60Co γ线照射血管内皮细胞外泌体中miRNA的鉴定分析

目的 利用微小RNA(miRNA)芯片和生物信息学技术,研究受照射人脐静脉血管内皮细胞(HUVEC)产生的外泌体miRNA组分的变化,为揭示血管组织放射损伤及其旁效应机制提供新的线索.方法 超高速离心法收集对照组和4 Gy剂量照射组的HUVEC外泌体,运用电镜及Western印迹技术对外泌体进行鉴定,miRNA芯片技术分析细胞内和外泌体中miRNA表达谱,qRT-PCR法验证部分差异miRNA,通过miRDB和TargetScan预测差异miRNA的靶基因,DAVID、KEGG等在线工具进行生物信息学分析.结果 HUVEC经4 Gy照射后外泌体miRNA与对照组相比,照后0.5 h共鉴定到18个发生表达变化的miRNA分子,5个表达上调,13个表达下调;照后2 h鉴定出16个表达上调、5个表达下调miRNA分子;细胞内miRNA与对照组相比,照射后0.5、2 h分别有38个和85个差异表达miRNA,且差异有统计学意义(P<0.01).生物信息学结果表明,这些表达变化的miRNA可能通过参与调控MAPK、Ras、PI3K-Akt信号通路等途径影响细胞辐射旁效应.结论 电离辐射损伤导致血管内皮细胞外泌体miRNA分子组分和表达水平发生显著改变,这些miRNA的靶基因组产物在细胞放射损伤反应的信号通路调节中发挥重要作用.     

60Co γ射线照射致小鼠肝脏的miRNAs表达改变

目的 应用miRNA芯片筛选4 Gy60Co γ射线照射后小鼠肝脏中差异表达的miRNAs,生物信息学方法探索差异表达miRNAs调控的主要功能.方法 SPF级C57BL/6J小鼠接受4 Gy60Co γ射线单次全身照射后,进行外周血白细胞计数和骨髓嗜多染红细胞微核计数.应用miRNA芯片筛选照射后小鼠肝脏中差异表达的miRNAs,用miRNA特异引物对部分差异表达miRNA进行实时定量PCR(real time PCR)验证.运用生物信息学方法,对差异miRNAs靶基因及调控功能进行预测.结果 4 Gyγ射线照射后,外周血白细胞总数与对照组相比显著减少(t=2.87,P＜0.05),而骨髓嗜多染红细胞微核率与对照组相比显著增加(t=-2.91,P＜0.05).miRNA芯片结果显示,照射组与对照组差异表达的miRNAs共17个,其中9个表达上调,8个表达下调.miR-124和miR-34a的实时荧光定量RT-PCR验证结果与芯片结果一致.GO分析发现,与黏附、细胞周期相关的通路被抑制,一些免疫相关通路被激活.结论 miR-34a和miR-194参与了急性辐射损伤的调控,起主要调控作用的miRNAs还有miR-124、miR-382和miR-92a*.

Abstract：

Objective To investigate the differential expression profiles of microRNAs in the liver of 60Co γ-ray irradiated mice using microRNA microarray and to explore their main functions by bioinformatic analysis.Methods After SPF C57BL/6J mice expose to 4 Gy-single whole body radiation,total number of peripheral WBC and the fMNPCE were measured at 3 d.The differentially expressed miRNAs in mouse liver were detected with miRNA microarray,miRNA-124 and miR-34a were confirmed by real time RT-PCR assay.Bioinformatic analysis was applied to explore target genes and the main functions of the differential expressed miRNAs.Results Compared with control group,the total number of peripheral WBC decreased( t = 2.87,P ＜ 0.05 ) ,while the fMNPCE in bone marrow increased ( t =-2.91,P ＜0.05) after 4 Gy γ-ray irradiation.miRNA microarray revealed that 17 miRNAs were differentially expressed,in which 9 up-regulated,8 down-regulated.The expression levels of miR-124 and miR-34a were coincident with the result of real time RT-PCR.GO analysis showed that some pathways including adherens junction and cell cycle were suppressed,while some immune-related pathways were activated.Conclusions miR-34a and miR-194 were involved in the regulation of acute radiation damage,some other miRNAs including miR-124、miR-382 and miR-92a* also played important roles in radiation process.     

