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Background Monocytes -key regulators of the innate immune response- are actively involved in the pathogenesis of systemic lupus erythematosus (SLE). We sought to identify novel compounds that might serve as monocyte-directed targeted therapies in SLE. Results We performed mRNA sequencing in monocytes from 15 patients with active SLE and 10 healthy individuals. Disease activity was assessed with the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2 K). Leveraging the drug repurposing platforms iLINCS, CLUE and L1000CDS2, we identified perturbagens capable of reversing the SLE monocyte signature. We identified transcription factors and microRNAs (miRNAs) that regulate the transcriptome of SLE monocytes, using the TRRUST and miRWalk databases, respectively. A gene regulatory network, integrating implicated transcription factors and miRNAs was constructed, and drugs targeting central components of the network were retrieved from the DGIDb database. Inhibitors of the NF-κB pathway, compounds targeting the heat shock protein 90 (HSP90), as well as a small molecule disrupting the Pim-1/NFATc1/NLRP3 signaling axis were predicted to efficiently counteract the aberrant monocyte gene signature in SLE. An additional analysis was conducted, to enhance the specificity of our drug repurposing approach on monocytes, using the iLINCS, CLUE and L1000CDS2 platforms on publicly available datasets from circulating B-lymphocytes, CD4+ and CD8+ T-cells, derived from SLE patients. Through this approach we identified, small molecule compounds, that could potentially affect more selectively the transcriptome of SLE monocytes, such as, certain NF-κB pathway inhibitors, Pim-1 and SYK kinase inhibitors. Furthermore, according to our network-based drug repurposing approach, an IL-12/23 inhibitor and an EGFR inhibitor may represent potential drug candidates in SLE. Conclusions Application of two independent - a transcriptome-reversal and a network-based -drug repurposing strategies uncovered novel agents that might remedy transcriptional disturbances of monocytes in SLE.

The pathogenic mechanisms determining the diverse clinical outcomes of HEV infection (e.g., self-limiting versus chronic or symptomatic versus asymptomatic) are not yet understood. Because specific microRNA signatures during viral infection inform the cellular processes involved in virus replication and pathogenesis, we investigated plasma microRNA profiles in 44 subjects, including patients with symptomatic acute (AHE, n = 7) and chronic (CHE, n = 6) hepatitis E, blood donors with asymptomatic infection (HEV BDs, n = 9), and anti-HEV IgG + IgM − (exposed BDs, n = 10) and anti-HEV IgG − IgM − (naive BDs, n = 12) healthy blood donors. By measuring the abundance of 179 microRNAs in AHE patients and naive BDs by reverse transcription-quantitative PCR (RT-qPCR), we identified 51 potential HEV-regulated microRNAs (P value adjusted for multiple testing by the Benjamini-Hochberg correction [ P ] < 0.05). Further analysis showed that HEV genotype 3 infection is associated with miR-122, miR-194, miR-885, and miR-30a upregulation and miR-221, miR-223, and miR-27a downregulation. AHE patients showed significantly higher levels of miR-122 and miR-194 and lower levels of miR-221, miR-27a, and miR-335 than HEV BDs. This specific microRNA signature in AHE could promote virus replication and reduce antiviral immune responses, contributing to the development of clinical symptoms. We found that miR-194, miR-335, and miR-221 can discriminate between asymptomatic HEV infections and those developing acute symptoms, whereas miR-335 correctly classifies AHE and CHE patients. Our data suggest that diverse outcomes of HEV infection result from different HEV-induced microRNA dysregulations. The specific microRNA signatures described offer novel information that may serve to develop biomarkers of HEV infection outcomes and improve our understanding of HEV pathogenesis, which may facilitate the identification of antiviral targets. IMPORTANCE There is increasing evidence that viruses dysregulate the expression and/or secretion of microRNAs to promote viral replication, immune evasion, and pathogenesis. In this study, we evaluated the change in microRNA abundance in patients with acute or chronic HEV infection and asymptomatic HEV-infected blood donors. Our results suggest that different HEV-induced microRNA dysregulations may contribute to the diverse clinical manifestations of HEV infection. The specific microRNA signatures identified in this study hold potential as predictive markers of HEV infection outcomes, which would improve the clinical management of hepatitis E patients, particularly of those developing severe symptoms or chronic infections. Furthermore, this study provides new insights into HEV pathogenesis that may serve to identify antiviral targets, which would have a major impact because no effective treatments are yet available

Background Planarians reliably regenerate all body parts after injury, including a fully functional head and central nervous system. But until now, the expression dynamics and functional role of miRNAs and other small RNAs during the process of head regeneration are not well understood. Furthermore, little is known about the evolutionary conservation of the relevant small RNAs pathways, rendering it difficult to assess whether insights from planarians will apply to other taxa. Results In this study, we applied high throughput sequencing to identify miRNAs, tRNA fragments and piRNAs that are dynamically expressed during head regeneration in Dugesia japonica. We further show that knockdown of selected small RNAs, including three novel Dugesia-specific miRNAs, during head regeneration induces severe defects including abnormally small-sized eyes, cyclopia and complete absence of eyes. Conclusions Our findings suggest that a complex pool of small RNAs takes part in the process of head regeneration in Dugesia japonica and provide novel insights into global small RNA expression profiles and expression changes in response to head amputation. Our study reveals the evolutionary conserved role of miR-124 and brings further promising candidate small RNAs into play that might unveil new avenues for inducing restorative programs in non-regenerative organisms via small RNA mimics based therapies.

