MicroRNAs miR-146-5p and let-7f as prognostic tools for aggressive papillary thyroid carcinoma: a case report

Papillary thyroid cancer (PTC) is the most incident histotype of thyroid cancer. A certain fraction of PTC cases (5%) are irresponsive to conventional treatment, and refractory to radioiodine therapy. The current prognostic factors for aggressiveness are mainly based on tumor size, the presence of lymph node metastasis, extrathyroidal invasion and, more recently, the presence of the BRAFT(1799A) mutation. MicroRNAs (miRNAs) have been described as promising molecular markers for cancer as their deregulation is observed in a wide range of tumors. Recent studies indicate that the over-expression of miR-146b-5p is associated with aggressiveness and BRAFT(1799A) mutation. Furthermore, down-regulation of let-7f is observed in several types of tumors, including PTC. In this study, we evaluated the miR146b-5p and let-7f status in a young male patient with aggressive, BRAFT(1799A)-positive papillary thyroid carcinoma, with extensive lymph node metastases and short-time recurrence. The analysis of miR-146b-5p and let-7f expression revealed a distinct pattern from a cohort of PTC patients, suggesting caution in evaluating miRNA expression data as molecular markers of PTC diagnosis and prognosis. Arq Bras Endocrinol Metab. 2012;56(8):552-7

Identifying common and specific microRNAs expressed in peripheral blood mononuclear cell of type 1, type 2, and gestational diabetes mellitus patients

Abstract Background Regardless the regulatory function of microRNAs (miRNA), their differential expression pattern has been used to define miRNA signatures and to disclose disease biomarkers. To address the question of whether patients presenting the different types of diabetes mellitus could be distinguished on the basis of their miRNA and mRNA expression profiling, we obtained peripheral blood mononuclear cell (PBMC) RNAs from 7 type 1 (T1D), 7 type 2 (T2D), and 6 gestational diabetes (GDM) patients, which were hybridized to Agilent miRNA and mRNA microarrays. Data quantification and quality control were obtained using the Feature Extraction software, and data distribution was normalized using quantile function implemented in the Aroma light package. Differentially expressed miRNAs/mRNAs were identified using Rank products, comparing T1DxGDM, T2DxGDM and T1DxT2D. Hierarchical clustering was performed using the average linkage criterion with Pearson uncentered distance as metrics. Results The use of the same microarrays platform permitted the identification of sets of shared or specific miRNAs/mRNA interaction for each type of diabetes. Nine miRNAs (hsa-miR-126, hsa-miR-1307, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-144, hsa-miR-199a-5p, hsa-miR-27a, hsa-miR-29b, and hsa-miR-342-3p) were shared among T1D, T2D and GDM, and additional specific miRNAs were identified for T1D (20 miRNAs), T2D (14) and GDM (19) patients. ROC curves allowed the identification of specific and relevant (greater AUC values) miRNAs for each type of diabetes, including: i) hsa-miR-1274a, hsa-miR-1274b and hsa-let-7f for T1D; ii) hsa-miR-222, hsa-miR-30e and hsa-miR-140-3p for T2D, and iii) hsa-miR-181a and hsa-miR-1268 for GDM. Many of these miRNAs targeted mRNAs associated with diabetes pathogenesis. Conclusions These results indicate that PBMC can be used as reporter cells to characterize the miRNA expression profiling disclosed by the different diabetes mellitus manifestations. Shared miRNAs may characterize diabetes as a metabolic and inflammatory disorder, whereas specific miRNAs may represent biological markers for each type of diabetes, deserving further attention.

