应用微孔板芯片分析机采血小板常温保存过程中凋亡相关miRNA的表达改变

目的分析机采血小板在常温保存过程中凋亡相关miRNA的表达改变。方法应用miRNA微孔板芯片技术研究常温保存机采血小板在不同保存时间凋亡相关miRNA的差异表达谱,采用生物信息学软件预测发生差异表达的miRNA可能的靶基因及相关生物学功能。结果与新鲜血小板相比,常温保存7 d的血小板,有7种miRNA发生了显著变化。其中miR-16、Let-7b、miR-26和Let-7c的表达上调,miR-7、miR-145和miR-197的表达下调。结论筛选出的差异表达miRNA可能参与了血小板的保存损伤过程。     

新发和完全缓解的重型再生障碍性贫血间microRNA差异表达谱（英文）

检测miRNA在新发与完全缓解SAA患者中的表达差异及预测其靶基因。方法:用miRNA芯片方法检测初发和完全缓解(CR)SAA患者骨髓单个核细胞中miRNA表达谱。结果:完全缓解SAA患者与初发SAA患者相比,上调miRNA为35个,下调miRNA为37个。此外,通过预测差异表达的miRNA靶点发现,其靶点与T细胞受体信号通路和细胞黏附分子相关。结论:某些miRNA可能与SAA患者发病机制相关。     

氧化低密度脂蛋白诱导人脐静脉内皮细胞凋亡中microRNA表达谱分析

目的:研究氧化低密度脂蛋白(ox-LDL)诱导人脐静脉内皮细胞(HUVECs)凋亡过程中micro-RNA(miRNA)表达谱的变化,对差异miRNA调控的靶基因进行初步预测。方法:建立体外ox-LDL诱导HUVECs凋亡模型,采用miRNA芯片技术筛选表达变化显著的miRNA,实时荧光定量PCR验证结果,并对差异miRNA调控的靶基因进行生物信息学分析。结果:miRNA芯片检测发现,在ox-LDL诱导HUVECs凋亡过程中有4个表达上调和11个表达下调的miRNA。实时荧光定量PCR对其中2个上调(hsa-miR-142-3p、hsa-miR-365)和2个下调(hsa-miR-590-5p、hsa-miR-33a)miRNA的验证结果与芯片检测所示有较好的一致性。生物信息学分析发现,差异miRNA调控的靶基因与细胞增殖、凋亡、代谢及原癌基因表达等生物学功能相关。结论:经ox-LDL诱导凋亡的HUVECs其miRNA表达谱发生明显改变,提示miRNA可能参与内皮细胞功能障碍和动脉粥样硬化的发生。     

大鼠心力衰竭细胞 microRNA 表达谱及生物信息学分析

目的：观察大鼠心力衰竭细胞中microRNA （ miRNA）表达变化,利用生物信息学分析技术探索差异miRNA靶基因的功能。方法18只成年雄性SD大鼠,体质量200～220g,随机数字表法随机分为两组：正常对照组（ CON组）和心力衰竭组（ HF组）。 HF组制备阿霉素致心力衰竭模型, CON组注射等量生理盐水。提取大鼠心脏, Largendorff逆行灌注法分离心室肌细胞,提取总RNA行miRNA表达谱检测,筛选两组差异表达的miRNA,荧光定量RT-PCR 技术验证结果, Targetscan、 miRanda软件预测差异表达miRNA的靶基因,并对靶基因行生物信息学分析。结果芯片检测结果显示,与CON组相比, HF组共有37个miRNA表达发生显著改变,其中22个miRNA上调,15个miRNA下调（均P＜0.01, FDR＜0.05）。荧光定量RT-PCR检测miR-133b-5p （t ＝14.56, P＜0.1）、 miR-6216（t＝9.32, P＜0.1）、 let-7e-5p （ t＝13.92, P＜0.1）表达水平,变化趋势与芯片结果一致。生物信息学分析显示,差异表达miRNA调控的靶基因显著富集于31个基因组（均P＜0.01, FDR＜0.05）和12条信号通路（均P＜0.05, FDR＜0.05）,其中泛素蛋白酶体系统、 MAPK信号通路、 Toll样受体信号通路富集程度较高。结论阿霉素诱导的心力衰竭模型大鼠心肌细胞中miRNA表达谱发生显著改变,这些差异表达miRNA可能通过调控靶基因功能参与心力衰竭的病理生理过程。     

