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MicroRNAs (miRNAs) are small noncoding RNAs that act as endogenous regulatory molecules targeting specific mRNAs for translational repression. Studies of breast cancer genomics indicate that breast cancer subtypes are distinguished and regulated by specific sets of miRNAs which affect activities such as tumor initiation, progression, and even drug response. Polo-like Kinase 1 (PLK1) is widely considered to be a proto-oncogene due to its increased expression in multiple tumor types, as well as its crucial role in regulating mitosis. Pharmacological inhibition of PLK1 can reduce tumor volume and induce tumor cell death in solid and hematologic malignancies. This prompted us to investigate how PLK1 inhibition with the target-specific inhibitor NMS-P937 would impact breast cancer cells, and how miRNAs may influence the overall response of these cells to this inhibition. We found that miR-183-5p targets PLK1 gene, effectively reducing its protein expression. Such miRNA-driven regulation of PLK1 expression sensitizes breast cancer cells to NMS-P937, resulting in synergistically increased apoptosis. We also show that the miRNA-regulated reduction of PLK1 influences the expression of apoptosis-related key proteins and possibly inducing further indirect PLK1 downmodulation through a DNMT1-p53 axis. These results suggest a potential biologically significant link between the expression of miR-183-5p and the efficacy of PLK1-specific inhibitors in breast cancer cells. Our work further elucidates how miR-183-5p regulates PLK1 gene while also enhancing NMS-P937 effect in breast cancer. Future studies assessing the role of miR-183-5p as a novel biomarker for anti-PLK1 chemotherapy agents are warranted.

MicroRNAs (miRNAs) have been extensively reported to be associated with hematological malignancies. The loss of miR-15a/16–1 at chromosome 13q14 is a hallmark of most of human chronic lymphocytic leukemia (CLL). Deletion of murine miR-15a/16–1 and miR-15b/16–2 has been demonstrated to promote B cell malignancies. Here, we evaluate the biological role of miR-15/16 clusters, crossbreeding miR-15a/16–1 and miR-15b/16–2 knockout mice. Unexpectedly, the complete deletion of both clusters promoted myeloproliferative disorders in the majority of the mice by the age of 5 months with a penetrance of 70%. These mice showed a significant enlargement of spleen and abnormal swelling of lymph nodes. Flow cytometry characterization demonstrated an expanded CD11b/Gr-1 double-positive myeloid population both in spleen and in bone marrow. The transplantation of splenocytes harvested from double-KO mice into wild-type recipient mice resulted in the development of myeloproliferative disorders, as observed in the donors. In vivo, miR-15/16 cluster deletion up-regulated the expression of Cyclin D1, Cyclin D2, and Bcl-2. Taken together, our findings identify a driver oncogenic role for miR-15/16 cluster deletion in different leukocytic cell lineages.

Despite recent advances in targeted therapies, the molecular mechanisms driving breast cancer initiation, progression, and metastasis are minimally understood. Growing evidence indicate that transfer RNA (tRNA)-derived small RNAs (tsRNA) contribute to biological control and aberrations associated with cancer development and progression. The runt-related transcription factor 1 (RUNX1) transcription factor is a tumor suppressor in the mammary epithelium whereas RUNX1 downregulation is functionally associated with breast cancer initiation and progression. We identified four tsRNA (ts-19, ts-29, ts-46, and ts-112) that are selectively responsive to expression of the RUNX1 tumor suppressor. Our finding that ts-112 and RUNX1 anticorrelate in normal-like mammary epithelial and breast cancer lines is consistent with tumor-related activity of ts-112 and tumor suppressor activity of RUNX1. Inhibition of ts-112 in MCF10CA1a aggressive breast cancer cells significantly reduced proliferation. Ectopic expression of a ts-112 mimic in normal-like mammary epithelial MCF10A cells significantly increased proliferation. These findings support an oncogenic potential for ts-112. Moreover, RUNX1 may repress ts-112 to prevent overactive proliferation in breast epithelial cells to augment its established roles in maintaining the mammary epithelium.