[eng] Venous Thrombosis (VT) is a frequent complex disease that involves a disruption of the balance of the hemostatic system. microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Promising findings in other complex diseases encourage their use as clinical biomarkers. However, up to now, the implication of miRNAs in VT has not been studied in-depth. The main objective of this Thesis was to study the regulatory mechanisms of the gene expression through miRNAs in VT. The specific objectives were (1) to identify a plasma miRNA profile associated with VT and to analyze its suitability as biomarker, (2) to dissect the interactions between biological layers in the biological context of VT and (3) to identify factors affecting either expression or quantification of miRNAs in plasma. In the first article, the differential expression of miRNAs in plasma was analyzed in the ‘Genetic Analysis of Idiopathic Thrombophilia’ 2 (GAIT-2) Project, which involves 935 individuals belonging to 35 extended Spanish families with idiopathic thrombophilia. First, we conducted a discovery phase, in which 752 miRNAs were measured in plasma by quantitative Polymerase Chain Reaction in 104 individuals of GAIT-2 (52 VT cases and 52 controls). Sixteen miRNAs were selected, which were measured in the entire GAIT-2 (n=935). Four of the miRNAs were significantly associated with VT (false discovery rate 0.7. The first model (VT ~ GATA2 + von Willebrand Factor) showed that the expression of GATA2 in blood was inversely correlated with the blood levels of von Willebrand Factor, and that the disruption of this relationship represents a prothrombotic phenotype. The second model (VT ~ Factor IX, ANXA2 + ENTPD1 + ILK + PDPK1 + PRKAR1A + STXBP3 +hsa-miR- 885-5p + hsa-miR-192-5p) represented an interaction between the fibrinolytic system and platelet activation through the αIIbβ3 signaling pathway. The third model (VT ~CSRP1+ LYN + hsa-miR-192-5p+ hsa-miR-885-5p) revealed the interaction between two group of genes involved in platelet activation, correlated with Protein S, and two miRNAs, in the biological context of VT. In the third article, we used the miRNA expression data of the discovery phase (103 miRNAs in 104 subjects) to analyze the effect of biological and technical factors in their expression. First, we found that the hemolysis marker represented ~10% of the shared variability of miRNA expression. Therefore, using this value as a continuous covariate, beyond as a categorical control, could help to increase consistency across miRNA studies. Second, we found that the expression of miRNAs in plasma was not biased by any blood cell count. Then, we identified 1,323 genetic variants associated with the expression of 16 miRNA genes, that represent 158 independent loci. Finally, we found that these loci were enriched in promoter regions from several tissues, though not in blood tissue. This finding is in agreement with the results regarding blood cell counts, and encourages the role of circulating miRNAs as biomarkers of tissue specific conditions., [spa] El objetivo general de esta Tesis fue estudiar los mecanismos de regulación de la expresión génica mediante microRNAs (miRNAs) en la trombosis venosa (VT). En el primer artículo, se explora la expresión diferencial de miRNAs en VT utilizando la población del proyecto ‘Genetic Analysis of Idiopathic Thrombophilia’ 2 (GAIT-2), compuesta por 935 individuos de 35 familias con trombosis idiopática. El diseño experimental implicó una fase de descubrimiento en la que 752 miRNAs plasmáticos se cuantificaron en 104 individuos del GAIT2 (52 casos y 52 controles) y se seleccionaron 16 miRNAs para la fase de validación, en la que se cuantificaron en toda la población GAIT-2 (n=935). Cuatro de los miRNAs se asociaron significativamente con la enfermedad (false discovery rate

[Background] MicroRNAs are small regulatory RNAs with important roles in carcinogenesis. Genetic variants in these regulatory molecules may contribute to disease. We aim to identify allelic variants in microRNAs as susceptibility factors to gastric cancer using association studies and functional approaches., [Methods] Twenty-one single nucleotide variants potentially functional, because of their location in either the seed, mature or precursor region of 22 microRNAs, were selected for association studies. Genetic association with gastric cancer in 365 cases and 1,284 matched controls (European Prospective Investigation into Cancer and Nutrition Cohort) was analysed using logistic regression. MicroRNA overexpression, transcriptome analysis, and target gene validation experiments were performed for functional studies., [Results] rs3746444:T>C, in the seed of MIR499A and mature MIR499B, associated with the cardia adenocarcinoma location; rs12416605:C>T, in the seed of MIR938, associated with the diffuse subtype; and rs2114358:T>C, in the precursor MIR1206, associated with the noncardia phenotype. In all cases, the association was inverse, indicating a protective affect against gastric cancer of the three minor allelic variants. MIR499 rs3746444:T>C and MIR1206 rs2114358:T>C are reported to affect the expression of these miRNAs, but the effect of MIR938 rs12416605:C>T is unknown yet. Functional approaches showed that the expression of MIR938 is affected by rs12416605:C>T and revealed that MIR938 could regulate a subset of cancer-related genes in an allele-specific fashion. Furthermore, we demonstrated that CXCL12, a chemokine participating in gastric cancer metastasis, is specifically regulated by only one of the rs12416605:C>T alleles., [Conclusion] rs12416605 appears to be involved in gastric cancer by affecting the regulatory function of MIR938 on genes related to this cancer type, particularly on CXCL12 posttranscriptional regulation., >la Caixa> Foundation. Grant Number: BM06-130-0; European Regional Development Fund. Grant Number: PI070130 and PI12/01187; Ministerio de Ciencia e Innovación. Grant Number: BFU2010-18477; Fondo de Fomento al Desarrollo Científico y Tecnológico. Grant Number: FONDECYT 1170446; Instituto de Salud Carlos III.

One largely unknown question in cell biology is the discrimination between inconsequential and functional transcriptional events with relevant regulatory functions. Here, we find that the oncofetal HMGA2 gene is aberrantly reexpressed in many tumor types together with its antisense transcribed pseudogene RPSAP52. RPSAP52 is abundantly present in the cytoplasm, where it interacts with the RNA binding protein IGF2BP2/IMP2, facilitating its binding to mRNA targets, promoting their translation by mediating their recruitment on polysomes and enhancing proliferative and self-renewal pathways. Notably, downregulation of RPSAP52 impairs the balance between the oncogene LIN28B and the tumor suppressor let-7 family of miRNAs, inhibits cellular proliferation and migration in vitro and slows down tumor growth in vivo. In addition, high levels of RPSAP52 in patient samples associate with a worse prognosis in sarcomas. Overall, we reveal the roles of a transcribed pseudogene that may display properties of an oncofetal master regulator in human cancers.