MicroRNA在垂体腺瘤中的诊断与治疗研究进展

MicroRNA(miRNA)是非编码的小分子RNA,参与靶基因转录后的表达调控,通过完全或不完全与mRNA对应的靶基因配对从而降解mRNA或干扰其翻译,负调控靶基因表达。目前研究表明miRNA的表达差异参与很多肿瘤性和非肿瘤性疾病的病理生理过程。垂体腺瘤是发生于腺垂体的肿瘤,近年来研究发现miRNA在不同激素类型、大小、侵袭性等的垂体腺瘤和垂体腺癌中表达各有差异,有可能成为垂体腺瘤的诊断标志物及治疗靶点。本文将对miRNA在垂体腺瘤中诊断与治疗的研究进展作相关的综述。     

青少年肥胖非酒精性脂肪肝患者血清外泌体源性miRNA表达谱的初步研究

目的分析非酒精性脂肪肝(NAFLD)与单纯肥胖对照者血清外泌体微小RNA(miRNA)表达谱的差异,为筛选外泌体源性miRNA作为NAFLD早期诊断标记物提供依据。方法选择5例NAFLD患者(NAFLD组)和5例单纯肥胖患者(对照组),采集外周血血清,使用高通量测序方法测定外泌体源性miRNA表达谱,并通过实时荧光定量PCR验证部分差异性表达的miRNA,预测靶基因及功能分析。结果NAFLD组与对照组相比差异表达的外泌体源性miRNA共30种(P<0.05),当倍比值>2且P<0.05的差异性miRNA有9种,其中5种表达上调(miR-122-5p,miR-3591-3p,miR-335-5p,let-7g-3p和miR-27a-5p),4种表达下调(miR-6753-3p,miR-129-2-3p,miR-6760-5p,miR-6516);对miR-122-5p、miR-335-5p、miR-27a-5p进行实时荧光定量PCR,结果与测序结果一致。差异表达miRNA的功能分析结果提示涉及葡萄糖稳态、脂质代谢、肝脏发育等。结论NAFLD组血清外泌体源性miRNA表达谱与对照组有明显差异,miR-122-5p、miR-335-5p、miR-27a-5p可能参与了NAFLD的发病机制,可以作为NAFLD早期诊断的标记物。     

深低温保存单采血小板凋亡相关miRNA的差异表达

目的了解单采血小板在深低温保存过程中凋亡相关microRNA(miRNA)的表达改变。方法应用miRNA微孔板芯片技术分析-80℃保存不同时间单采血小板凋亡相关miRNA表达谱的差异,同时利用qRT-PCR技术验证该结果的准确性;采用生物信息学软件预测差异表达miRNA可能的靶基因。结果与新鲜单采血小板相比,凋亡相关miRNA表达谱在血小板-80℃保存5 d时无明显变化、保存7 d时仅有2种明显上调变化的miRNA:miR-216和miR-296。选择表达谱中miR-216 miR-296 miR-7和miR-16共4种miRNA做qRT-PCR验证:以新鲜血小板表达水平为基准值1,-80℃保存5、7 d后miR-216的相对表达量分别为1.81±0.21和2.88±0.23,miR-296的相对表达量分别为2.27±0.20和3.85±0.17,miR-7的相对表达量分别为0.89±0.06和0.76±0.03,miR-16的相对表达量分别为1.12±0.35和1.40±0.32,miR-216和miR-296在7 d保存过程中表达上调(P0.05),miR-7和miR-16的表达未发生明显变化(P0.05)。结论短期(7 d)深低温保存单采血小板凋亡相关miRNA的表达谱接近新鲜单采血小板。     

肺腺癌miR-195的表达及靶基因筛选

目的研究miR-195在肺腺癌中的表达差异,筛选miR-195靶基因,分析靶基因作用网络及其对患者生存预后的影响。方法利用TCGA数据库,比较miR-195在肺腺癌癌组织与癌旁正常组织间的表达差异;采用实时荧光定量PCR检测miR-195在临床标本中的相对表达水平。设计miR-195的Hybrid PCR引物,扩增出靶基因的mRNA 3′-非翻译区(3′UTR),菌落PCR筛选靶基因的3′UTR片段进行测序。美国国家生物信息中心网站比对测序结果,得到靶基因的基因注释和全长序列,构建靶基因作用网络并进行生存分析。结果miR-195在肺腺癌癌组织中的相对表达水平低于癌旁正常组织,差异有统计学意义(P<0.01)。Hybrid PCR筛选miR-195候选靶基因显示多个条带。miR-195与靶基因的结合位点在基因3′UTR上有19个。靶基因GCLC、NOB1的低表达与肺腺癌患者的良好预后相关。结论肺腺癌癌组织中miR-195呈低表达。miR-195可能通过直接抑制靶基因GCLC、NOB1的功能,进而影响肺腺癌的发生、发展。     