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and dysregulation of tRNA-derived short noncoding RNA (tsRNA) (tRF-1) expression is an accompanying event in the development of this disease. tsRNAs are fragments originating from the 3′ end of tRNA precursors and do not contain mature tRNA sequences. In contrast to tsRNAs, mature tRFs (tRF-3s, tRF-5s, and internal tRFs) are produced from mature tRNA sequences and are redundant fragments. We investigated tsRNA expression in CLL and determined tsRNA signatures in indolent CLL and aggressive CLL vs. normal B cells. We noticed that both ts-43 and ts-44 are derived from distinct genes of pre-tRNA His , and are down-regulated in CLL 3- to 5-fold vs. normal B cells. Thus, we investigated expression levels of tRF-5 fragments from tRNA His in CLL samples and healthy controls, and determined that such fragments are down-regulated by 5-fold in CLLs vs. normal controls. Given these results, we investigated the expression of all mature tRFs in CLLs vs. normal controls. We found a drastic dysregulation of the expression of mature tRFs in CLL. In aggressive CLL, for the top 15 up-regulated fragments, linear fold change varied from 2,053- to 622-fold. For the top 15 down-regulated fragments in CLL, linear fold change varied from 314- to 52-fold. In addition, 964 mature tRFs were up-regulated at least 2-fold in CLL, while 701 fragments were down-regulated at least 2-fold. Similar results were obtained for indolent CLL. Our results suggest that mature tRFs may have oncogenic and/or tumor suppressor function in CLL.

// Salvatore Alaimo 2 , Rosalba Giugno 1 , Mario Acunzo 3 , Dario Veneziano 3 , Alfredo Ferro 2 , Alfredo Pulvirenti 2 1 Department of Computer Science, University of Verona, Verona, Italy 2 Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy 3 Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA Correspondence to: Alfredo Pulvirenti, email: apulvirenti@dmi.unict.it Keywords: pathway analysis, microRNAs, phenotype classification, RNA-Seq Received: October 21, 2015 Accepted: May 11, 2016 Published: June 02, 2016 ABSTRACT Motivation: Prediction of phenotypes from high-dimensional data is a crucial task in precision biology and medicine. Many technologies employ genomic biomarkers to characterize phenotypes. However, such elements are not sufficient to explain the underlying biology. To improve this, pathway analysis techniques have been proposed. Nevertheless, such methods have shown lack of accuracy in phenotypes classification. Results: Here we propose a novel methodology called MITHrIL (Mirna enrIched paTHway Impact anaLysis) for the analysis of signaling pathways, which extends the work of Tarca et al., 2009. MITHrIL augments pathways with missing regulatory elements, such as microRNAs, and their interactions with genes. The method takes as input the expression values of genes and/or microRNAs and returns a list of pathways sorted according to their degree of deregulation, together with the corresponding statistical significance (p-values). Our analysis shows that MITHrIL outperforms its competitors even in the worst case. In addition, our method is able to correctly classify sets of tumor samples drawn from TCGA. Availability: MITHrIL is freely available at the following URL: http://alpha.dmi.unict.it/mithril/

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and transgenic mouse studies indicate that activation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease. While studying the regulation of TCL1 expression, we identified the microRNA cluster miR-4521/3676 and discovered that these two microRNAs are associated with tRNA sequences and that this region can produce two small RNAs, members of a recently identified class of small noncoding RNAs, tRNA-derived small RNAs (tsRNAs). We further proved that miR-3676 and miR-4521 are tsRNAs using Northern blot analysis. We found that, like ts-3676, ts-4521 is down-regulated and mutated in CLL. Analysis of lung cancer samples revealed that both ts-3676 and ts-4521 are down-regulated and mutated in patient tumor samples. Because tsRNAs are similar in nature to piRNAs [P-element-induced wimpy testis (Piwi)-interacting small RNAs], we investigated whether ts-3676 and ts-4521 can interact with Piwi proteins and found these two tsRNAs in complexes containing Piwi-like protein 2 (PIWIL2). To determine whether other tsRNAs are involved in cancer, we generated a custom microarray chip containing 120 tsRNAs 16 bp or more in size. Microarray hybridization experiments revealed tsRNA signatures in CLL and lung cancer, indicating that, like microRNAs, tsRNAs may have an oncogenic and/or tumor-suppressor function in hematopoietic malignancies and solid tumors. Thus, our results show that tsRNAs are dysregulated in human cancer.