Ovarian cancer is the most lethal gynecological malignancy due to the silent nature on its early onset and the rapid acquisition of drug resistance. Histologically heterogeneous, it includes several subtypes with different mutational landscapes, hampering the development of effective targeted therapies. Non-coding RNAs are emerging as potential new therapeutic targets in cancer. To search for a microRNA signature related to ovarian carcinomas and study its potential as effective targeted therapy, we examined the expression of 768 miRNA in a large collection of tumor samples and found miR-654-5p to be infraexpressed in ovarian serous carcinomas, the most common and aggressive type. Restoration of miR-654-5p levels reduced tumor cell viability in vitro and in vivo and impaired sphere formation capacity and viability of ovarian cancer patient-derived ascitic cells ex vivo. CDCP1 and PLAGL2 oncogenes were found to be the most relevant direct miR-654-5p targets and both genes convey in a molecular signature associated with key cancer pathways relevant to ovarian tumorigenesis, such as MYC, WNT and AKT pathways. Together, we unveiled the tumor suppressor function of miR-654-5p, suggesting that its restoration or co-targeting of CDCP1 and PLAGL2 may be an effective therapeutic approach for ovarian cancer., This work was supported in part by grants from Instituto de la Mujer Dexeus (DEXEUS-B29/012), CIBER (CB16/12/00328), SGR (2017 SGR 1661), the Ministerio de Economia y Competitividad and Fondos FEDER (RTC-2015-3821-1), Instituto Carlos III (PI15/00238 to A.S. and PI17/00564 to M.F.S) and the Miguel Servet Program (CP13/00158 and CPII18/00027 to AS. and CPII16/00006 to MFS). AP and LS were supported by predoctoral VHIR fellowships and CJ by an AGAUR predoctoral fellowship (VHIR: PRED-VHIR-2014-11 and PRED-VHIR-2017; AGAUR: 2017FI_B_00095, respectively).

Fluoxetine (FLX) is a pharmaceutical used to treat affective disorders in humans, but as environmental contaminant also affects inadvertently exposed fish in urban watersheds. In humans and fish, acute FLX treatment and exposure are linked to endocrine disruption, including effects on the reproductive and stress axes. Using the zebrafish model, we build on the recent finding that developmental FLX exposure reduced cortisol production across generations, to determine possible parental and/or life-stage-dependent (age and/or breeding experience) contributions to this phenotype. Specifically, we combined control and developmentally FLX-exposed animals of both sexes (F 0 ) into four distinct breeding groups mated at 5 and 9 months, and measured offspring (F 1 ) basal cortisol at 12 dpf. Basal cortisol was lower in F 1 descended from developmentally FLX-exposed F 0 females bred at 5, but not 9 months, revealing a maternal, life-stage dependent effect. To investigate potential molecular contributions to this phenotype, we profiled maternally deposited transcripts involved in endocrine stress axis development and regulation, epigenetic (de novo DNA methyltransferases) and post-transcriptional (miRNA pathway components and specific miRNAs) regulation of gene expression in unfertilized eggs. Maternal FLX exposure resulted in decreased transcript abundance of glucocorticoid receptor, dnmt3 paralogues and miRNA pathway components in eggs collected at 5 months, and increased transcript abundance of miRNA pathway components at 9 months. Specific miRNAs predicted to target stress axis transcripts decreased (miR-740) or increased (miR-26, miR-30d, miR-92a, miR-103) in eggs collected from FLX females at 5 months. Increased abundance of miRNA-30d and miRNA-92a persisted in eggs collected from FLX females at 9 months. Clustering and principal component analyses of egg transcript profiles separated eggs collected from FLX-females at 5 months from other groups, suggesting that oocyte molecular signatures, and miRNAs in particular, may serve as predictive tools for the offspring phenotype of reduced basal cortisol in response to maternal FLX exposure. © 2019 Martinez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., RM was supported also by a FPU predoctoral fellowship (www.ciencia.gob.es), grant #FPU15/03332, and research mobility grants from the Spanish Ministry of Education, Culture and Sport (www.mecd.gob.es), grant #EST16/00695 and grant #EST17/00830, respectively. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (www.nserc-crsng.gc.ca) under the Discovery Grant program, grant #2114456-2017, and The Canada Foundation of Innovation (www.innovation. ca), under the John R. Evans Leaders Fund (https://www.innovation.ca) program, grant #148035, awarded to JAM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

microRNAs (miRNAs) play key roles in cancer, but their propensity to couple their targets as competing endogenous RNAs (ceRNAs) has only recently emerged. Multiple models have studied ceRNA regulation, but these models did not account for the effects of co-regulation by miRNAs with many targets. We modeled ceRNA and simulated its effects using established parameters for miRNA/mRNA interaction kinetics while accounting for co-regulation by multiple miRNAs with many targets. Our simulations suggested that co-regulation by many miRNA species is more likely to produce physiologically relevant context-independent couplings. To test this, we studied the overlap of inferred ceRNA networks from four tumor contexts—our proposed pan-cancer ceRNA interactome (PCI). PCI was composed of interactions between genes that were co-regulated by nearly three-times as many miRNAs as other inferred ceRNA interactions. Evidence from expression-profiling datasets suggested that PCI interactions are predictive of gene expression in 12 independent tumor- and non-tumor contexts. Biochemical assays confirmed ceRNA couplings for two PCI subnetworks, including oncogenes CCND1, HIF1A and HMGA2, and tumor suppressors PTEN, RB1 and TP53. Our results suggest that PCI is enriched for context-independent interactions that are coupled by many miRNA species and are more likely to be context independent.

