Horizontal transfer of microRNAs: molecular mechanisms and clinical applications

A new class of RNA regulatory genes known as microRNAs (miRNAs) has been found to introduce a whole new layer of gene regulation in eukaryotes. The intensive studies of the past several years have demonstrated that miRNAs are not only found intracellularly, but are also detectable outside cells, including in various body fluids (e.g. serum, plasma, saliva, urine and milk). This phenomenon raises questions about the biological function of such extracellular miRNAs. Substantial amounts of extracellular miRNAs are enclosed in small membranous vesicles (e.g. exosomes, shedding vesicles and apoptotic bodies) or packaged with RNA-binding proteins (e.g. high-density lipoprotein, Argonaute 2 and nucleophosmin 1). These miRNAs may function as secreted signaling molecules to influence the recipient cell phenotypes. Furthermore, secreted extracellular miRNAs may reflect molecular changes in the cells from which they are derived and can therefore potentially serve as diagnostic indicators of disease. Several studies also point to the potential application of siRNA/miRNA delivery as a new therapeutic strategy for treating diseases. In this review, we summarize what is known about the mechanism of miRNA secretion. In addition, we describe the pathophysiological roles of secreted miRNAs and their clinical potential as diagnostic biomarkers and therapeutic drugs. We believe that miRNA transfer between cells will have a significant impact on biological research in the coming years.     

Ion channels/transporters as epigenetic regulators? —a microRNA perspective

MicroRNA (miRNA) alterations in response to changes in an extracellular microenvironment have been observed and considered as one of the major mechanisms for epigenetic modifications of the cell. While enormous efforts have been made in the understanding of the role of miRNAs in regulating cellular responses to the microenvironment, the mechanistic insight into how extracellular signals can be transduced into miRNA alterations in cells is still lacking. Interestingly, recent studies have shown that ion channels/transporters, which are known to conduct or transport ions across the cell membrane, also exhibit changes in levels of expression and activities in response to changes of extracellular microenvironment. More importantly, alterations in expression and function of ion channels/transporters have been shown to result in changes in miRNAs that are known to change in response to alteration of the microenvironment. In this review, we aim to summarize the recent data demonstrating the ability of ion channels/transporters to transduce extracellular signals into miRNA changes and propose a potential link between cells and their microenvironment through ion channels/transporters. At the same time, we hope to provide new insights into epigenetic regulatory mechanisms underlying a number of physiological and pathological processes, including embryo development and cancer metastasis.     

Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation

We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change ≥ 1.5, false discovery rate (FDR) ≤ 0.3; p < 0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling.     

microRNA deregulation in keloids: an opportunity for clinical intervention?

Keloids are defined as benign dermal scars invading adjacent healthy tissue, characterized by aberrant fibroblast dynamics and overproduction of extracellular matrix. However, the aetiology and molecular mechanism of keloid production remain poorly understood. Recent discoveries have shed new light on the involvement of a class of non‐coding RNAs known as microRNAs (miRNA), in keloid formation. A number of miRNAs have differential expression in keloid tissues and keloid‐derived fibroblasts. These miRNAs have been characterized as novel regulators of cellular processes pertinent to wound healing, including extracellular matrix deposition and fibroblast proliferation. Delineating the functional significance of miRNA deregulation may help us better understand pathogenesis of keloids, and promote development of miRNA‐directed therapeutics against this condition.     

MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins

Circulating microRNAs (miRNA) are relatively stable in plasma and are a new class of disease biomarkers. Here we present evidence that high-density lipoprotein (HDL) transports endogenous miRNAs and delivers them to recipient cells with functional targeting capabilities. Cellular export of miRNAs to HDL was demonstrated to be regulated by neutral sphingomyelinase. Reconstituted HDL injected into mice retrieved distinct miRNA profiles from normal and atherogenic models. HDL delivery of both exogenous and endogenous miRNAs resulted in the direct targeting of messenger RNA reporters. Furthermore, HDL-mediated delivery of miRNAs to recipient cells was demonstrated to be dependent on scavenger receptor class B type I. The human HDL-miRNA profile of normal subjects is significantly different from that of familial hypercholesterolemia subjects. Notably, HDL-miRNA from atherosclerotic subjects induced differential gene expression, with significant loss of conserved mRNA targets in cultured hepatocytes. Collectively, these observations indicate that HDL participates in a mechanism of intercellular communication involving the transport and delivery of miRNAs.     