IL-33在结直肠癌中的表达及其临床意义

目的研究白细胞介素33(IL-33)在结直肠癌中的表达情况及其临床意义,为其在结直肠癌早期诊断和预后方面的应用提供依据。方法应用免疫组织化学染色法检测IL-33在癌旁组织、结直肠腺瘤和腺癌中的表达情况,并分析其表达与临床特征的关系。结果 IL-33在癌旁组织、结直肠腺瘤和腺癌中的表达有显著性差异,其中在癌旁组织中表达量最低。在结直肠腺瘤中主要分布于表面上皮,与癌旁组织相比无显著性差异(χ~2=0.488,P0.05)。结直肠腺癌组织中IL-33阳性表达率显著高于癌旁黏膜组(χ~2=23.188,P0.01)和结直肠腺瘤组(χ~2=17.647,P0.01),且阳性信号富集于细胞浆和表面上皮。IL-33表达与结直肠癌患者的年龄、性别、分化程度无显著相关性,而与肿瘤浸润肠壁深度、淋巴结转移和临床分期显著相关。此外IL-33表达情况与患者的病死率无相关性。结论 IL-33表达与结直肠癌发生和发展密切相关,可能作为结直肠癌诊断的标志物和治疗靶点。     

结直肠腺癌、腺瘤组织及正常组织中黑色素瘤缺乏因子2表达差异的研究

目的通过免疫组化方法比较结直肠癌患者的癌组织、腺瘤组织、癌旁正常组织及单纯结直肠腺瘤患者的腺瘤组织中黑色素瘤缺乏因子2(AIM2)基因的表达差异。方法收集结直肠癌合并肠道息肉的患者,每例患者手术切除标本中均同时有腺癌病灶及独立的腺瘤病灶。同时收集单纯结直肠腺瘤患者,均通过门诊内镜或外科手术获取腺瘤组织标本。对样本进行AIM2蛋白免疫组化染色(En Vision法),对比分析不同的样本组织间AIM2表达情况。结果研究纳入的52例腺癌合并腺瘤的结直肠癌患者,获取癌组织、腺瘤组织及癌旁正常组织标本各52份,纳入78例结直肠腺瘤患者,获取单纯腺瘤标本78例。秩和检验发现,AIM2基因的表达情况,结直肠癌患者的正常组织中最强(秩均值151. 37),单纯结直肠腺瘤患者的腺瘤组织次之(秩均值121. 88),结直肠癌患者的腺瘤组织再次之(秩均值103. 11),结直肠癌患者的癌组织中AIM2表达最弱(秩均值91. 45),差异有统计学意义(χ~2=26. 048,P 0. 01)。结论 AIM2在正常组织、腺瘤组织、腺癌组织中的表达逐步下调,提示AIM2可能是一种抑癌基因,它的缺失,可能导致结直肠肿瘤的发生及进展。     

MicroRNAs 的失调在高转移肝细胞癌中的作用

目的：筛选在高转移肝细胞癌中差异表达的miRNAs图谱。方法利用Agilent miRNA array芯片技术平台分析比较了4对高转移肝细胞患者的癌组织样本与其相对应的正常组织之间的差异miRNAs。通过生物信息学方法分析了这些候选差异 miRNAs。结果与正常组织相比,我们在高转移肝细胞癌中发现了22个失调的miRNAs,其中包括13个上调的miRNAs（miR-200a、miR-425、miR-221和miR-20b等）和9个下调的miRNAs（miR-762、miR-638和miR-1305等）。其中,一些miRNAs已经被报道与肿瘤的发生和转移相关。靶基因预测分析也表明,这些基因在肿瘤的发生和转移过程中扮演着重要的作用。结论我们在高转移肝细胞癌中发现了一些表达失调的 miRNAs,生物信息学分析也发现这些 miRNAs 在肿瘤的发生和转移中发挥着重要的作用,可以作为肝细胞癌在临床治疗上的一个新的肿瘤标志物。     

MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors

MicroRNAs (miRNAs) regulate gene expression. It has been suggested that obtaining miRNA expression profiles can improve classification, diagnostic, and prognostic information in oncology. Here, we sought to comprehensively identify the miRNAs that are overexpressed in lung cancer by conducting miRNA microarray expression profiling on normal lung versus adjacent lung cancers from transgenic mice. We found that miR-136, miR-376a, and miR-31 were each prominently overexpressed in murine lung cancers. Real-time RT-PCR and in situ hybridization (ISH) assays confirmed these miRNA expression profiles in paired normal-malignant lung tissues from mice and humans. Engineered knockdown of miR-31, but not other highlighted miRNAs, substantially repressed lung cancer cell growth and tumorigenicity in a dose-dependent manner. Using a bioinformatics approach, we identified miR-31 target mRNAs and independently confirmed them as direct targets in human and mouse lung cancer cell lines. These targets included the tumor-suppressive genes large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A), and expression of each was augmented by miR-31 knockdown. Their engineered repression antagonized miR-31–mediated growth inhibition. Notably, miR-31 and these target mRNAs were inversely expressed in mouse and human lung cancers, underscoring their biologic relevance. The clinical relevance of miR-31 expression was further independently and comprehensively validated using an array containing normal and malignant human lung tissues. Together, these findings revealed that miR-31 acts as an oncogenic miRNA (oncomir) in lung cancer by targeting specific tumor suppressors for repression.     

A miRNA Signature of Prion Induced Neurodegeneration

MicroRNAs (miRNAs) are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. Compelling evidence links miRNAs to the control of neuronal development and differentiation, however, little is known about their role in neurodegeneration. We used microarrays and RT-PCR to profile miRNA expression changes in the brains of mice infected with mouse-adapted scrapie. We determined 15 miRNAs were de-regulated during the disease processes; miR-342-3p, miR-320, let-7b, miR-328, miR-128, miR-139-5p and miR-146a were over 2.5 fold up-regulated and miR-338-3p and miR-337-3p over 2.5 fold down-regulated. Only one of these miRNAs, miR-128, has previously been shown to be de-regulated in neurodegenerative disease. De-regulation of a unique subset of miRNAs suggests a conserved, disease-specific pattern of differentially expressed miRNAs is associated with prion–induced neurodegeneration. Computational analysis predicted numerous potential gene targets of these miRNAs, including 119 genes previously determined to be also de-regulated in mouse scrapie. We used a co-ordinated approach to integrate miRNA and mRNA profiling, bioinformatic predictions and biochemical validation to determine miRNA regulated processes and genes potentially involved in disease progression. In particular, a correlation between miRNA expression and putative gene targets involved in intracellular protein-degradation pathways and signaling pathways related to cell death, synapse function and neurogenesis was identified.     

Three new circulating microRNAs may be associated with wet age-related macular degeneration

Abstract

This study investigates the circulating microRNA (miRNA) expression profiles in patients with age-related macular degeneration (AMD) and the role of miRNA in wet AMD and its pathways. Exosomes were extracted from serum samples of AMD patients (n?=?70) and a control group (n?=?50). After isolating miRNA from the exosomes, miRNAs were transformed into cDNA. In the control and AMD samples, the expression was compared with a panel including 175 genes using the PCR array method. Target genes and pathways of miRNAs were detected by KEGG and Biocarta signaling pathway enrichments. Comparing the serum samples between groups revealed that the expression levels of 15 microRNAs within 175 genes had significantly changed. In the validation studies, miR-129-3p and miR-132-3p had no significant expression in AMD group compared to the controls. miR-486-5p and miR-626 had higher expression in AMD patients compared to the control group, while miR-885-5p showed significantly lower expression. Pathway analysis revealed that these miRNAs may have critical roles in the apoptosis and neovascularization pathways. The data suggest that some miRNAs within the serum may have a role in the pathogenesis of wet AMD. Further studies are needed to examine the use of these miRNAs as biomarkers.     

Genetic Variation That Predicts Platinum Sensitivity Reveals the Role of miR-193b* in Chemotherapeutic Susceptibility

Platinum agents are the backbone of cancer chemotherapy. Recently, we identified and replicated the role of a single nucleotide polymorphism (SNP, rs1649942) in predicting platinum sensitivity both in vitro and in vivo. Using the CEU samples from the International HapMap Project, we found the same SNP to be a master regulator of multiple gene expression phenotypes, prompting us to investigate whether rs1649942-mediated regulation of miRNAs may in part contribute to variation in platinum sensitivity. To these ends, 60 unrelated HapMap CEU I/II samples were used for our discovery-phase study using high-throughput genome-wide miRNA and gene expression profiling. Examining the relationships among rs1649942, its gene expression targets, genome-wide miRNA expression, and cellular sensitivity to carboplatin and cisplatin, we identified 2 platinum-associated miRNAs (miR-193b* and miR-320) that inhibit the expression of 5 platinum-associated genes (CRIM1, IFIT2, OAS1, KCNMA1, and GRAMD1B). We further replicated the relationship between the expression of miR-193b*, CRIM1, IFIT2, KCNMA1, and GRAMD1B, and platinum sensitivity in a separate HapMap CEU III dataset. We then showed that overexpression of miR-193b* in a randomly selected HapMap cell line results in resistance to both carboplatin and cisplatin. This relationship was also found in 7 ovarian cancer cell lines from NCI60 dataset and confirmed in an OVCAR-3 that overexpression of miR-193b* leads to increased resistance to carboplatin. Our findings highlight a potential mechanism of action for a previously observed genotype-survival outcome association. Further examination of miR-193b* in platinum sensitivity in ovarian cancer is warranted. Mol Cancer Ther; 11(9); 2054-61. ?2012 AACR.     