贾第虫microRNA 及其靶基因的预测与验证

MicroRNAs (miRNAs)是一类长约21-25nt 的非编码小分子RNAs，通过与靶基因的互补结合在转录水平及转录后水平来负调控基因表达。人们已在众多高等多细胞生物中如人、果蝇、线虫、拟南芥等鉴定出众多microRNAs 分子。近来报道单细胞原生生物衣藻中也存在大量microRNAs。然而到目前为止，在被很多证据证实是最原始的单细胞真核生物贾第虫中却仍未有microRNAs 的报道。那么到底贾第虫这种具有特殊进化地位的单细胞原生动物是否存在有microRNAs 呢？如果存在的话，其microRNAs 的特点是什么？与高等多细胞生物及单细胞衣藻的 microRNA 相比又有何异同点呢？贾第虫的microRNAs 是否与其致病性相关呢？已有研究表明，贾第虫基因组中存在与RNAi 相关的Argonaute（AGO）家族蛋白和Dicer 酶。有意思的是，这些与siRNA 引起RNAi 作用关键的蛋白AGO 和Dicer 同样也是miRNA 系统的关键成份，这就提示我们在贾第虫中很有可能也存在有miRNA 并发挥功能。有研究发现在贾第虫基因组中存在大量的非编码转录物，这些大量的非编码转录物中，是否都是后来所认为的为双向启动子转录有用基因时的副产物，还是也存在一些起调控作用的RNA 分子（如miRNAs 等），需要进一步的研究。本文利用生物信息学的手段，依据miRNAs 的生物学特征，结合多种计算机预测的方法，在贾第虫基因组中筛选可能的microRNAs 分子，结果共鉴定出50 个miRNAs 候选分子，这50 个可能的贾第虫miRNAs 不具有保守性，在已知的其他物种的miRNAs 中找不到同源物。用这50 个microRNAs BLASTN 贾第虫的蛋白质编码序列及其相邻5’端和3’端各200bp 的序列，来寻找这些microRNAs 所调控的靶基因。结果表明，寻找到的贾第虫miRNA 的靶基因除很大一部分未知功能的蛋白外，还包括了很多涉及不同功能的蛋白，如VSP 蛋白（various surface proteins）这样一类表面抗原蛋白，提示我们贾第虫miRNA 可能与其致病性相关。接下来我们对其中14 个预测的贾第虫microRNAs 进行了RT-PCR 检测并克隆测序，结果表明gla-mir-6, gla-mir-35 在贾第虫滋养体细胞中稳定表达。我们的研究第一次用生物信息学结合实验的方法在贾第虫寻找到了microRNAs，为下一步深入研究这些microRNAs 在贾第虫中的功能提供了可能。

Roles of microRNA in plant defense and virus offense interaction

MicroRNAs (miRNA) that are around 22 nucleotides long non-protein-coding RNAs, play key regulatory roles in plants. Recent research findings show that miRNAs are involved in plant defense and viral offense systems. Advances in understanding the mechanism of miRNA biogenesis and evolution are useful for elucidating the complicated roles they play in viral infection networks. In this paper a brief summary of evolution of plant anti-virus defense is given and the function of miRNAs involved in plant-virus competition is highlighted. It is believed that miRNAs have several advantages over homology-dependent and siRNA-mediated gene silencing when they are applied biotechnologically to promote plant anti-virus defense. miRNA-mediated anti-virus pathway is an ancient mechanism with a promising future. However, using miRNAs as a powerful anti-virus tool will be better realized only if miRNA genomics and functions in plant viral infection are fully understood.

Identification de caractéristiques communes et rares dans les ARN structurés dans la base de données Rfam