MicroRNAs (miRNAs) are small noncoding RNA molecules (sncRNAs) involved in gene expression regulation. Having been widely studied during last two decades, they have been associated with several diseases, including cancer. Recent improvements in high throughput sequencing technologies have revealed a more complex miRNAome, due to miRNA sequence modification phenomena, such as RNA editing and isomiRs. As a result, a new class of tools is necessary in order to appropriately investigate this emerging complexity. To address such need, we developed isoTar, a high-performance Web-based containerized application designed for miRNA consensus targeting prediction and functional enrichment analyses. In the present chapter, we provide an overview of isoTar ( https://ncrnaome.osumc.edu/isotar/ ), as well as benchmarks and a guide to its usage.

Noncoding RNAs (ncRNAs) have received much attention due to their central role in gene expression and translational regulation as well as due to their involvement in several biological processes and disease development. Small noncoding RNAs (sncRNAs), such as microRNAs and piwiRNAs, have been thoroughly investigated and functionally characterized. Long noncoding RNAs (lncRNAs), known to play an important role in chromatin-interacting transcription regulation, posttranscriptional regulation, cell-to-cell signaling, and protein regulation, are also being investigated to further elucidate their functional roles.Next-generation sequencing (NGS) technologies have greatly aided in characterizing the ncRNAome. Moreover, the coupling of NGS technology together with bioinformatics tools has been essential to the genome-wide detection of RNA modifications in ncRNAs. RNA editing, a common human co-transcriptional and posttranscriptional modification, is a dynamic biological phenomenon able to alter the sequence and the structure of primary transcripts (both coding and noncoding RNAs) during the maturation process, consequently influencing the biogenesis, as well as the function, of ncRNAs. In particular, the dysregulation of the RNA editing machineries have been associated with the onset of human diseases.In this chapter we discuss the potential functions of ncRNA editing and describe the knowledge base and bioinformatics resources available to investigate such phenomenon.

In the last two decades noncoding RNAs have been the recipients of increasing scientific interest. In particular, miRNAs, short (~22 nts) noncoding transcripts, have been thoroughly investigated since their essential role in posttranscriptional gene expression regulation had been established in the early 2000s. With the advent and the advancements of high-throughput sequencing technologies in recent years, long noncoding RNAs have also started to emerge as important actors in cellular functions and processes. Such transcripts, on average longer than 200 nt, whose functions have yet to be fully characterized, have recently been identified as regulatory elements of the RNAi pathway, harboring several miRNA response elements, uncovering the phenomena of competing endogenous RNAs (ceRNAs), or "sponge RNAs." The present chapter aims to provide a brief update on the actual biomedical relevance of ceRNAs, together with a summary of resources, tools, and practical examples of their application to aid researchers in the discovery and further elucidation of lncRNA-miRNA interactions.

Next-generation sequencing is increasing our understanding and knowledge of non-coding RNAs (ncRNAs), elucidating their roles in molecular mechanisms and processes such as cell growth and development. Within such a class, tRNA-derived ncRNAs have been recently associated with gene expression regulation in cancer progression. In this paper, we characterize, for the first time, tRNA-derived ncRNAs in NCI-60. Furthermore, we assess their expression profile in The Cancer Genome Atlas (TCGA). Our comprehensive analysis allowed us to report 322 distinct tRNA-derived ncRNAs in NCI-60, categorized in tRNA-derived fragments (11 tRF-5s, 55 tRF-3s), tRNA-derived small RNAs (107 tsRNAs) and tRNA 5′ leader RNAs (149 sequences identified). In TCGA, we were able to identify 232 distinct tRNA-derived ncRNAs categorized in 53 tRF-5s, 58 tRF-3s, 63 tsRNAs and 58 5′ leader RNAs. This latter group represents an additional evidence of tRNA-derived ncRNAs originating from the 5′ leader region of precursor tRNA. We developed a public database, tRFexplorer, which provides users with the expression profile of each tRNA-derived ncRNAs in every cell line in NCI-60 as well as for each TCGA tumor type. Moreover, the system allows us to perform differential expression analyses of such fragments in TCGA, as well as correlation analyses of tRNA-derived ncRNAs expression in TCGA and NCI-60 with gene and miRNA expression in TCGA samples, in association with all omics and compound activities data available on CellMiner. Hence, the tool provides an important opportunity to investigate their potential biological roles in absence of any direct experimental evidence. Database URL: https://trfexplorer.cloud/