Circulating small RNAs, including miRNAs but also isomiRs and other RNA species, have the potential to be used as non-invasive biomarkers for communicable and non-communicable diseases. This study aims to characterize and compare small RNA profiles in human biofluids. For this purpose, RNA was extracted from plasma and breast milk samples from 15 healthy postpartum mothers. Small RNA libraries were prepared with the NEBNext(R) small RNA library preparation kit and sequenced in an Illumina HiSeq2000 platform. miRNAs, isomiRs and clusters of small RNAs were annotated using seqBuster/seqCluster framework in 5 plasma and 10 milk samples that passed the initial quality control. The RNA yield was 81 ng/mL [standard deviation (SD): 41] and 3985 ng/mL (SD: 3767) for plasma and breast milk, respectively. Mean number of good quality reads was 4.04 million (M) (40.01% of the reads) in plasma and 12.5M (89.6%) in breast milk. One thousand one hundred eighty two miRNAs, 12,084 isomiRs and 1,053 small RNA clusters that included piwi-interfering RNAs (piRNAs), tRNAs, small nucleolar RNAs (snoRNA) and small nuclear RNAs (snRNAs) were detected. Samples grouped by biofluid, with 308 miRNAs, 1,790 isomiRs and 778 small RNA clusters differentially detected. In summary, plasma and milk showed a different small RNA profile. In both, miRNAs, piRNAs, tRNAs, snRNAs, and snoRNAs were identified, confirming the presence of non-miRNA species in plasma, and describing them for the first time in milk.

Increasing evidence indicates that microRNAs (miRNAs) are contributing factors to neurodegeneration. Alterations in miRNA signatures have been reported in several neurodegenerative dementias, but data in prion diseases are restricted to ex vivo and animal models. The present study identified significant miRNA expression pattern alterations in the frontal cortex and cerebellum of sporadic Creutzfeldt-Jakob disease (sCJD) patients. These changes display a highly regional and disease subtype-dependent regulation that correlates with brain pathology. We demonstrate that selected miRNAs are enriched in sCJD isolated Argonaute(Ago)-binding complexes in disease, indicating their incorporation into RNA-induced silencing complexes, and further suggesting their contribution to disease-associated gene expression changes. Alterations in the miRNA-mRNA regulatory machinery and perturbed levels of miRNA biogenesis key components in sCJD brain samples reported here further implicate miRNAs in sCJD gene expression (de)regulation. We also show that a subset of sCJD-altered miRNAs are commonly changed in Alzheimer’s disease, dementia with Lewy bodies and fatal familial insomnia, suggesting potential common mechanisms underlying these neurodegenerative processes. Additionally, we report no correlation between brain and cerebrospinal fluid (CSF) miRNA-profiles in sCJD, indicating that CSF-miRNA profiles do not faithfully mirror miRNA alterations detected in brain tissue of human prion diseases. Finally, utilizing a sCJD MM1 mouse model, we analyzed the miRNA deregulation patterns observed in sCJD in a temporal manner. While fourteen sCJD-related miRNAs were validated at clinical stages, only two of those were changed at early symptomatic phase, suggesting that the miRNAs altered in sCJD may contribute to later pathogenic processes. Altogether, the present work identifies alterations in the miRNA network, biogenesis and miRNA-mRNA silencing machinery in sCJD, whereby contributions to disease mechanisms deserve further investigation., Author summary miRNAs are small non-coding RNAs that regulate gene expression through complementary binding to their mRNA targets. Specific miRNA signatures have been proposed for several neurodegenerative diseases supporting the idea that miRNA deregulation is a common disease hallmark. Here we present the comprehensive miRNA signature in sporadic Creutzfeldt-Jakob disease (sCJD). Our study unravels the complex network of regional and disease-subtype miRNA alterations, and the presence of a disturbed miRNA biogenesis pathway and miRNA-mRNA silencing machinery. We also highlight the existence of time-dependent miRNA profiles and identify commonly regulated miRNAs between several dementias with cortical pathology sharing a partial clinical overlap and pathological involvement with sCJD. The present data shed light on the potential role of miRNAs as a contributing factor of pathogenic molecular traits associated with sCJD.

A high percentage of the mammalian genome consists of non-coding RNAs (ncRNAs). Among ncRNAs two main subgroups have been identified: long ncRNAs (lncRNAs) and micro RNAs (miRNAs). ncRNAs have been demonstrated to play a role in a vast variety of diseases, since they regulate gene transcription and are involved in post-transcriptional regulation. They have the potential to function as molecular signals or as guides for transcription factors and to regulate epigenetic modifiers. In this literature review we have summarized data on miRNAs and lncRNAs and their involvement in dyslipidaemia, atherosclerosis, insulin resistance and adipogenesis. Outlining certain ncRNAs as disease biomarkers and/or therapeutic targets, and testing them in vivo, will be the next steps in future research.

Micro RNAs (miRNAs) are small single strand non-coding RNAs that regulate geneexpression at the post-transcriptional level, either by translational inhibition or mRNA degradation based on the extent of complementarity between the miRNA and its target mRNAs. Potato (Solanum tuberosum L.) is the most important horticultural crop in Argentina. Achieving an integrated control of diseases is crucial for this crop, where frequent agrochemical applications, particularly fungicides, are carried out. A promising strategy is based on promoting induced resistance through the application ofenvironmentally friendly compounds such as phosphites, inorganic salts of phosphorous acid. The use of phosphites in disease control management has provento be effective. Although the mechanisms underlying their effect remain unclear, we postulated that miRNAs could be involved. Therefore we performed next generation sequencing (NGS) in potato leaves treated and non treated with potassium phosphite (KPhi). We identified 25 miRNAs that were expressed differentially, 14 already annotated in miRBase and 11 mapped to the potato genome as potential new miRNAs. A prediction of miRNA targets showed genes related to pathogen resistance,transcription factors, and oxidative stress. We also analyzed in silico stress and phytohormone responsive cis-acting elements on differentially expressed pre miRNAs. Despite the fact that some of the differentially expressed miRNAs have been already identified, this is to our knowledge the first report identifying miRNAs responsive to abiocompatible stress resistance inducer such as potassium phosphite, in plants.Further characterization of these miRNAs and their target genes, might help toelucidate the molecular mechanisms underlying KPhi-induced resistance. Fil: Rey Burusco, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Daleo, Gustavo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Feldman, Mariana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

During the development of the nervous system the regulation of cell cycle, differentiation, and survival is tightly interlinked. Newly generated neurons must keep cell cycle components under strict control, as cell cycle re-entry leads to neuronal degeneration and death. However, despite their relevance, the mechanisms controlling this process remain largely unexplored. Here we show that Scratch2 is involved in the control of the cell cycle in neurons in the developing spinal cord of the zebrafish embryo. scratch2 knockdown induces postmitotic neurons to re-enter mitosis. Scratch2 prevents cell cycle re-entry by maintaining high levels of the cycle inhibitor p57 through the downregulation of miR-25. Thus, Scratch2 appears to safeguard the homeostasis of postmitotic primary neurons by preventing cell cycle re-entry.