高通量测序技术分析急性髓系白血病血浆外泌体微RNA表达谱差异

急性髓系白血病（acute myeloid leukemia,AML）是一类造血干细胞的恶性克隆性疾病,目前的诊断方法不利于疾病的早期发现,且诊断结果重复性较差。已有大量研究显示,细胞外microRNA（miRNA或miR）富集在外泌体（exosome）中,且受其表面膜的保护而具有很好的稳定性,是理想的分子标志物。目前,多种实体肿瘤均已检测到肿瘤特异性外泌体miRNA(exosomal miRNA)。然而,在AML患者中未见此外泌体miRNA报道。本研究探讨急性髓系白血病血浆外泌体miRNA表达谱差异及新miRNA序列。采用solexa高通量测序技术对7例AML患者（AML组）及7例健康对照者（对照组）血浆外泌体miRNAs进行测序,利用Mireap预测软件进行新miRNAs分析,通过edger差异分析软件筛选组间差异miRNA,获得211个已知的差异表达miRNAs以及2个新miRNAs,选择4个差异表达的miRNAs：miR-155-5p、miR-335-5p、miR-451a及xxx-m0038 5p（新miRNA）,在两组（各23例）的血浆外泌体样本中,进行实时荧光定量PCR（qRT-PCR）验证,验证结果与测序结果一致。对差异表达的外泌体miRNA进行靶基因预测及其GO（Gene Ontology）和信号通路富集分析,发现靶基因聚集的生物学功能多数参与生物进展过程的调控。靶基因主要富集在FoxO、MAPK、Hippo信号通路以及HTLV-I感染等。结果显示,AML患者与健康对照者的血浆外泌体miRNA存在着差异性表达。差异性表达的miRNA特异性很高,对进一步阐明AML白血病发生与发展的分子机制、研发新的无创诊断方法、新的诊断标记物和有效治疗AML的方法具有十分重要和深远的意义。     

Massive analysis of cDNA Ends (MACE) and miRNA expression profiling identifies proatherogenic pathways in chronic kidney disease

Epigenetic dysregulation contributes to the high cardiovascular disease burden in chronic kidney disease (CKD) patients. Although microRNAs (miRNAs) are central epigenetic regulators, which substantially affect the development and progression of cardiovascular disease (CVD), no data on miRNA dysregulation in CKD-associated CVD are available until now. We now performed high-throughput miRNA sequencing of peripheral blood mononuclear cells from ten clinically stable hemodialysis (HD) patients and ten healthy controls, which allowed us to identify 182 differentially expressed miRNAs (e.g., miR-21, miR-26b, miR-146b, miR-155). To test biological relevance, we aimed to connect miRNA dysregulation to differential gene expression. Genome-wide gene expression profiling by MACE (Massive Analysis of cDNA Ends) identified 80 genes to be differentially expressed between HD patients and controls, which could be linked to cardiovascular disease (e.g., KLF6, DUSP6, KLF4), to infection / immune disease (e.g., ZFP36, SOCS3, JUND), and to distinct proatherogenic pathways such as the Toll-like receptor signaling pathway (e.g., IL1B, MYD88, TICAM2), the MAPK signaling pathway (e.g., DUSP1, FOS, HSPA1A), and the chemokine signaling pathway (e.g., RHOA, PAK1, CXCL5). Formal interaction network analysis proved biological relevance of miRNA dysregulation, as 68 differentially expressed miRNAs could be connected to 47 reciprocally expressed target genes. Our study is the first comprehensive miRNA analysis in CKD that links dysregulated miRNA expression with differential expression of genes connected to inflammation and CVD. After recent animal data suggested that targeting miRNAs is beneficial in experimental CVD, our data may now spur further research in the field of CKD-associated human CVD.     

HPat a Decapping Activator Interacting with the miRNA Effector Complex

Animal miRNAs commonly mediate mRNA degradation and/or translational repression by binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). The CCR4-NOT1 complex required for the deadenylation of target mRNAs is directly recruited to the miRNA effector complex. However, it is unclear whether the following decapping step is only a consequence of deadenylation occurring independent of the miRNA effector complex or e.g. decapping activators can get recruited to the miRNA effector complex. In this study we performed split-affinity purifications in Drosophila cells and provide evidence for the interaction of the decapping activator HPat with the miRNA effector complex. Furthermore, in knockdown analysis of various mRNA degradation factors we demonstrate the importance of NOT1 for this interaction. This suggests that deadenylation and/or the recruitment of NOT1 protein precedes the association of HPat with the miRNA effector complex. Since HPat couples deadenylation and decapping, the recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA target for degradation.     