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia. It is characterized by the accumulation of CD19+/CD5+ lymphocytes and can have variable outcomes. Richter syndrome (RS) is a lethal complication in CLL patients that results in aggressive B-cell lymphomas, and there are no tests to predict its occurrence. Because alterations in microRNA expression can predict the development and progression of several cancers, we investigated whether dysregulation of specific microRNAs can predict RS in CLL patients. Thus, we compared microRNA expression levels in samples from 49 CLL patients who later developed RS with samples from 59 CLL patients who did not. We found that high expression of miR-125a-5p or low expression of miR-34a-5p can predict ∼50% of RS with a false positive rate of ∼9%. We found that CLL patients predicted to develop RS show either an increase of miR-125a-5p expression (∼20-fold) or a decrease of miR-34a-5p expression (∼21-fold) compared with CLL patients that are not predicted to develop RS. Thus, miR-125a-5p and miR-34a-5p can be valuable predictor markers of RS and have the potential to provide physicians with information that can indicate the best therapeutic strategy for CLL patients.

RNA editing in microRNAs has been recently proposed as a novel biomarker in cancer. Here, we investigated RNA editing by leveraging small-RNA sequencing data from 87 NSCLC (Non-Small Cell Lung Cancer) samples paired with normal lung tissues from The Cancer Genome Atlas (TCGA) combined with 26 plasma-derived exosome samples from an independent cohort. Using both the editing levels and microRNA editing expression, we detected deregulated microRNA editing events between NSCLC tumor and normal tissues. Interestingly, and for the first time, we also detected editing sites in the microRNA cargo of circulating exosomes, providing the potential to non-invasively discriminate between normal and tumor samples. Of note, miR-411-5p edited in position 5 was significantly dysregulated in tissues as well as in exosomes of NSCLC patients, suggesting a potential targetome shift relevant to lung cancer biology.

Nearly all classes of coding and non-coding RNA undergo post-transcriptional modification, as more than 150 distinct modification types have been reported. Since RNA modifications were first described over 50 years ago, our understanding of their functional relevance in cellular control mechanisms and phenotypes has truly progressed only in the last 15 years due to advancements in detection and experimental techniques. Specifically, the phenomenon of RNA methylation in the context of ncRNA has emerged as a novel process in the arena of epitranscriptomics. Methylated ncRNA molecules may indeed contribute to a potentially vast functional panorama, from regulation of post-transcriptional gene expression to adaptive cellular responses. Recent discoveries have uncovered novel dynamic mechanisms and new layers of complexity, paving the way to a greater understanding of the role of such phenomena within the broader molecular cellular context of human disease.

Oncogenic KRAS induces tumor onset and development by modulating gene expression via different molecular mechanisms. MicroRNAs (miRNAs) are small non-coding RNAs that have been established as main players in tumorigenesis. By overexpressing wild type or mutant KRAS (KRASG12D) and using inducible human and mouse cell lines, we analyzed KRAS-regulated microRNAs in non-small-cell lung cancer (NSCLC). We show that miR-30c and miR-21 are significantly upregulated by both KRAS isoforms and induce drug resistance and enhance cell migration/invasion via inhibiting crucial tumor suppressor genes, such as NF1, RASA1, BID, and RASSF8. MiR-30c and miR-21 levels were significantly elevated in tumors from patients that underwent surgical resection of early stages NSCLC compared to normal lung and in plasma from the same patients. Systemic delivery of LNA-anti-miR-21 in combination with cisplatin in vivo completely suppressed the development of lung tumors in a mouse model of lung cancer. Mechanistically, we demonstrated that ELK1 is responsible for miR-30c and miR-21 transcriptional activation by direct binding to the miRNA proximal promoter regions. In summary, our study defines that miR-30c and miR-21 may be valid biomarkers for early NSCLC detection and their silencing could be beneficial for therapeutic applications.

Small, noncoding RNAs are short untranslated RNA molecules, some of which have been associated with cancer development. Recently we showed that a class of small RNAs generated during the maturation process of tRNAs (tRNA-derived small RNAs, hereafter “tsRNAs”) is dysregulated in cancer. Specifically, we uncovered tsRNA signatures in chronic lymphocytic leukemia and lung cancer and demonstrated that the ts-4521/3676 cluster (now called “ts-101” and “ts-53,” respectively), ts-46, and ts-47 are down-regulated in these malignancies. Furthermore, we showed that tsRNAs are similar to Piwi-interacting RNAs (piRNAs) and demonstrated that ts-101 and ts-53 can associate with PiwiL2, a protein involved in the silencing of transposons. In this study, we extended our investigation on tsRNA signatures to samples collected from patients with colon, breast, or ovarian cancer and cell lines harboring specific oncogenic mutations and representing different stages of cancer progression. We detected tsRNA signatures in all patient samples and determined that tsRNA expression is altered upon oncogene activation and during cancer staging. In addition, we generated a knocked-out cell model for ts-101 and ts-46 in HEK-293 cells and found significant differences in gene-expression patterns, with activation of genes involved in cell survival and down-regulation of genes involved in apoptosis and chromatin structure. Finally, we overexpressed ts-46 and ts-47 in two lung cancer cell lines and performed a clonogenic assay to examine their role in cell proliferation. We observed a strong inhibition of colony formation in cells overexpressing these tsRNAs compared with untreated cells, confirming that tsRNAs affect cell growth and survival.