International audience; The female mammary gland is a very dynamic organ that undergoes continuous tissue remodeling during adulthood. Although it is well established that the number of menstrual cycles and pregnancy (in this case transiently) increase the risk of breast cancer, the reasons are unclear. Growing clinical and experimental evidence indicates that improper involution plays a role in the development of this malignancy. Recently, we described the miR-424(322)/503 cluster as an important regulator of mammary epithelial involution after pregnancy. Here, through the analysis of ∼3000 primary tumors, we show that miR-424(322)/503 is commonly lost in a subset of aggressive breast cancers and describe the genetic aberrations that inactivate its expression. Furthermore, through the use of a knockout mouse model, we demonstrate for the first time that loss of miR-424(322)/503 promotes breast tumorigenesis in vivo. Remarkably, we found that loss of miR-424(322)/503 promotes chemoresistance due to the up-regulation of two of its targets: BCL-2 and insulin-like growth factor-1 receptor (IGF1R). Importantly, targeted therapies blocking the aberrant activity of these targets restore sensitivity to chemotherapy. Overall, our studies reveal miR-424(322)/ 503 as a tumor suppressor in breast cancer and provide a link between mammary epithelial involution, tumori-genesis, and the phenomenon of chemoresistance.

Research in RF laboratory is currently supported by the Spanish Ministerio de Economía y Competitividad (grant BFU2014-56812-P).

Recently, microRNAs (miRNA) have been proposed as regulators in the different processes involved in alcohol intake, and differences have been found in the miRNA expression profile in alcoholics. However, no study has focused on analyzing polymorphisms in genes encoding miRNAs and daily alcohol consumption at the population level. Our aim was to investigate the association between a functional polymorphism in the pre-miR-27a (rs895819 A>G) gene and alcohol consumption in an elderly population. We undertook a cross-sectional study of PREvención con DIeta MEDiterránea (PREDIMED)-Valencia participants (n = 1007, including men and women aged 67 7 years) and measured their alcohol consumption (total and alcoholic beverages) through a validated questionnaire. We found a strong association between the pre-miR-27a polymorphism and total alcohol intake, this being higher in GG subjects (5.2 0.4 in AA, 5.9 0.5 in AG and 9.1 1.8 g/day in GG; padjusted = 0.019). We also found a statistically-significant association of the pre-miR-27a polymorphism with the risk of having a high alcohol intake (>2 drinks/day in men and >1 in women): 5.9% in AA versus 17.5% in GG; padjusted < 0.001. In the sensitivity analysis, this association was homogeneous for sex, obesity and Mediterranean diet adherence. In conclusion, we report for the first time a significant association between a miRNA polymorphism (rs895819) and daily alcohol consumption. This study was funded, by the Spanish Ministry of Health (Instituto de Salud Carlos III) and the Ministerio de Economía y Competitividad-Fondo Europeo de Desarrollo Regional (Projects CNIC-06/2007, RTIC G03/140, CIBER 06/03, PI06-1326, PI07-0954, PI11/02505, SAF2009-12304, AGL2010-22319-C03-03 and PRX14/00527), by the University Jaume I (Project P1-1B2013-54), by Contracts 53-K06-5-10 and 58-1950-9-001 from the U.S. Department of Agriculture Research Service, USA, by the Generalitat Valenciana (ACOMP2010-181, AP111/10, AP-042/11, ACOM2011/145, ACOMP/2012/190, ACOMP/2013/159 and ACOMP/213/165), and with the collaboration of the Real Colegio Complutense at Harvard University, Cambridge. MA, USA. Rocío Barragán’s contract is funded by the Ayudas para la contratación de personal investigador en formación de caracter predoctoral, Programa “VALencia Investigación más Desarrollo” (VALi+d). Conselleria d’Educació, Investigació, Cultura i Esport. Generalitat Valenciana, Spain (ACIF/2013/168).

Most diseases, including human cancer, are frequently associated with an altered transcription pattern. The alteration of the transcriptome is not restricted to the production of aberrant levels of protein-coding RNAs, but also refers to the dysregulation of the expression of the multiple noncoding members that comprise the human genome. Unexpectedly, recent RNA-seq data of the human transcriptome have revealed that less than 2% of the genome encodes protein-coding transcripts, even though the vast majority of the genome is actively transcribed into non-coding RNAs (ncRNAs) under different conditions. In this review, we present an updated version of the mechanistic aspects of some long non-coding RNAs (lncRNAs) that play critical roles in human cancer. Most importantly, we focus on the interplay between lncRNAs and microRNAs, and the importance of such interactions during the tumorigenic process, providing new insight into the regulatory mechanisms underlying several ncRNA classes of importance in cancer, particularly transcribed ultraconserved regions (T-UCRs). This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway., An interferon-induced miRNA suppresses the sterol biosynthesis pathway via multiple targets, thereby helping establish broad cellular resistance to unrelated clinically significant viruses., Author Summary How infected cells respond to a virus during the first minutes to hours after infection can determine whether a disease develops and influences the host’s long-term survival. In mammals, unlike plants and flies that use small RNAs to fight viral infections, virus-induced interferon responses are a critical early event resulting in broad protection against infection. Interferon is a secreted host protein that binds to receptors on the surface of infected and uninfected cells and activates biochemical pathways that profoundly change the expression of hundreds of cellular genes, including those encoding microRNAs. The antiviral functions of only a handful of these genes are understood, and it is not known how the majority contribute to broadly protect against many different viruses. In this study, we uncover an interferon-regulated microRNA (miR-342-5p) that contributes to broad host cell immunity against infection through the cholesterol biosynthesis pathway. We show that miR-342-5p does this through a multihit strategy, turning down the master regulator of sterol biosynthesis as well as several specifically targeted enzymes within the pathway. A wide range of viruses depend on a number of the metabolite side-branches of the sterol biosynthesis pathway for their replication. Notably, our study reveals that by utilising multihit targeting of key branch-points in a single pathway, miR-342-5p is able to inhibit the replication of unrelated, clinically significant pathogens ranging from Herpes to Flu viruses.