Using information theory to assess the communicative capacity of circulating microRNA

The discovery of extracellular microRNAs (miRNAs) and their transport modalities (i.e., microparticles, exosomes, proteins and lipoproteins) has sparked theories regarding their role in intercellular communication. Here, we assessed the information transfer capacity of different miRNA transport modalities in human serum by utilizing basic principles of information theory. Zipf Statistics were calculated for each of the miRNA transport modalities identified in human serum. Our analyses revealed that miRNA-mediated information transfer is redundant, as evidenced by negative Zipf’s Statistics with magnitudes greater than one. In healthy subjects, the potential communicative capacity of miRNA in complex with circulating proteins was significantly lower than that of miRNA encapsulated in circulating microparticles and exosomes. Moreover, the presence of coronary heart disease significantly lowered the communicative capacity of all circulating miRNA transport modalities. To assess the internal organization of circulating miRNA signals, Shannon’s zero- and first-order entropies were calculated. Microparticles (MPs) exhibited the lowest Shannon entropic slope, indicating a relatively high capacity for information transfer. Furthermore, compared to the other miRNA transport modalities, MPs appeared to be the most efficient at transferring miRNA to cultured endothelial cells. Taken together, these findings suggest that although all transport modalities have the capacity for miRNA-based information transfer, MPs may be the simplest and most robust way to achieve miRNA-based signal transduction in sera. This study presents a novel method for analyzing the quantitative capacity of miRNA-mediated information transfer while providing insight into the communicative characteristics of distinct circulating miRNA transport modalities.     

Nature and consequences of mammalian brain and CSF efflux transporters: four decades of progress

In the last 40 years, especially with the application of new neurochemical and molecular biological techniques, there has been explosive progress in understanding how certain ligands and drugs are transported across the blood-brain barrier and choroid plexus out of brain and CSF. In the CNS, there are several separate efflux transporters with very broad specificity that are responsible for much of the efflux transport. This review focuses on three such transporters: organic acid transporter-3, peptide transporter-2 and P-glycoprotein for which there is substantial new information including 'knockout' models in mice and, in one case, dogs. Moreover, the structural biology and transport mechanism of P-glycoprotein at 3.8 angstroms is described. The overall objective is to show how this new knowledge provides a more thorough understanding (e.g., of molecular mechanisms) of efflux transport and in several cases leads to clinically relevant information that allows better treatment of certain CNS disorders (e.g., meningitis and brain cancer).     

Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) are endogenous regulators of a broad range of physiological processes and act by either degrading mRNA or blocking its translation. Oilseed rape (Brassica napus) is one of the most important crops in China, Europe and other Asian countries with publicly available expressed sequence tags (ESTs) and genomic survey sequence (GSS) databases, but little is known about its miRNAs and their targets. To date, only 46 miRNAs have been identified in B. napus. RESULTS: Forty-one conserved and 62 brassica-specific candidate B. napus miRNAs, including 20 miRNA* sequences, were identified using Solexa sequencing technology. Furthermore, 33 non-redundant mRNA targets of conserved brassica miRNAs and 19 new non-redundant mRNA targets of novel brassica-specific miRNAs were identified by genome-scale sequencing of mRNA degradome. CONCLUSIONS: This study describes large scale cloning and characterization of B. napus miRNAs and their potential targets, providing the foundation for further characterization of miRNA function in the regulation of diverse physiological processes in B. napus.     

Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures

Three-dimensional organotypic culture using reconstituted basement membrane matrix (rBM 3-D) is an invaluable tool to characterize morphogenesis of epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix. microRNAs (miRNA) are a novel class of tumor modulating genes. A substantial amount of investigation of miRNAs in cancer is carried out using monolayer 2-D culture on plastic substratum, which lacks a consideration of the matrix-mediated regulation of miRNAs. In the current study we compared the expression of miRNAs in rBM 3-D and 2-D cultures of two lung adenocarcinoma cell lines. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures of lung adenocarcinoma cells. The rBM 3-D culture-specific miRNA profile was highlighted with higher expression of the tumor suppressive miRNAs (i.e., miR-200 family) and lower expression of the oncogenic miRNAs (i.e., miR-17–92 cluster and miR-21) than that of 2-D culture. Moreover, the expression pattern of miR-17, miR-21, and miR-200a in rBM 3-D culture correlated with the expression of their targets and acinar morphogenesis, a differentiation behavior of lung epithelial cells in rBM 3-D culture. Over-expression of miR-21 suppressed its target PTEN and disrupted acinar morphogenesis. In summary, we provide the first miRNA profile of lung adenocarcinoma cells in rBM 3-D culture with respect to acinar morphogenesis. These results indicate that rBM 3-D culture is essential to a comprehensive understanding of the miRNA biology in lung epithelial cells pertinent to lung adenocarcinoma.