Characterization of microRNA expression profiles in blood and saliva using the Ion Personal Genome Machine® System (Ion PGM™ System)

MicroRNA (miRNA) expression profiling is gaining interest in the forensic community because the intrinsically short fragment and tissue-specific expression pattern enable miRNAs as a useful biomarker for body fluid identification. Measuring the quantity of miRNAs in forensically relevant body fluids is an important step to screen specific miRNAs for body fluid identification. The recent introduction of massively parallel sequencing (MPS) has the potential for screening miRNA biomarkers at the genome-wide level, which allows both the detection of expression pattern and miRNA sequences. In this study, we employed the Ion Personal Genome Machine^® System (Ion PGM™ System, Thermo Fisher) to characterize the distribution and expression of 2588 human mature miRNAs (miRBase v21) in 5 blood samples and 5 saliva samples. An average of 1,885,000 and 1,356,000 sequence reads were generated in blood and saliva respectively. Based on miRDong, a Perl-based tool developed for semi-automated miRNA distribution designations, and manually ascertained, 6 and 19 miRNAs were identified respectively as potentially blood and saliva-specific biomarkers. Herein, this study describes a complete and reliable miRNA workflow solution based on Ion PGM™ System, starting from efficient RNA extraction, followed by small RNA library construction and sequencing. With this workflow solution and miRDong analysis it will be possible to measure miRNA expression pattern at the genome-wide level in other forensically relevant body fluids.     

乙型肝炎病毒基因组转染HepG2细胞的microRNA表达研究

目的 检测乙型肝炎病毒(HBV)全基因组转染对人肝母细胞瘤细胞系HepG2细胞中microRNA(miRNA)表达的影响,为进一步探讨肝细胞中HBV复制的分子生物学机制提供平台.方法 分别提取HepG2.2.15细胞(转染HBV全基因组的HepG2细胞,实验组)和其亲本HepG2细胞(对照组)的总RNA并分离miRNA,采用含509条探针的哺乳动物miRNA表达谱芯片对两组之间差异表达的miRNA进行分析.用SAM软件针对差异miRNA筛选的标准为在两组之间荧光强度差异4倍以上,差异miRNA的整体假阳性率为0.选择其中2个miRNA,应用实时荧光定量RT-PCR方法对芯片结果进行验证.结果 HepG2.2.15细胞与HepG2细胞间差异表达的miRNA共27个(占5.3%),其中7个表达上调,20个表达下调.miR-181d和miR-15a的实时荧光定量RT-PCR验证结果与芯片表达结果基本一致.结论 肝细胞中存在着与HBV复制相关的miRNA,这些上调和下调表达的miRNAs可能参与了HBV在肝细胞中的复制.     

基于microRNA差异表达的精神分裂症与抑郁症共病研究

目的 观察精神分裂症(SZ)患者中差异表达的microRNA(miRNA),从miRNA表达水平上分析SZ与抑郁症的关系.方法 通过基因芯片筛选在SZ患者中差异表达的miRNA.采用实时定量PCR(qRT-PCR)在40例SZ患者外周血单核细胞中验证芯片筛查结果,并进行精神分裂症阳性和阴性症状量表(队NSS)评定,同时检测抑郁症中差异表达的5种miRNA(miR-1972、miR-26b、miR-4485、miR-4498、miR-4743)的表达改变及其与SZ中差异表达的miRNA及PANSS评分的相关性.结果 与正常人相比,SZ患者中33种miRNA存在差异表达(32种表达上调,1种表达下调),且其中8种上调的miRNA(miR-1273d、miR-1303、miR-3064-5p、miR-3131、miR-3687、miR-4428、miR-4725-3p、miR-5096)表达差异有统计学意义(P＜0.05).在抑郁症中差异表达的5种miRNA在SZ患者中差异表达也有统计学意义(P＜0.05),且与SZ中差异表达的8种miRNA呈中、高度相关(r=0.607～0.909,P＜0.01),其中miR-1972与PANSS量表阳性症状得分呈显著正相关(r=0.339,P＜0.05),miR-26b与复合量表因子分呈显著正相关(r=0.342,p＜0.05).结论 SZ与抑郁症不仅具有某些共同的临床表现,二者在分子遗传学方面也可能有共同的病理基础.     