Background Micro RNAs are a class of small non coding RNAs of 20–24 nucleotides transcribed as single stranded precursors from MIR gene loci. Initially described as post-transcriptional regulators involved in development, two decades ago, miRNAs have been proven to regulate a wide range of processes in plants such as germination, morphology and responses to biotic and abiotic stress. Despite wide conservation in plants, a number of miRNAs are lineage specific. We describe the first genome wide survey of Eucalyptus miRNAs based on high throughput sequencing. Results In addition to discovering small RNA sequences, MIR loci were mapped onto the reference genome and interspecific variability investigated. Sequencing was carried out for the two most world widely planted species, E. grandis and E. globulus. To maximize discovery, E. grandis samples were from BRASUZ1, the same tree whose genome provided the reference sequence. Interspecific analysis reinforces the variability in small RNA repertoire even between closely related species. Characterization of Eucalyptus small RNA sequences showed 95 orthologous to conserved miRNAs and 193 novel miRNAs. In silico target prediction confirmed 163 novel miRNAs and degradome sequencing experimentally confirmed several hundred targets. Experimental evidence based on the exclusive expression of a set of small RNAs across 16 species within Myrtaceae further highlighted variable patterns of conservation and diversity of these regulatory elements. Conclusions The description of miRNAs in Eucalyptus contributes to scientific knowledge of this vast genre, which is the most widely planted hardwood crop in the tropical and subtropical world, adding another important element to the annotation of Eucalyptus grandis reference genome. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2322-6) contains supplementary material, which is available to authorized users.

Long non-coding RNAs (lncRNAs) are deregulated in several tumors, although their role in acute myeloid leukemia (AML) is mostly unknown. We have examined the expression of the lncRNA HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in 241 AML patients. We have correlated HOTAIRM1 expression with a miRNA expression profile. We have also analyzed the prognostic value of HOTAIRM1 expression in 215 intermediate-risk AML (IR-AML) patients. The lowest expression level was observed in acute promyelocytic leukemia (P < 0.001) and the highest in t(6; 9) AML (P = 0.005). In 215 IR-AML patients, high HOTAIRM1 expression was independently associated with shorter overall survival OR: 2.04; P = 0.001), shorter leukemia-free survival (OR: 2.56; P < 0.001) and a higher cumulative incidence of relapse (OR: 1.67; P = 0.046). Moreover, HOTAIRM1 maintained its independent prognostic value within the favorable molecular subgroup (OR: 3.43; P = 0.009). Interestingly, HOTAIRM1 was overexpressed in NPM1-mutated AML (P < 0.001) and within this group retained its prognostic value (OR: 2.21; P = 0.01). Moreover, HOTAIRM1 expression was associated with a specific 33- microRNA signature that included miR-196b (P < 0.001). miR-196b is located in the HOX genomic region and has previously been reported to have an independent prognostic value in AML. miR-196b and HOTAIRM1 in combination as a prognostic factor can classify patients as high-, intermediate-, or low-risk (5-year OS: 24% vs 42% vs 70%; P = 0.004). Determination of HOTAIRM1 level at diagnosis provided relevant prognostic information in IR-AML and allowed refinement of risk stratification based on common molecular markers. The prognostic information provided by HOTAIRM1 was strengthened when combined with miR-196b expression. Furthermore, HOTAIRM1 correlated with a 33-miRNA signature., Marina Diaz-Beya is supported by ISCII (Rio Hortega CM13/00205). This research was in part supported by Fundacion Espanola de Hematologia Hemoterapia (beca de investigacion MDB). This research is also supported by grants from Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III PI13/00999 (IP: Dr. Jordi Esteve), RETICS RD12/0036/0010 (JE; MDB) and SDCSD from School of Medicine, University of Barcelona, AECC-Catalunya 2013 (AN) (sponsored by Mat Holding). Anna Cordeiro is an APIF fellow of the University of Barcelona

Saliva is a complex body fluid that comprises secretions from the major and minor salivary glands, which are extensively supplied by blood. Therefore, molecules such as proteins, DNA, RNA, etc., present in plasma could be also present in saliva. Many studies have reported that saliva body fluid can be useful for discriminating several oral diseases, but also systemic diseases including cancer. Most of these studies revealed messenger RNA (mRNA) and proteomic biomarker signatures rather than specific non-coding RNA (ncRNA) profiles. NcRNAs are emerging as new regulators of diverse biological functions, playing an important role in oncogenesis and tumor progression. Indeed, the small size of these molecules makes them very stable in different body fluids and not as susceptible as mRNAs to degradation by ribonucleases (RNases). Therefore, the development of a non-invasive salivary test, based on ncRNAs profiles, could have a significant applicability to clinical practice, not only by reducing the cost of the health system, but also by benefitting the patient. Here, we summarize the current status and clinical implications of the ncRNAs present in human saliva as a source of biological information.

Review on MIR135A1, with data on DNA/RNA and where the gene is implicated.