The ENCODE project has reported that at least 80% of the human genome is biologically active, yet only a small part of human DNA encodes for protein. The massive amount of RNA transcribed but not translated into protein can be classified as housekeeping RNA (such as rRNA, tRNA) and regulatory RNA (such as miRNA, piRNA, lncRNA). Small non-coding RNAs, in particular, have been the focus of many studies in the last 20 years and their fundamental role in many human diseases is currently well established. Inter alia, their role in cancer development and progression, as well as in drug resistance, is being increasingly investigated. In this review, focusing our attention on recent research results, we provide an overview of the four large classes of small non-coding RNAs, namely, miRNAs, piRNAs, snoRNA and the new class of tRNA-derived fragments, highlighting their fundamental role in cancer and their potential as diagnostic and prognostic biomarkers.

Mutated protein-coding genes drive the molecular pathogenesis of many diseases, including cancer. Specifically, mutated KRAS is a documented driver for malignant transformation, occurring early during the pathogenesis of cancers such as lung and pancreatic adenocarcinomas. Therapeutically, the indiscriminate targeting of wild-type and point-mutated transcripts represents an important limitation. Here, we leveraged on the design of miRNA-like artificial molecules (amiRNAs) to specifically target point-mutated genes, such as KRAS, without affecting their wild-type counterparts. Compared with an siRNA-like approach, the requirement of perfect complementarity of the microRNA seed region to a given target sequence in the microRNA/target model has proven to be a more efficient strategy, accomplishing the selective targeting of point-mutated KRAS in vitro and in vivo.

One of the most significant biological discoveries of the last decade is represented by the reality that the vast majority of the transcribed genomic output comprises diverse classes of noncoding RNAs (ncRNAs) that may play key roles and/or be affected by many biochemical cellular processes (i.e., RNA editing), with implications in human health and disease. With 90% of the human genome being transcribed and novel classes of ncRNA emerging (tRNA-derived small RNAs and circular RNAs among others), the great majority of the human transcriptome suggests that many important ncRNA functions/processes are yet to be discovered. An approach to filling such vast void of knowledge has been recently provided by the increasing application of next-generation sequencing (NGS), offering the unprecedented opportunity to obtain a more accurate profiling with higher resolution, increased throughput, sequencing depth, and low experimental complexity, concurrently posing an increasing challenge in terms of efficiency, accuracy, and usability of data analysis software. This review provides an overview of ncRNAs, NGS technology, and the most recent/popular computational approaches and the challenges they attempt to solve, which are essential to a more sensitive and comprehensive ncRNA annotation capable of furthering our understanding of this still vastly uncharted genomic territory.

RNA editing is a finely tuned, dynamic mechanism for post-transcriptional gene regulation that has been thoroughly investigated in the last decade. Nevertheless, RNA editing in non-coding RNA, such as microRNA (miRNA), have caused great debate and have called for deeper investigation. Until recently, in fact, inadequate methodologies and experimental contexts have been unable to provide detailed insights for further elucidation of RNA editing affecting miRNAs, especially in cancer. In this work, we leverage on recent innovative bioinformatics approaches applied to a more informative experimental context in order to analyze the variations in miRNA seed region editing activity during a time course of a hypoxia-exposed breast cancer cell line. By investigating its behavior in a dynamic context, we found that miRNA editing events in the seed region are not depended on miRNA expression, unprecedentedly providing insights on the targetome shifts derived from these modifications. This reveals that miRNA editing acts under the influence of environmentally induced stimuli. Our results show a miRNA editing activity trend aligning with cellular pathways closely associated to hypoxia, such as the VEGF and PI3K/Akt pathways, providing important novel insights on this poorly elucidated phenomenon.