Les ARN non codants (ARNnc) sont des transcrits d'ARN qui ne sont pas traduits en protéines et qui pourtant ont des fonctions clés et variées dans la cellule telles que la régulation des gènes, la transcription et la traduction. Parmi les nombreuses catégories d'ARNnc qui ont été découvertes, on trouve des ARN bien connus tels que les ARN ribosomiques (ARNr), les ARN de transfert (ARNt), les snoARN et les microARN (miARN). Les fonctions des ARNnc sont étroitement liées à leurs structures d’où l’importance de développer des outils de prédiction de structure et des méthodes de recherche de nouveaux ARNnc. Les progrès technologiques ont mis à la disposition des chercheurs des informations abondantes sur les séquences d'ARN. Ces informations sont accessibles dans des bases de données telles que Rfam, qui fournit des alignements et des informations structurelles sur de nombreuses familles d'ARNnc. Dans ce travail, nous avons récupéré toutes les séquences des structures secondaires annotées dans Rfam, telles que les boucles en épingle à cheveux, les boucles internes, les renflements « bulge », etc. dans toutes les familles d'ARNnc. Une base de données locale, RNAstem, a été créée pour faciliter la manipulation et la compilation des données sur les motifs de structure secondaire. Nous avons analysé toutes les boucles terminales et internes ainsi que les « bulges » et nous avons calculé un score d’abondance qui nous a permis d’étudier la fréquence de ces motifs. Tout en minimisant le biais de la surreprésentation de certaines classes d’ARN telles que l’ARN ribosomal, l’analyse des scores a permis de caractériser les motifs rares pour chacune des catégories d’ARN en plus de confirmer des motifs communs comme les boucles de type GNRA ou UNCG. Nous avons identifié des motifs abondants qui n’ont pas été étudiés auparavant tels que la « tetraloop » UUUU. En analysant le contenu de ces motifs en nucléotides, nous avons remarqué que ces régions simples brins contiennent beaucoup plus de nucléotides A et U. Enfin, nous avons exploré la possibilité d’utiliser ces scores pour la conception d’un filtre qui permettrait d’accélérer la recherche de nouveaux ARN non-codants. Nous avons développé un système de scores, RNAscore, qui permet d’évaluer un ARN en se basant sur son contenu en motifs et nous avons testé son applicabilité avec différents types de contrôles.

MicroRNA miR-146b-5p regulates signal transduction of TGF-beta by repressing SMAD4 in thyroid cancer

MicroRNAs (miRNA) are small non-coding RNAs involved in post-transcriptional gene regulation that have crucial roles in several types of tumors, including papillary thyroid carcinoma (PTC). miR-146b-5p is overexpressed in PTCs and is regarded as a relevant diagnostic marker for this type of cancer. A computational search revealed that miR-146b-5p putatively binds to the 3' untranslated region (UTR) of SMAD4, an important member of the transforming growth factor beta (TGF-beta) signaling pathway. The TGF-beta pathway is a negative regulator of thyroid follicular cell growth, and the mechanism by which thyroid cancer cells evade its inhibitory signal remains unclear. We questioned whether the modulation of the TGF-beta pathway by miR-146b-5p can contribute to thyroid tumorigenesis. Luciferase reporter assay confirmed the direct binding of miR-146b-5p on the SMAD4 3'UTR. Specific inhibition of miR-146b-5p with a locked nucleic acid-modified anti-miR-146b oligonucleotide significantly increased SMAD4 levels in the human papillary carcinoma cell lines, TPC-1 and BCPAP. Moreover, suppression of miR-146b-5p increased the cellular response to the TGF-beta anti-proliferative signal, significantly decreasing the proliferation rate. The overexpression of miR-146b-5p in normal rat follicular PCCL3 cells decreased SMAD4 levels and disrupted TGF-beta signal transduction. MiR-146b-5p overexpression in PCCL3 cells also significantly increased cell proliferation in the absence of thyroid-stimulating hormone and conferred resistance to TGF-beta-mediated cell-cycle arrest. Additionally, the activation of thyroid most common oncogenes RET/PTC3 and BRAF in PCCL3 cells upregulated miR-146b-5p expression. Our results confirm the oncogenic role of miR-146b-5p in thyroid follicular cells and contribute to knowledge regarding the modulation of TGF-beta signal transduction by miRNAs in PTCs. Oncogene (2012) 31, 1910-1922; doi:10.1038/onc.2011.381; published online 29 August 2011