Tesis para obtener el grado de Doctor en el área de Ciencias Biológicas, de la Universidad de Buenos Aires, en 2013 Las pérdidas en cultivos ocasionadas por virus dependen mayormente de la severidad de los síntomas producidos. La comprensión de los mecanismos moleculares que subyacen a la producción de síntomas es de importancia para el desarrollo de estrategias que permitan reducir las pérdidas ocasionadas por fitovirus. Mediante el uso de cepas de virus de los géneros Tobamovirus y Potyvirus, el presente trabajo analizó la relación entre la severidad de sus síntomas en Nicotiana tabacum y Arabidopsis thaliana con su capacidad para alterar diferencialmente parámetros morfológicos, fisiológicos y transcriptómicos relevantes para los procesos de desarrollo y respuestas a estrés, hormonas y defensa. Los resultados indicaron que a tiempos tempranos de la infección en tejidos sistémicos, cuando aún no son visibles los síntomas característicos de la infección, se producen cambios moleculares importantes. Éstos incluyen alteraciones en la acumulación de metabolitos vinculados con procesos de señalización y defensa, así como cambios transcriptómicos en genes clave en redes de senescencia, defensa y respuesta a hormonas. La respuesta de microRNAs con importantes roles en el desarrollo ante tratamientos con hormonas y virus exhibió un componente transcripcional y muchos de estos genes reguladores evidenciaron un patrón bifásico de cambios, sugiriendo que los procesos que conducen a su alteración en etapas tempranas de la infección difieren de los que condicionan la misma a etapas avanzadas. En Arabidopsis, gran parte de las diferencias en alteraciones fisiológicas y de síntomas inducidas por distintas cepas de un Potyvirus se perdieron en plantas mutantes para la vía del ácido salicílico. A la luz de estos hallazgos, se propone un mecanismo por el cual la retroalimentación entre ácido salicílico y especies reactivas de oxígeno determina la activación de genes clave en los procesos de desarrollo y senescencia. Losses on crops caused by virus mostly depend on symptoms severity. The understanding of the molecular mechanisms underlying the production of symptoms is important for the development of strategies that will reduce the losses caused by plant virus. By using viral strains of virus of the genus Tobamovirus and Potyvirus, this study analyzed the relationship between the severity of their symptoms in Nicotiana tabacum and Arabidopsis thaliana with their ability to differentially alter physiological, morphological and transcriptomic parameters relevant to the processes of development and responses to stress, hormones, and defense. The results indicated that at early times of infection in systemic tissues, when the characteristic symptoms of the infection are still not visible, important molecular changes occur. These include alterations in the accumulation of metabolites linked to processes of signaling and defense, as well as transcriptomic changes in key genes in senescence, defense and response to hormones networks. The response of microRNAs with important roles in development following treatment with hormones and viruses exhibited a transcriptional component and many of these regulatory genes showed a biphasic pattern of changes, suggesting that the processes that lead to their alteration at early stages of infection differ from which condition it to advanced stages. In Arabidopsis, much of the differences in physiological changes and symptoms induced by different strains of a Potyvirus were lost in mutant plants for salicylic acid pathway. In the light of these findings, a mechanism is proposed by which the feedback between salicylic acid and reactive oxygen species determines the activation of key gen es in the processes of development and senescence. Instituto de Biotecnología Fil: Manacorda, Carlos Augusto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina

BackgroundIn recent years, microRNA (miRNA) pathways have emerged as a crucial system for the regulation of tumorogenesis. miR-SNPs are a novel class of single nucleotide polymorphisms that can affect miRNA pathways.Design and methodsWe analyzed eight miR-SNPs by allelic discrimination in 141 patients with Hodgkin lymphoma and correlated the results with treatment-related toxicity, response, disease-free survival (DFS) and overall survival (OS).ResultsThe KRT81 (rs3660) GG genotype was associated with an increased risk of neurological toxicity (P = 0.016), while patients with XPO5 (rs11077) AA or CC genotypes had a higher rate of bleomycin-associated pulmonary toxicity (P = 0.048). Both miR-SNPs emerged as independent factors in the multivariate analysis. The XPO5 AA and CC genotypes were also associated with a lower response rate (P = 0.036). XPO5 (P = 0.039) and TRBP (rs784567) (P = 0.022) genotypes emerged as prognostic markers for DFS, and XPO5 was also associated with OS (P = 0.033). In the multivariate analysis, only XPO5 emerged as an independent prognostic factor for DFS (HR: 2.622; 95%CI 1.039-6.620; P = 0.041). Given the influence of XPO5 and TRBP as individual markers, we then investigated the combined effect of these miR-SNPs. Patients with both the XPO5 AA/CC and TRBP TT/TC genotypes had the shortest DFS (P = 0.008) and OS (P = 0.008).ConclusionmiR-SNPs can add useful prognostic information on treatment-related toxicity and clinical outcome in Hodgkin lymphoma and can be used to identify patients likely to be chemoresistant or to relapse.

Prostate cancer (PCa) is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.

Phenotypic plasticity is often postulated as a principal characteristic of tuber-bearing wild Solanum species. The hypotheses to explore this observation have been developed based on the presence of genetic variation. In this context, evolutionary changes and adaptation are impossible without genetic variation. However, epigenetic effects, which include DNA methylation and microRNAs expression control, could be another source of phenotypic variation in ecologically relevant traits. To achieve a detailed mechanistic understanding of these processes, it is necessary to separate epigenetic from DNA sequence-based effects and to evaluate their relative importance on phenotypic variability. We explored the potential relevance of epigenetic effects in individuals with the same genotype. For this purpose, a clone of the wild potato Solanum ruiz-lealii, a non-model species in which natural methylation variability has been demonstrated, was selected and its DNA methylation was manipulated applying 5-Azacytidine (AzaC), a demethylating agent. The AzaC treatment induced early flowering and changes in leaf morphology. Using quantitative real-time PCR, we identified four miRNAs up-regulated in the AzaC-treated plants. One of them, miRNA172, could play a role on the early flowering phenotype. In this work, we showed that the treatment with AzaC could provide meaningful results allowing to study both the phenotypic plasticity in tuber-bearing Solanum species and the inter-relation between DNA methylation and miRNA accumulations in a wide range of species. Fil: Marfil, Carlos Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Masuelli, Ricardo Williams. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Mendoza-San Juan. Estación Experimental Agropecuaria La Consulta; Argentina