The majority of the human transcriptome is defined as non-coding RNA (ncRNA), since only a small fraction of human DNA encodes for proteins, as reported by the ENCODE project. Several distinct classes of ncRNAs, such as transfer RNA (tRNA), microRNA (miRNA), and long non-coding RNA (lncRNA), have been classified, each with its own three-dimensional folding and specific function. As ncRNAs are highly abundant in living organisms and have been discovered to play important roles in many biological processes, there has been an ever increasing need to investigate the entire ncRNAome in further unbiased detail.Recently, the advent of Next-Generation Sequencing (NGS) technologies has substantially increased the throughput of transcriptome studies, allowing an unprecedented investigation of ncRNAs, as regulatory pathways and novel functions involving ncRNAs are now also emerging. The huge amount of transcript data produced by NGS has progressively required the development and implementation of suitable bioinformatics workflows, complemented by knowledge-based approaches, to identify, classify, and evaluate the expression of hundreds of ncRNAs in normal and pathological states, such as cancer.In this mini-review, we present and discuss current bioinformatics advances in the development of such computational approaches to analyze and classify the non-coding RNA component of human transcriptome sequence data obtained from NGS technologies.

RNA editing is a dynamic mechanism for gene regulation attained through the alteration of the sequence of primary RNA transcripts. A-to-I (Adenosine-to-Inosine) RNA editing, which is catalyzed by members of the Adenosine Deaminase Acting on RNA (ADAR) family of enzymes, is the most common post-transcriptional modification in humans. The ADARs bind double-stranded regions and deaminate adenosine (A) into inosine (I), which in turn is interpreted by the translation and splicing machineries as guanosine (G). In recent years, this modification has been discovered to occur not only in coding RNAs but also in non-coding RNAs (ncRNA), such as microRNAs (miRNAs), small interfering RNAs (siRNAs), transfer RNAs (tRNAs), and long non-coding RNAs (lncRNAs). This may have several consequences, such as the creation or disruption of microRNA/mRNA binding sites, and thus affect the biogenesis, stability, and target recognition properties of ncRNAs. The malfunction of the editing machinery is not surprisingly associated with various human diseases, such as neurodegenerative, cardiovascular and carcinogenic diseases.Despite the enormous efforts made so far, the real biological function of this phenomenon, as well as the features of the ADAR substrate, in particular in non-coding RNAs, has still not been fully understood. In this work we focus on the current knowledge of RNA editing on ncRNA molecules and provide a few examples of computational approaches to elucidate its biological function.

MicroRNAs (miRNAs or miRs) are small regulatory RNAs crucial for modulation of signaling pathways in multiple organs. While the link between miRNAs and heart disease has grown more readily apparent over the past three years, these data are primarily limited to small animal models or cell-based systems. Here, we performed a high-throughput RNA sequencing (RNAseq) analysis of left ventricle and other tissue from a pre-clinical ovine model. We identified 172 novel miRNA precursors encoding a total of 264 mature miRNAs. Notably, 84 precursors were detected in both the left ventricle and other tissues. However, 10 precursors, encoding 11 mature sequences, were specific to the left ventricle. Moreover, the total 168 novel miRNA precursors included 22 non-conserved ovine-specific sequences. Our data identify and characterize novel miRNAs in the left ventricle of sheep, providing fundamental new information for our understanding of protein regulation in heart and other tissues.

RNAi is a powerful tool for the regulation of gene expression. It is widely and successfully employed in functional studies and is now emerging as a promising therapeutic approach. Several RNAi-based clinical trials suggest encouraging results in the treatment of a variety of diseases, including cancer. Here we present miR-Synth, a computational resource for the design of synthetic microRNAs able to target multiple genes in multiple sites. The proposed strategy constitutes a valid alternative to the use of siRNA, allowing the employment of a fewer number of molecules for the inhibition of multiple targets. This may represent a great advantage in designing therapies for diseases caused by crucial cellular pathways altered by multiple dysregulated genes. The system has been successfully validated on two of the most prominent genes associated to lung cancer, c-MET and Epidermal Growth Factor Receptor (EGFR). (See http://microrna.osumc.edu/mir-synth).