Molekulare Ursachen und therapeutische Intervention bei der Alzheimer-Demenz

Ein charakteristisches, neuropathologisches Merkmal der Alzheimer-Demenz (AD), der am häufigsten vorkommenden Demenz-Form des Menschen, ist das Auftreten von senilen Plaques im Gehirn der Patienten. Hierbei stellt das neurotoxische A-beta Peptid den Hauptbestandteil dieser Ablagerungen dar. Einen Beitrag zu der pathologisch erhöhten A-beta Generierung liefert das verschobene Expressionsgleichgewicht der um APP-konkurrierenden Proteasen BACE-1 und ADAM10 zu Gunsten der beta-Sekretase BACE-1. In der vorliegenden Dissertation sollten molekulare Mechanismen identifiziert werden, die zu einem pathologisch veränderten Gleichgewicht der APP-Spaltung und somit zum Entstehen und Fortschritt der AD beitragen. Des Weiteren sollten Substanzen identifiziert werden, die durch Beeinflussung der Genexpression einer der beiden Proteasen das physiologische Gleichgewicht der APP-Prozessierung wiederherstellen können und somit therapeutisch einsetzbar sind.rnAnhand eines „Screenings“ von 704 Transkriptionsfaktoren wurden 23 Faktoren erhalten die das Verhältnis ADAM10- pro BACE-1-Promotor Aktivität beeinflussten. Exemplarisch wurden zwei der molekularen Faktoren auf ihren Wirkmechanismus untersucht: Der TF „X box binding protein-1“ (XBP-1), der die so genannte „unfolded protein response“ (UPR) reguliert, erhöhte die Expression von ADAM10 in Zellkultur-Experimenten. Die Menge dieses Faktors war in AD-Patienten im Vergleich zu gesunden, Alters-korrelierten Kontrollen signifikant erniedrigt. Im Gegensatz dazu verminderte der Seneszenz-assoziierte TF „T box 2“ (Tbx2) die Menge an ADAM10 in SH-SY5Y Zellen. Die Expression des Faktors selbst war in post-mortem Kortexgewebe von AD-Patienten erhöht. Zusätzlich zu den TFs konnten in einer Kooperation mit dem Helmholtz Zentrum München drei microRNAs (miRNA 103, 107, 1306) bioinformatisch prädiziert und experimentell validiert werden, die die Expression des humanen ADAM10 reduzierten.rnIm Rahmen dieser Arbeit konnten damit körpereigene Faktoren identifiziert werden, die die Menge an ADAM10 regulieren und folglich potenziell an der Entstehung der gestörten Homöostase der APP-Prozessierung beteiligt sind. Somit ist die AD auch im Hinblick auf eine A-beta-vermittelte Pathologie als multifaktorielle Krankheit zu verstehen, in der verschiedene Regulatoren zur gestörten APP-Prozessierung und somit zur pathologisch gesteigerten A-beta Generierung beitragen können. rnEine pharmakologische Erhöhung der ADAM10 Genexpression würde zu der Freisetzung von neuroprotektivem APPs-alpha und gleichzeitig zu einer reduzierten A-beta Generierung führen. Deshalb war ein weiteres Ziel dieser Arbeit die Evaluierung von Substanzen mit therapeutischem Potenzial im Hinblick auf eine erhöhte ADAM10 Expression. Von 640 FDA-zugelassenen Medikamenten einer Substanz-Bibliothek wurden 23 Substanzen identifiziert, die die Menge an ADAM10 signifikant steigerten während die Expression von BACE-1 und APP unbeeinflusst blieb. In Zusammenarbeit mit dem Institut für Pathologie (Johannes Gutenberg Universität Mainz) wurde ein Zellkultur-basiertes Modell etabliert, um die Permeationsfähigkeit der potenziellen Kandidaten-Substanzen über die Blut-Hirn Schranke (BHS) zu untersuchen. Von den 23 Medikamenten konnten neun im Rahmen des etablierten Modells als BHS-gängig charakterisiert werden. Somit erfüllen diese verbleibenden Medikamente die grundlegenden Anforderungen an ein AD-Therapeutikum. rnADAM10 spaltet neben APP eine Vielzahl anderer Substrate mit unterschiedlichen Funktionen in der Zelle. Zum Beispiel reguliert das Zelladhäsionsmolekül Neuroligin-1 (NL-1), das von ADAM10 prozessiert wird, die synaptische Funktion exzitatorischer Neurone. Aus diesem Grund ist die Abschätzung potenzieller, Therapie-bedingter Nebenwirkungen sehr wichtig. Im Rahmen eines Forschungsaufenthalts an der Universität von Tokio konnte in primären, kortikalen Neuronen der Ratte bei einer Retinoid-induzierten Erhöhung von ADAM10 neben einer vermehrten alpha-sekretorischen APP-Prozessierung auch eine gesteigerte Spaltung von NL-1 beobachtet werden. Dies lässt vermuten, dass bei einer Behandlung mit dem Retinoid Acitretin neben einer vermehrten APP-Spaltung durch ADAM10 auch die Regulation glutamaterger Neurone durch die Spaltung von NL-1 betroffen ist. Anhand eines geeigneten Alzheimer-Tiermodells sollten diese Befunde weiter analysiert werden, um so auf einen sicheren therapeutischen Ansatz bezüglich einer vermehrten ADAM10 Genexpression schließen zu können.rn