[Background]: The molecular mechanisms by which PROS1 mutations result in protein S deficiency are still unknown for many of the mutations, particularly for those that result in a premature termination codon. The aim of this study was to analyze the functional relevance on mRNA and protein expression of 12 natural PROS1 mutations associated with protein S deficiency., [Design and methods]: Five mutations were nonsense, three were small frameshift deletions, one was c.258,259AG>GT at the 3' end of exon 3, one was p.M640T and the last two were c.-7C>G and p.L15H, found in double heterozygosis as [c.-7C>G;44T>A]. The apparently neutral variant p.R233K was also analyzed. PROS1 cDNA was assessed by reverse transcriptase polymerase chain reaction of platelet mRNA. Expression of mutant proteins was determined by site-directed mutagenesis and analyses of transiently transfected PROS1 mutants in COS-7 cells., [Results]: Only cDNA from the normal allele was observed from the five nonsense mutations, the frameshift deletion c.1731delT and from c.258,259AG>GT. Both the normal and the mutated alleles were observed from [c.-7C>G;44T>A], c.187,188delTG and p.M640T. Transient expression analyses of PROS1 mutants whose mRNA was normally expressed revealed greatly reduced secretion of p.L15H and c.1272delA, mild secretion values of p.M640T and normal secretion levels of c.-7C>G and, as expected, p.R233K., [Conclusions]: Whereas the main cause of quantitative protein S deficiency associated with missense mutations is defective synthesis, stability or secretion of the mutated protein, the main mechanism for the deficiency associated with mutations that generate a premature termination codon is not the synthesis of a truncated protein, but the exclusion of the mutated allele, probably by nonsense-mediated mRNA decay., Funding: we acknowledge grants ISCIII 01/1468, ISCIII network C03/07 and ISCIII PI051149, from the Instituto de Salud Carlos III; SAF 2001-1059 and SAF2004-07539, from the Spanish Ministry of Education and Science; and grant 2002-PIR-00333 from the AGAUR, Generalitat de Catalunya. BH is the recipient of a grant from the Institut d’Investigació Biomèdica de Bellvitge (IDIBELL06/IDB-001).

Learning and memory difficulties observed in patients with fragile X syndrome, as well as in a mouse model for the syndrome, are partially due to impaired translational regulation of neuronal mRNAs encoding key molecules for the synaptic structure and function. There has been intense interest in characterizing the mRNAs that are regulated by the fragile X mental retardation protein (FMRP) in the neuronal cell. A large number of candidate FMRP-interacting mRNAs has been identified over the last few years and three models have been described so far to explain the specificity of these interactions. Here, we report our vision on how they could work in the same and/or in different pathways and suggest that the three mechanisms may not be mutually exclusive. ispartof: Neuroreport vol:15 issue:16 pages:2447-2450 ispartof: location:England status: published

In primary culture of adult rat hepatocytes, vanadate in the presence of glucose stimulates the expression of the liver (L-type) pyruvate kinase gene. Glucose by itself was inactive, and vanadate, like insulin, was also inefficient in the absence of glucose. Similar results were obtained on glucokinase gene expression. An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.

Intermittent hypoxia (IH) during sleep is one of the major abnormalities occurring in patients suffering from obstructive sleep apnea (OSA), a highly prevalent disorder affecting 6–15% of the general population, particularly among obese people. IH has been proposed as a major determinant of oncogenetically-related processes such as tumor growth, invasion and metastasis. During the growth and expansion of tumors, fragmented DNA is released into the bloodstream and enters the circulation. Circulating tumor DNA (cirDNA) conserves the genetic and epigenetic profiles from the tumor of origin and can be isolated from the plasma fraction. Here we report a microarray-based epigenetic profiling of cirDNA isolated from blood samples of mice engrafted with TC1 epithelial lung cancer cells and controls, which were exposed to IH during sleep (XenoIH group, n=3) or control conditions, (i.e., room air (RA); XenoRA group, n=3) conditions. To prepare the targets for microarray hybridization, we applied a previously developed method that enriches the modified fraction of the cirDNA without amplification of genomic DNA. Regions of differential cirDNA modification between the two groups were identified by hybridizing the enriched fractions for each sample to Affymetrix GeneChip Human Promoter Arrays 1.0R. Microarray raw and processed data were deposited in NCBI's Gene Expression Omnibus (GEO) database (accession number: GSE61070).

Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.

INTRODUCTION: Preoperative chemoradiotherapy (CRT) is a standard treatment for locally advanced rectal cancer patients. Despite the benefits of CRT, its use in non-responder patients can be associated with increased toxicities and surgical resection delay. The identification of CRT response biomarkers, such as microRNAs, could improve the management of these patients. We have studied the microRNA expression in pretreatment endoscopy biopsies from rectal cancer patients treated with CRT to identify potential microRNAs able to predict CRT response and clinical outcome of these patients. MATERIAL AND METHODS: RNA from pretreatment endoscopy biopsies from 96 rectal cancer patients treated with preoperative CRT were studied. Pathological response was graded according to the tumor regression grade (TRG) Dworak classification. In the screening phase, 377 miRNAs were studied in 12 patients with extreme responses (TRG0-1 vs TRG4). The potential role as predictive biomarkers for CRT response, disease-free survival (DFS) and overall survival (OS) of the miRNAs identified in the screening phase were validated in the whole cohort. RESULTS: In the screening phase, an 8-miRNAs CRT-response signature was identified: let-7b, let-7e, miR-21, miR-99b, miR-183, miR-328, miR-375 and miR-483-5p. In the validation phase, miR-21, miR-99b and miR-375 emerged as CRT response-related miRNAs while miR-328 and let-7e emerged as prognostic markers for DFS and OS. Interestingly, ROC curve analysis showed that the combination of miR-21, miR-99b and miR-375 had the best capacity to distinguish patients with maximum response (TRG4) from others. CONCLUSIONS: miR-21, miR-99b and miR-375 could add valuable information for individualizing treatment in locally advanced rectal cancer patients.