结合基因组分析预测炎症相关miRNA及其靶标

目的：预测炎症相关miRNA及其靶基因。方法：将基因组分析结果和miRNA靶标预测结果融合。结果：通过两种方法所产生数据的叠加,可以挑选出候选的关键miRNA和关键靶基因作为进一步实验研究的线索。结论：将基因组分析与靶标预测相结合可以有效减少假阳性预测结果,缩小实验验证的范围。     

Massively parallel sequencing of microRNA in bloodstains and evaluation of environmental influences on miRNA candidates using realtime polymerase chain reaction

MicroRNAs (miRNA) are small (22–24 nucleotides) non-coding RNAs with potential application in forensic science because of their anti-degradation property and tissue specificity. Recent studies on the use of miRNA in forensic applications have mainly focused on body fluid identification using realtime polymerase chain reaction or microarray analysis. However, the exploration of miRNA in bloodstains, which are the most valuable source of biological evidence during case investigations, is currently lacking, particularly for aged and environmentally compromised forensic samples. Recent developments in massively parallel sequencing (MPS) technology provide the opportunity to establish a whole-genome miRNA profile with high throughput and efficiency. However, MPS analysis of genome-wide miRNA profiles from bloodstains has not been reported to date. In this study, the whole-genome miRNA profiles of bloodstains were examined using MPS, revealing 633 known miRNAs and 266 novel miRNAs. To further explore the stability of miRNAs in bloodstains under various circumstances, the expression levels of six miRNAs (miR-16-5p, miR-20a-5p, miR-486-5p, miR-148a-3p, miR-151a-3p, and miR-451a) that were abundant in blood/bloodstains were examined. The results showed that freezing/thawing and a high concentration of oxidant solution affects the absolute expression of miRNA significantly, while storage for up to 5 months and a temperature of 37 °C did not have any observed effects. This study not only provides a novel method to explore miRNA profiles in bloodstains using MPS, but also points to the circumstantial influences on miRNA expression, which are an important consideration for practical application. Collectively, our work may shed light on MPS-based approaches with miRNA analysis of bloodstains in forensics.     

X射线诱导非小细胞肺癌A549细胞凋亡的适应性反应及相关miRNA筛选的研究

目的:研究X射线辐射诱导非小细胞肺癌（NSCLC) A549细胞凋亡的适应性反应,并筛选适应性反应相关的微RNA (miRNA）。方法:将NSCLC A549细胞分为6组,包括50 mGy+20 Gy、200 mGy+20 Gy、20 Gy、50 mGy、200 mGy照射组及对照组（0 Gy）,前2组细胞分别用50、...     

临床膀胱癌组织中miRNA的差异表达研究

目的寻找并鉴定在膀胱癌及癌旁组织中差异表达的微RNA（miRNA）。方法结合miRNA芯片技术寻找膀胱癌及癌旁组织中差异表达的miRNA,并通过荧光定量PCR进行验证。结果在癌与癌旁组织中共检测到115条存在显著性差异的miRNA,其中膀胱癌组织中下调42条,上调73条。结论膀胱癌组织与癌旁组织中miRNA表达谱存在较大差异,miRNA与膀胱癌的发生发展存在一定的相关性。     

心房颤动患者循环microRNAs表达谱的初步研究

目的 利用microRNA(miRNA)芯片研究持续性和阵发性心房颤动(房颤)患者循环miRNAs表达谱的改变,为进一步探讨miRNA对房颤的调控机制提供依据.方法 解放军总医院心内科2010年11月-2011年2月持续性房颤患者、阵发性房颤患者和健康对照者各5例,取其全血标本,提取血清总RNA,采用microRNA芯片进行杂交,得到miRNA表达谱,通过Volcano Plot方法寻找差异表达的miRNAs,并采用MEV软件进行聚类分析.结果 和健康对照者相比,阵发性和持续性房颤患者血清中表达都有明显差异的miRNA共有13个,其中表达上调的有8个:miR-3169,miR-3612,miR-634,miR-376a,miR-517b,miR-377*,miR-590-3p,miR-664;表达下调的有5个:miR-1,kshv-miR-K12-5,miR-378c,miR-204,miR-27a.阵发性房颤和持续性房颤患者间miRNA的表达谱也有显著差异.结论 持续性和阵发性房颤患者循环miRNAs表达谱有显著改变,提示循环miRNAs可用于房颤发生和发展过程中调控机制的研究.     

In vitro and in vivo direct monitoring of miRNA-22 expression in isoproterenol-induced cardiac hypertrophy by bioluminescence imaging

Purpose

Growing evidence suggests that microRNAs (miRNAs) play key roles in cardiac hypertrophy. To measure the expression of endogenous miRNAs is very conducive to understanding the importance of miRNAs in cardiac hypertrophy. However, current methods to monitor endogenous miRNA levels, such as Northern blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and microarrays cannot provide real-time information on miRNA biogenesis in vivo.