miR-15a and miR-16 are implicated in cell cycle regulation in a Rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer

MicroRNAs (miRNA) are negative regulators of gene expression at the posttranscriptional level, which are involved in tumorigenesis. Two miRNAs, miR-15a and miR-16, which are located at chromosome 13q14, have been implicated in cell cycle control and apoptosis, but little information is available about their role in solid tumors. To address this question, we established a protocol to quantify miRNAs from laser capture microdissected tissues. Here, we show that miR-15a/miR-16 are frequently deleted or down-regulated in squamous cell carcinomas and adenocarcinomas of the lung. In these tumors, expression of miR-15a/miR-16 inversely correlates with the expression of cyclin D1. In non-small cell lung cancer (NSCLC) cell lines, cyclins D1, D2, and E1 are directly regulated by physiologic concentrations of miR-15a/miR-16. Consistent with these results, overexpression of these miRNAs induces cell cycle arrest in G(1)-G(0). Interestingly, H2009 cells lacking Rb are resistant to miR-15a/miR-16-induced cell cycle arrest, whereas reintroduction of functional Rb resensitizes these cells to miRNA activity. In contrast, down-regulation of Rb in A549 cells by RNA interference confers resistance to these miRNAs. Thus, cell cycle arrest induced by these miRNAs depends on the expression of Rb, confirming that G(1) cyclins are major targets of miR-15a/miR-16 in NSCLC. Our results indicate that miR-15a/miR-16 are implicated in cell cycle control and likely contribute to the tumorigenesis of NSCLC.

