iPSC-astrocytes from people with schizophrenia

Despite the high heritability of schizophrenia (SCZ), details of its pathophysiology and etiology are still unknown. Recent findings suggest that aberrant inflammatory regulation and microRNAs (miRNAs) are involved. Here we performed a comparative analysis of the global miRNome of human induced pluripotent stem cell (iPSC)-astrocytes, derived from SCZ patients and healthy controls (CTRLs), at baseline and following inflammatory modulation using IL-1β. We identified four differentially expressed miRNAs (miR-337-3p, miR-127-5p, miR-206, miR-1185-1-3p) in SCZ astrocytes that exhibited significantly lower baseline expression relative to CTRLs. Group-specific differential expression (DE) analyses exploring possible distinctions in the modulatory capacity of IL-1β on miRNA expression in SCZ versus CTRL astroglia revealed trends toward altered miRNA expressions. In addition, we analyzed peripheral blood samples from a large cohort of SCZ patients (n = 484) and CTRLs (n = 496) screening for the expression of specific gene targets of the four DE miRNAs that were identified in our baseline astrocyte setup. Three of these genes, LAMTOR4, IL23R, and ERBB3, had a significantly lower expression in the blood of SCZ patients compared to CTRLs after multiple testing correction. We also found nominally significant differences for ERBB2 and IRAK1, which similarly displayed lower expressions in SCZ versus CTRL. Furthermore, we found matching patterns between the expressions of identified miRNAs and their target genes when comparing our in vitro and in vivo results. The current results further our understanding of the pathobiological basis of SCZ.     

MicroRNA Expression Aberration in Chinese Patients with Relapsing Remitting Multiple Sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease that is caused by an abnormal immune system that attacks the central nervous system. MicroRNAs (miRNAs) are known to play essential roles in the immune system. Most of the previous studies about miRNA dysregulation in MS have focused on European populations. In the present study, the miRNA expression profiles associated with Chinese MS patients are investigated. Here, human miRNA expression profiling experiments were performed on the peripheral blood mononuclear leukocytes from ten MS patients and ten healthy controls. Nine overexpressed and six underexpressed miRNAs were found. The 15 miRNAs were validated independently in a second cohort of 40 MS patients and 40 controls by real-time PCR; six miRNAs were significantly abnormally expressed, and principal component analysis of six miRNAs indicated that the MS patients could be clearly differentiated from the healthy controls based on the miRNA expression patterns. This study provided the indications of abnormal miRNA expression patterns in the peripheral blood mononuclear leukocytes of Chinese MS patients. The potential roles of these differentially expressed miRNAs as MS biomarker and in pathogenesis need to be investigated in future studies.     

Integrated analysis of microRNA and mRNA expression profiles in the rat spinal cord under inflammatory pain conditions

Recent studies using microarray‐based approaches have demonstrated that microRNAs (miRNAs) are involved in pain processing pathways. However, a significant proportion of computational predictions of miRNA targets are false‐positive interactions. To increase the chance of identifying biologically relevant targets, we performed an integrated analysis of both miRNA and mRNA expression profiles in the rat spinal cord during complete Freund's adjuvant (CFA)‐induced inflammatory pain. We generated miRNA and mRNA arrays from the same corresponding samples on days 5 and 14 after CFA injection. Five miRNAs and 1096 mRNAs in the CFA 5d group and 16 miRNAs and 647 mRNAs in the CFA 14d group were differentially expressed based on a filter of at least a 1.5‐fold change in either direction. An integrated analysis revealed 54 mRNA targets with an inverse correlation to the expression patterns of three miRNAs in the CFA 5d group. Seventy‐five targets were inversely correlated to six miRNAs in the CFA 14d group. The miRNA–mRNA interaction networks revealed significant changes in miR‐124, miR‐149, miR‐3584 and their target genes, IL‐6R, ADAM19, LAMC1 and CERS2, in the CFA 5d group. In the CFA 14d group, significant changes were noted in miR‐124, miR‐29, miR‐34, miR‐30, miR‐338 and their target genes, TIMP2, CREB5 and EFNB1. We also investigated an interaction pair, miR‐124‐3p and IL‐6R, and the results showed that miR‐124‐3p could attenuate inflammatory pain and decrease IL‐6R expression in the spinal cord. These specific miRNAs and their target genes provide possible avenues for the diagnosis and treatment of inflammatory pain.