Methods

We constructed a miRNA reporter imaging system to monitor miR-22 expression in isoproterenol-induced cardiac hypertrophy repetitively and noninvasively. There were three copies of the antisense of miR-22 (3×PT_miR-22) cloned into the 3′ untranslated region (UTR) of the Gaussia luciferase (Gluc) reporter genes under the control of the cytomegalovirus (CMV) promoter in this miRNA reporter system (CMV/Gluc/3×PT_miR-22). CMV/firefly luciferase (Fluc) was used as a positive control for imaging of miR-22 expression. Meanwhile, quantifications of miR-22 in cardiomyocyte hypertrophy and in mouse cardiac hypertrophy induced by isoproterenol stimulation were measured by qRT-PCR. Furthermore, we used this miRNA reporter imaging system to appraise the antihypertrophic effect of antagomir-22 in vitro and in vivo.

Results

The bioluminescence signals of the CMV/Gluc/3×PT_miR-22 were gradually decreased with prolongation of isoproterenol intervention in vitro and in vivo. Overexpression of miR-22 was observed in cardiac hypertrophy, and markedly administration of antagomir-22 could reverse the upregulation of miR-22 and its prohypertrophic effects. Furthermore, knockdown of miR-22 by antagomir-22 could markedly reverse the repressed Gluc activities in vitro and in vivo. However, the Fluc activity of CMV/Fluc was not affected with isoproterenol treatment.

Conclusion

This study elucidates the feasibility of using our constructed miRNA reporter imaging system to monitor the location and magnitude of expression levels of miR-22 in cardiac hypertrophy in vitro and in vivo.     

Predicting the origin of stains from whole miRNome massively parallel sequencing data

In this study, we have screened the six most relevant forensic body fluids / tissues, namely blood, semen, saliva, vaginal secretion, menstrual blood and skin, for miRNAs using a whole miRNome massively parallel sequencing approach. We applied partial least squares (PLS) and linear discriminant analysis (LDA) to predict body fluids based on the expression of the miRNA markers. We estimated the prediction accuracy for models including different subsets of miRNA markers to identify the minimum number of markers needed for sufficient prediction performance. For one selected model consisting of 9 miRNA markers we calculated their importance for prediction of each of the six different body fluid categories.     

γ线照射小鼠胸腺组织基因表达转录谱分析

目的 探讨6 Gy γ线照射后不同时间小鼠胸腺组织基因表达转录谱的动态变化。方法 应用快速高通量的cDNA基因芯片技术进行研究。结果 1随照射后时间推移,差异表达基因数目和种类逐渐减少,如照射后1、6、14和28 d差异基因数量分别为878、318、162和25;2辐射所诱导的胸腺组织差异表达基因广泛涉及细胞周期、免疫和应激、细胞凋亡、细胞信号转导、转录调节、DNA合成修复和重组、细胞骨架、离子通道和运输、代谢、蛋白翻译和合成、发育、细胞分化多个方面;3照射后对某些重要差异表达基因的分析表明,与细胞周期相关的基因有5个(上调3个:Cyclin G、Anxa1、Fgf1,下调2个:Cdc2a、Cdc25b);与免疫应激相关的5个(上调4个:IL-18、Casp1、IL-15、IL-7,下调1个:Cd28);与细胞凋亡相关的7个(上调4个:Casp1、Anxa1、Perp、IL-7,下调3个:Pten、Api5、Fas)。结论 6 Gy γ线照射后,小鼠胸腺组织差异表达基因随照射后时间延长不仅涉及多个靶点、多个层次,显示出多样性变化的特点,而且显示出时间上的明显差异;从基因水平上揭示了中等剂量照射小鼠胸腺组织损伤修复和重建基因的变化规律,为辐射免疫损伤与修复的分子机制和防治措施的研究提供了新的启示。     

14 q32 miRNA基因簇与肝细胞癌的发生发展

肝细胞癌（肝癌,hepatocellular carcinoma ,HCC ）是全球范围内发生率和死亡率较高的恶性肿瘤之一。微小RNA（ microRNA,miRNA）是一类内源性、非编码、高度保守的单链小分子RNA,主要在转录后水平抑制靶基因的表达。有些位置相近的miRNA基因在染色体上成簇排列,在一个多顺反子内形成miRNA基因簇,通常以共表达的形式协同作用。在人类14号染色体长臂端的14q32印迹基因区域约215 kb的基因组范围内,聚集了52个miRNA基因。已有研究发现此miRNA基因簇的异常表达与肝癌的发生发展密切相关。该文概括了14 q32 miRNA基因簇的结构特点,并对其在肝癌发生发展过程中所发挥的作用进行了综述。