Genetic Predictors of Clinical Outcomes in Non-Small Cell Lung Cancer Patients

Lung cancer is the leading cause of cancer-related mortality in the US. Emerging evidence has shown that host genetic factors can interact with environmental exposures to influence patient susceptibility to the diseases as well as clinical outcomes, such as survival and recurrence. We aimed to identify genetic prognostic markers for non-small cell lung cancer (NSCLC), a major (85%) subtype of lung cancer, and also in other subgroups. With the fast evolution of genotyping technology, genetic association studies have went through candidate gene approach, to pathway-based approach, to the genome wide association study (GWAS). Even in the era of GWAS, pathway-based approach has its own advantages on studying cancer clinical outcomes: it is cost-effective, requiring a smaller sample size than GWAS easier to identify a validation population and explore gene-gene interactions. In the current study, we adopted pathway-based approach focusing on two critical pathways - miRNA and inflammation pathways. MicroRNAs (miRNA) post-transcriptionally regulate around 30% of human genes. Polymorphisms within miRNA processing pathways and binding sites may influence patients’ prognosis through altered gene regulation. Inflammation plays an important role in cancer initiation and progression, and also has shown to impact patients’ clinical outcomes. We first evaluated 240 single nucleotide polymorphisms (SNPs) in miRNA biogenesis genes and predicted binding sites in NSCLC patients to determine associations with clinical outcomes in early-stage (stage I and II) and late-stage (stage III and IV) lung cancer patients, respectively. First, in 535 early-stage patients, after correcting multiple comparisons, FZD4:rs713065 (hazard ratio [HR]:0.46, 95% confidence interval [CI]:0.32-0.65) showed a significant inverse association with survival in early stage surgery-only patients. SP1:rs17695156 (HR:2.22, 95% CI:1.44-3.41) and DROSHA:rs6886834 (HR:6.38, 95% CI:2.49-16.31) conferred increased risk of progression in the all patients and surgery-only populations, respectively. FAS:rs2234978 was significantly associated with improved survival in all patients (HR:0.59, 95% CI:0.44-0.77) and in the surgery plus chemotherapy populations (HR:0.19, 95% CI:0.07-0.46).. Functional genomics analysis demonstrated that this variant creates a miR-651 binding site resulting in altered miRNA regulation of FAS, providing biological plausibility for the observed association. We then analyzed these associations in 598 late-stage patients. After multiple comparison corrections, no SNPs remained significant in the late stage group, while the top SNP NAT1:rs15561 (HR=1.98, 96%CI=1.32-2.94) conferred a significantly increased risk of death in the chemotherapy subgroup. To test the hypothesis that genetic variants in the inflammation-related pathways may be associated with survival in NSCLC patients, we first conducted a three-stage study. In the discovery phase, we investigated a comprehensive panel of 11,930 inflammation-related SNPs in three independent lung cancer populations. A missense SNP (rs2071554) in HLA-DOB was significantly associated with poor survival in the discovery population (HR: 1.46, 95% CI: 1.02-2.09), internal validation population (HR: 1.51, 95% CI: 1.02-2.25), and external validation (HR: 1.52, 95% CI: 1.01-2.29) population. Rs2900420 in KLRK1 was significantly associated with a reduced risk for death in the discovery (HR: 0.76, 95% CI: 0.60-0.96) and internal validation (HR: 0.77, 95% CI: 0.61-0.99) populations, and the association reached borderline significance in the external validation population (HR: 0.80, 95% CI: 0.63-1.02). We also evaluated these inflammation-related SNPs in NSCLC patients in never smokers. Lung cancer in never smokers has been increasingly recognized as distinct disease from that in ever-smokers. A two-stage study was performed using a discovery population from MD Anderson (411 patients) and a validation population from Mayo Clinic (311 patients). Three SNPs (IL17RA:rs879576, BMP8A:rs698141, and STK:rs290229) that were significantly associated with survival were validated (pCD74:rs1056400 and CD38:rs10805347) were borderline significant (p=0.08) in the Mayo Clinic population. In the combined analysis, IL17RA:rs879576 resulted in a 40% reduction in the risk for death (p=4.1 × 10-5 [p=0.61, heterogeneity test]). We also validated a survival tree created in MD Anderson population in the Mayo Clinic population. In conclusion, our results provided strong evidence that genetic variations in specific pathways that examined (miRNA and inflammation pathways) influenced clinical outcomes in NSCLC patients, and with further functional studies, the novel loci have potential to be translated into clinical use.

miRNAs as therapeutic agents in neurodegeneration, a pilot study

L’échec des différents essais cliniques souligne la nécessité de développer des nouvelles thérapies pour la maladie d’Alzheimer (MA), la cause la plus commune de démence. Les microARNs (miARNs) sont les ARNs non-codants les plus étudiés et ils jouent un rôle important dans la modulation de l’expression des gènes et de multiples voies de signalisation. Des études antérieures, dont celles de mon laboratoire d’accueil, ont permis de développer l’hypothèse que certains membres de la famille miR-15/107 (c.-à-d. miR-15ab, miR-16, miR-195, miR-424, and miR-497) pourraient être utilisés comme agents thérapeutiques dans MA. En effet, cette famille avait le potentiel de réguler de multiples gènes associés à MA, tels que la protéine précurseur de l’amyloïde (APP), la β-secrétase (BACE1), et la protéine Tau. Tel que démontré dans ce projet de thèse, j’ai choisi miR-16 comme cible thérapeutique potentielle pour MA parmi tous les membres de la famille. L’essai luciférase dans ce projet confirme que miR-16 peut réguler simultanément APP et BACE1, directement par une interaction avec la région non-codante en 3’ de l’ARNm). Notamment, nous observons aussi une réduction de la production des peptides amyloïdes et de la phosphorylation de Tau après une augmentation de miR-16 en cellule. J’ai ensuite validé mes résultats in vivo dans la souris en utilisant une méthode de livraison de miR-16 via une pompe osmotique implanté dans le cerveau. Dans ce cas, l’expression des protéines d’intérêts (APP, BACE1, Tau) a été mesurée par immunobuvardage et PCR à temps réel. Après validation, ces résultats ont été complémentés par une étude protéomique (iTRAQ) du tronc cérébral et de l’hippocampe, deux régions associées à la maladie. Ces données m’ont permis d’identifier d’autres protéines régulées par miR-16 in vivo, incluant α-Synucléine, Transferrine receptor1, et SRm300. Une autre observation intéressante : les voies régulées par miR-16 in vivo sont directement en lien avec le stress oxydatif et la neurodégénération. En résumé, ce travail démontre l’efficacité et la faisabilité d’utiliser un miARN comme outil thérapeutique pour la maladie d’Alzheimer. Ces résultats rentrent dans un cadre plus vaste de découvrir de nouvelles cibles pour MA, et en particulier la forme sporadique de la maladie qui représente plus de 95% de tous les cas. Évidemment, la découverte d’une molécule pouvant cibler simultanément les deux pathologies de la maladie (plaques amyloïdes et hyper phosphorylation de tau) est nouvelle et intéressante, et ce domaine de recherche ouvre la porte aux autres petits ARNs non-codants dans MA et les maladies neurodégénératives connexes.