MicroRNAs (miRNAs) are small non-coding RNAs responsible of post-transcriptional regulation of gene expression through interaction with messenger RNAs (mRNAs). They are involved in important biological processes and are often dysregulated in a variety of diseases, including cancer and infections. Viruses also encode their own sets of miRNAs, which they use to control the expression of either the host's genes and/or their own. In the past few years evidence of the presence of cellular miRNAs in extracellular human body fluids such as serum, plasma, saliva, and urine has accumulated. They have been found either cofractionate with the Argonaute2 protein or in membrane-bound vesicles such as exosomes. Although little is known about the role of circulating miRNAs, it has been demonstrated that miRNAs secreted by virus-infected cells are transferred to and act in uninfected recipient cells. In this work we summarize the current knowledge on viral circulating miRNAs and provide a few examples of computational prediction of their function. © 2013 Laganà, Russo, Veneziano, Di Bella, Giugno, Pulvirenti, Croce and Ferro.

Chronic lymphocytic leukemia (CLL) is the most common human leukemia. This disease occurs in two forms, aggressive and indolent, both characterized by clonal expansion of CD5+ B-lymphocytes. Hallmarks of aggressive CLL are the high expression level of ZAP-70, the unmutated sequence of IgVH genes, and activation of the TCL1 oncogene. While investigating whether the expression of miR-3676/4521 cluster in CLL is correlated with TCL1 regulation and ZAP-70 methylation in aggressive CLLs, we found that these two microRNAs are associated with tRNA sequences and that this region produces two small RNAs, members of a novel class of small non-coding RNAs (tsRNAs). Ts-3676/4521 cluster is located on the short arm of chromosome 17 and is co-deleted with p53 in 17p deleted CLL patients. We previously demonstrated that ts-3676 and ts-4521 are down-regulated and mutated in all CLL types. Furthermore, ts-3676 targets TCL1 3'UTR, indicating that tsRNAs can interfere with gene expression at a post-transcriptional level in a micoRNA-like manner. However, tsRNAs do not share the biogenesis mechanism of microRNAs but are cleaved from the 3' ends of pre-tRNAs by the endonuclease RNase Z and represent unique sequences starting at the 3' ends of the tRNAs and ending at a sequence of 4 consecutive T nucleotides. Thus tsRNAs are single stranded RNAs, more similar to piRNAs than miRNAs. Since piRNAs interact with Piwi proteins to affect gene methylation, we hypothesized that tsRNAs could also interact with Piwi proteins. By performing a series of RNA immunoprecipitation experiments, we verified the interaction of ts-3676 and ts-4521 with Piwi proteins, suggesting that these molecules could be involved in gene promoter methylation, hence affecting gene expression at a pre-transcriptional level in a piRNA-like manner. Since ts-3676 and ts-4521 are deregulated in CLLs, we studied if other tsRNAs are differentially regulated CLL. We retrieved the tsRNAs sequences from 3' ends of tRNA genes, obtaining a total of 120 tsRNA sequences, and designed a tsRNA microarray chip to investigate signatures of tsRNAs that can distinguish different classes of CLLs. We hybridized total RNAs extracted from 23 CLL samples (11 indolent, 12 aggressive) and 8 CD19+ B cells of healthy donors and found 17 tsRNA differentially expressed in these cohorts of samples. 9 tsRNAs are differentially expressed in aggressive CLL vs. normal B-cells, 10 tsRNAs are differentially expressed in indolent CLL vs. normal B-cells, 15 tsRNAs are differentially expressed in indolent vs. aggressive CLL. In these signatures, we identified ts-46 and ts-47 strongly downregulated in aggressive CLL, suggesting these ts-RNAs as potential tumor-suppressors. Finally, we found that ts-3676 and ts-4521 have a prognostic value for particular subgroups of CLLs. Patients with low ZAP-70 expression and mutated IgVH generally show indolent disease, while patients with high ZAP-70 expression and un-mutated IgVH show aggressive disease. However, a relatively small cohort of patients shows aggressive prognostic markers but clinically indolent disease. Our analysis revealed that expression of ts-3676 is lower in aggressive CLLs vs indolent CLLs, although, we did not test these tsRNAs in cohort of patients with aggressive prognostic markers but indolent disease. Currently there is no good marker to discriminate these patients from aggressive CLLs. Thus, we selected 8 patients with unfavorable prognostic markers but indolent disease and 12 patients with both aggressive clinical course and unfavorable prognosis markers, to test for differences in the expression level of ts-3676 and ts-4521. We found that in patients with unfavorable prognostic markers but indolent disease, expression of ts-3676 and ts-4521 was almost double of that in patients displaying both aggressive clinical course and markers. In conclusion, we provided an evidence that tsRNAs deregulation can affect gene expression by interfering with the epigenetic machinery and/or with the post-transcriptional stability of target genes. We also identified the first tsRNAs important in CLL as new markers and/or targets for therapy: ts-3676, ts-4521, ts-46 and ts-47. Disclosures Kipps: Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau.