Intratumoral heterogeneity of microRNA expression in breast cancer

Profiling studies have identified specific microRNA (miRNA) signatures in malignant tumors including breast cancer. Our aim was to assess intratumoral heterogeneity in miRNA expression levels within primary breast cancers and between axillary lymph node metastases from the same patient. Specimens of 16 primary breast cancers were sampled in 8-10 distinct locations including the peripheral, intermediate, and central tumor zones, as well as two to five axillary lymph node metastases (n = 9). Total RNA was extracted from 132 paraffin-embedded samples, and the expression of miR-10b, miR-210, miR-31, and miR-335 was assessed as well as the reproducibility of RNA extraction and miRNA analysis by quantitative RT-PCR. Considerable intratumoral heterogeneity existed for all four miRNAs within primary breast cancers (CV 40%). No significant differences within (CV 34%) or between different tumor zones (CV 33%) were found. A similar variation in miRNA expression was observed between corresponding lymph node metastases (mean CV 40%). In comparison, the variation among different patients showed a CV of 80% for primary tumors and 103% for lymph node metastases. Both miRNA extraction procedures and quantitative RT-PCR showed high reproducibility (CV ≤ 2%). Thus, the intratumoral heterogeneity of miRNA expression in breast cancers can lead to significant sampling bias. Assessment of breast cancer miRNA profiles may require sampling at several different tumor locations and of several tumor-involved lymph nodes when deriving miRNA expression profiles of metastases.     

MicroRNAs as potential target gene in cancer gene therapy of gastrointestinal tumors

INTRODUCTION: MicroRNA (miRNA) is a small non-coding RNA, which negatively regulates the expression of many target genes, thereby contributing to the modulation of diverse cell fates. Recent advances in molecular biology have revealed the potential role of miRNAs in tumor initiation, progression and metastasis. Aberrant regulation of miRNAs has been frequently reported in a variety of cancers, including gastrointestinal tumors, suggesting that cancer-related miRNAs are promising as novel biomarkers for tumor diagnosis and are potential target genes for cancer gene therapy against gastrointestinal tumors. AREAS COVERED: The review focuses on the role of specific miRNAs (miR-192/194/215 and miR-7) in the differentiation of gastrointestinal epithelium and on the role of tumor-suppressive (miR-34, miR-143, miR-145) and oncogenic miRNAs (miR-21, miR-17-92 cluster) in gastrointestinal tumors. Furthermore, the potential role of miRNAs as novel biomarkers and target genes for cancer gene therapy against gastrointestinal tumors are discussed. We will also outline the potential clinical application of miRNAs for tumor diagnosis and cancer gene therapy against gastrointestinal tumors. EXPERT OPINION: Exploration of tumor-related miRNAs would provide important opportunities for the development of novel cancer gene therapies aimed at normalizing the critical miRNAs that are deregulated in gastrointestinal tumors.     

MicroRNA-182 drives metastasis of primary sarcomas by targeting multiple genes

Metastasis causes most cancer deaths, but is incompletely understood. MicroRNAs can regulate metastasis, but it is not known whether a single miRNA can regulate metastasis in primary cancer models in vivo. We compared the expression of miRNAs in metastatic and nonmetastatic primary mouse sarcomas and found that microRNA-182 (miR-182) was markedly overexpressed in some tumors that metastasized to the lungs. By utilizing genetically engineered mice with either deletion of or overexpression of miR-182 in primary sarcomas, we discovered that deletion of miR-182 substantially decreased, while overexpression of miR-182 considerably increased, the rate of lung metastasis after amputation of the tumor-bearing limb. Additionally, deletion of miR-182 decreased circulating tumor cells (CTCs), while overexpression of miR-182 increased CTCs, suggesting that miR-182 regulates intravasation of cancer cells into the circulation. We identified 4 miR-182 targets that inhibit either the migration of tumor cells or the degradation of the extracellular matrix. Notably, restoration of any of these targets in isolation did not alter the metastatic potential of sarcoma cells injected orthotopically, but the simultaneous restoration of all 4 targets together substantially decreased the number of metastases. These results demonstrate that a single miRNA can regulate metastasis of primary tumors in vivo by coordinated regulation of multiple genes.