MicroRNA-23a has minimal effect on endurance exercise-induced adaptation of mouse skeletal muscle

Skeletal muscles contain several subtypes of myofibers that differ in contractile and metabolic properties. Transcriptional control of fiber-type specification and adaptation has been intensively investigated over the past several decades. Recently, microRNA (miRNA)-mediated posttranscriptional gene regulation has attracted increasing attention. MiR-23a targets key molecules regulating contractile and metabolic properties of skeletal muscle, such as myosin heavy-chains and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α). In the present study, we analyzed the skeletal muscle phenotype of miR-23a transgenic (miR-23a Tg) mice to explore whether forced expression of miR-23a affects markers of mitochondrial content, muscle fiber composition, and muscle adaptations induced by 4 weeks of voluntary wheel running. When compared with wild-type mice, protein markers of mitochondrial content, including PGC-1α, and cytochrome c oxidase complex IV (COX IV), were significantly decreased in the slow soleus muscle, but not the fast plantaris muscle of miR-23a Tg mice. There was a decrease in type IId/x fibers only in the soleus muscle of the Tg mice. Following 4 weeks of voluntary wheel running, there was no difference in the endurance exercise capacity as well as in several muscle adaptive responses including an increase in muscle mass, capillary density, or the protein content of myosin heavy-chain IIa, PGC-1α, COX IV, and cytochrome c. These results show that miR-23a targets PGC-1α and regulates basal metabolic properties of slow but not fast twitch muscles. Elevated levels of miR-23a did not impact on whole body endurance capacity or exercise-induced muscle adaptations in the fast plantaris muscle.

Wolbachia uses host microRNAs to manipulate host gene expression and facilitate colonization of the dengue vector Aedes aegypti

The obligate endosymbiont Wolbachia pipientis is found in a wide range of invertebrates where they are best known for manipulating host reproduction. Recent studies have shown that Wolbachia also can modulate the lifespan of host insects and interfere with the development of human pathogens in mosquito vectors. Despite considerable study, very little is known about the molecular interactions between Wolbachia and its hosts that might mediate these effects. Using microarrays, we show that the microRNA (miRNA) profile of the mosquito, Aedes aegypti, is significantly altered by the wMelPop-CLA strain of W. pipientis. We found that a host miRNA (aae-miR-2940) is induced after Wolbachia infection in both mosquitoes and cell lines. One target of aae-miR-2940 is the Ae. aegypti metalloprotease gene. Interestingly, expression of the target gene was induced after Wolbachia infection, ectopic expression of the miRNA independent of Wolbachia, or transfection of an artificial mimic of the miRNA into mosquito cells. We also confirmed the interaction of aae-miR-2940 with the target sequences using GFP as a reporter gene. Silencing of the metalloprotease gene in both Wolbachia-infected cells and adult mosquitoes led to a significant reduction in Wolbachia density, as did inhibition of the miRNA in cells. These results indicate that manipulation of the mosquito metalloprotease gene via aae-miR-2940 is crucial for efficient maintenance of the endosymbiont. This report shows how Wolbachia alters the host miRNA profile and provides insight into the mechanisms of host manipulation used by this widespread endosymbiont.