RNA editing is a finely tuned, dynamic mechanism for post-transcriptional gene regulation that has been thoroughly investigated in the last decade. In this work we analyse the variations in microRNA seed region editing activity during a time course of a hypoxia-exposed breast cancer cell line. By leveraging on recent innovative bioinformatics approaches applied to a more informative experimental context, we have analysed small RNA-seq (sRNA-seq) data obtained from a hypoxia-treated breast cancer cell line over time, focusing specifically on miRNA post-transcriptional modifications occurring in the miRNA seed region (MSR), with particular attention to A-to-I editing events. To estimate the impact of editing on miRNA function during hypoxia progression, we decided to investigate A-to-G miRNA modifications, as these are expected to be the result of A-to-I editing. Specifically, we decided to consider all A-to-I editing events occurring in MSRs (2-8 nt seed region). Subsequently, in order to compare the target sets of the wild type (WT) and edited (ED) versions of each miRNA, we performed binding site prediction on gene 3’-UTRs by multiple prediction tool consensus. Furthermore, our target prediction results were validated experimentally. Finally, employing gene microarray data associated to the sRNA-seq data, we performed differential gene expression analysis followed by functional enrichment, focusing on hypoxia-related pathways, such as VEGF and PI3K/Akt, to evaluate how the miRNA editing phenomenon could affect these pathways during the hypoxic time course. We identified a total of 29 statistically significant canonical and non-canonical editing sites in 20 different miRNAs, with 8 sites previously known. The average modification levels of the majority of MSRs generally increased with time of exposure to hypoxia. We found that microRNA editing events in the seed region are not depended on microRNA expression. Additionally, the evidence that emerges from our functional data shows, indeed, how the editing of certain miRNAs is significantly associated with the cellular response to hypoxic stimulus, unprecedentedly providing insights on the meaning of the target set shift derived from these modifications. This reveals that microRNA editing acts under the influence of environmentally induced stimuli. Our results, thus, show a microRNA editing activity trend in favour of cellular pathways closely associated to hypoxia, providing novel insights on the functional role of miRNA seed region editing. This provides a foundation for further elucidation of the miRNA editing phenomenon, giving a direction to future innovative investigation of its biological importance and potential involvement in physiological and pathological cellular changes. Citation Format: Mario Acunzo, Giovanni Nigita, Giulia Romano, Dario Veneziano, Alessandro Lagana’, Dorothee Wernicke, Alfredo Ferro, Carlo Croce. miRNA editing in seed region is in synergy with cellular changes in hypoxic conditions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-166.

microRNAs (miRNAs) have been shown to play a crucial role in the most important biological processes and their dysregulation has been connected to a variety of diseases, including cancer. The number of computational tools for the analysis of miRNA related data is continuously increasing. They range from simple look-up resources to more sophisticated tools for functional analysis of miRNAs. These systems may help to investigate the role of miRNAs in key biological processes and their involvement in diseases. The ultimate goal is to allow the development of regulatory models describing complex processes and the effects of their dysregulation.

Motivation: A-to-I RNA editing is an important mechanism that consists of the conversion of specific adenosines into inosines in RNA molecules. Its dysregulation has been associated to several human diseases including cancer. Recent work has demonstrated a role for A-to-I editing in microRNA (miRNA)-mediated gene expression regulation. In fact, edited forms of mature miRNAs can target sets of genes that differ from the targets of their unedited forms. The specific deamination of mRNAs can generate novel binding sites in addition to potentially altering existing ones. Results: This work presents miR-EdiTar, a database of predicted A-to-I edited miRNA binding sites. The database contains predicted miRNA binding sites that could be affected by A-to-I editing and sites that could become miRNA binding sites as a result of A-to-I editing. Availability: miR-EdiTar is freely available online at http://microrna.osumc.edu/mireditar. Contact:alessandro.lagana@osumc.edu or carlo.croce@osumc.edu Supplementary information:Supplementary data are available at Bioinformatics online.