Circulating microRNAs involved in multiple sclerosis

Multiple sclerosis (MS) is an immune-mediated, demyelinating and neurodegenerative disease of the central nervous system. After traumatic brain injury, it is the leading cause of neurology disability in young adults. Considerable advances have been made in identifying genes involved in MS but the genetic and phenotypic complexity associated with this disease significantly hinders any progress. A novel class of small RNA molecules, microRNAs (miRNAs) has acquired much attention because they regulate the expression of up to 30% of protein-coding genes and may play a pivotal role in the development of many, if not all, complex diseases. Seven published studies investigated miRNAs from peripheral blood mononuclear cells, CD4+, CD8+ T cell, B lymphocytes, peripheral blood leukocytes, whole blood and brain astrocytes with MS risk. The absence of MS studies investigating plasma miRNA prompted the current investigation of identifying a circulating miRNA signature in MS. We conducted a microarray analysis of over 900 known miRNA transcripts from plasma samples collected from four MS individuals and four sex-aged and ethnicity matched healthy controls. We identified six plasma miRNA (miR-614, miR-572, miR-648, miR-1826, miR-422a and miR-22) that were significantly up-regulated and one plasma miRNA (miR-1979) that was significantly down-regulated in MS individuals. Both miR-422a and miR-22 have previously been implicated in MS. The present study is the first to show a circulating miRNA signature involved in MS that could serve as a potential prognostic and diagnostic biomarker for MS.

The role of EBV-encoded microRNAs in B cell differentiation

Epstein Barr virus (EBV) is a common γ-herpes virus, infecting approximately 90% of the world‟s population. It is also one of the first known viruses known to be oncogenic, and is associated with a number of tumour types, primarily lymphomas. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and many human miRNAs have been associated with the development of malignancies including cancer. EBV was the first human virus identified to express miRNAs and encodes more than 40 miRNAs within its genome. Yet, an understanding of the targets of EBV-miRNAs, and thereby the function of them in pathogenesis remains sadly limited. This study identifies a potential novel target of EBV-miRNAs, MECP2 and characterises the miRNA:mRNA interactions between two previously identified novel targets; Bim and EBF1. In particular, this study focuses upon the interaction between EBF1 and the EBV-miRNA BART11-5p, demonstrating a 151bp region of the EBF1 3‟UTR that is capable of mediating the silencing of luciferase expression by BART11-5p but is not capable of silencing a full length EBF1-3‟UTR luciferase construct. This study provides evidence that EBF1 may be a target of one or more EBV-miRNAs.

Efficient silencing of endogenous microRNAs using artificial microRNAs in Arabidopsis thaliana

We report here that the expression of endogenous microRNAs (miRNAs) can be efficiently silenced in Arabidopsis thaliana (Arabidopsis) using artificial miRNA (amiRNA) technology. We demonstrate that an amiRNA designed to target a mature miRNA directs silencing against all miRNA family members, whereas an amiRNA designed to target the stem-loop region of a miRNA precursor transcript directs silencing against only the individual family member targeted. Furthermore, our results indicate that amiRNAs targeting both the mature miRNA and stem-loop sequence direct RNA silencing through cleavage of the miRNA precursor transcript, which presumably occurs in the nucleus of a plant cell during the initial stages of miRNA biogenesis. This suggests that small RNA (sRNA)-guided RNA cleavage in plants occurs not only in the cytoplasm, but also in the nucleus. Many plant miRNA gene families have been identified via sequencing and bioinformatic analysis, but, to date, only a small tranche of these have been functionally characterized due to a lack of effective forward or reverse genetic tools. Our findings therefore provide a new and powerful reverse-genetic tool for the analysis of miRNA function in plants. © The Author 2010. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.