Function Of Small RNAs Research Articles

Engineered exosomes for targeted co-delivery of miR-21 inhibitor and chemotherapeutics to reverse drug resistance in colon cancer

Background5-Fluorouracil (5-FU) has been commonly prescribed for patients with colorectal cancer (CRC), but resistance to 5-FU is one of the main reasons for failure in CRC. Recently, microRNAs (miRNAs) have been established as a means of reversing the dilemma by regulating signaling pathways involved in initiation and progression of CRC. However, how to safely and effectively deliver miRNA to target cells becomes a main challenge.ResultsIn this study, Engineered exosomes were exploited to simultaneously deliver an anticancer drug 5-FU and miR-21 inhibitor oligonucleotide (miR-21i) to Her2 expressing cancer cells. Purified engineered exosomes from the donor cells loaded with 5-FU and miR-21i via electroporation to introduce into 5-FU-resistant colorectal cancer cell line HCT-1165FR. Furthermore, systematic administration of 5-FU and miR-21i loaded exosomes in tumor bearing mice indicated a significantly anti-tumor effect. The results showed that the engineered exosome-based 5-FU and miR-21i co-delivery system could efficiently facilitate cellular uptake and significantly down-regulate miR-21 expression in 5-FU resistant HCT-1165FR cell lines. Consequently, the down-regulation of miR-21 induced cell cycle arrest, reduced tumor proliferation, increased apoptosis and rescued PTEN and hMSH2 expressions, regulatory targets of miR-21. Of particular importance was the significant reduction in tumor growth in a mouse model of colon cancer with systematic administration of the targeting miR-21i. More excitedly, the combinational delivery of miR-21i and 5-FU with the engineered exosomes effectively reverse drug resistance and significantly enhanced the cytotoxicity in 5-FU-resistant colon cancer cells, compared with the single treatment with either miR-21i or 5-FU.ConclusionThe strategy for co-delivering the functional small RNA and anticancer drug by exosomes foreshadows a potential approach to reverse the drug resistance in CRC and thus to enhance the efficacy of the cancer treatment.

Small RNAs With a Big Impact on Horticultural Traits

The role of protein-encoding genes in determining phenotype is well established, whereas the impact of small RNAs in regulating horticultural traits is being gradually recognized. Small RNAs are small noncoding RNAs of 20–24-nt, which regulate target genes by post-transcriptional regulation or de-novo DNA methylation. In plants, small RNAs are classified into the following two major categories, microRNA (miRNA) and short interfering RNA (siRNA). Most small RNA research focuses on model plants (e.g. Arabidopsis, tomato and rice), and has identified the function of small RNAs on plant development (e.g. meristem organization, leaf development, flower organ identity and transition to flowering). Recently, small RNA sequencing has been applied to study gene regulation in horticultural crops. Plant specific small RNA (miRNAs and phasiRNAs) families have similar function across model plants to horticultural crops. In addition, most plants have unique nonconserved small RNAs, which might affect specific traits. This review focuses on small RNA biogenesis, function prediction and the roles of small RNAs in horticultural traits (e.g. flowering, fruit quality, abiotic and biotic stress), and summarizes small RNA information for future horticultural crop research and breeding.

Small RNAs in parasitic nematodes - forms and functions.

Small RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes - microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) - and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host-parasite interface.

The green peach aphid gut contains host plant microRNAs identified by comprehensive annotation of Brassica oleracea small RNA data

Like all organisms, aphids, plant sap-sucking insects that house a bacterial endosymbiont called Buchnera, are members of a species interaction network. Ecological interactions across such networks can result in phenotypic change in network members mediated by molecular signals, like microRNAs. Here, we interrogated small RNA data from the aphid, Myzus persicae, to determine the source of reads that did not map to the aphid or Buchnera genomes. Our analysis revealed that the pattern was largely explained by reads that mapped to the host plant, Brassica oleracea, and a facultative symbiont, Regiella. To start elucidating the function of plant small RNA in aphid gut, we annotated 213 unique B. oleracea miRNAs; 32/213 were present in aphid gut as mature and star miRNAs. Next, we predicted targets in the B. oleracea and M. persicae genomes for these 32 plant miRNAs. We found that plant targets were enriched for genes associated with transcription, while the distribution of targets in the aphid genome was similar to the functional distribution of all genes in the aphid genome. We discuss the potential of plant miRNAs to regulate aphid gene expression and the mechanisms involved in processing, export and uptake of plant miRNAs by aphids.

Levels of sdRNAs in cytoplasm and their association with ribosomes are dependent upon stress conditions but independent from snoRNA expression

In recent years, a number of small RNA molecules derived from snoRNAs have been observed. Findings concerning the functions of snoRNA-derived small RNAs (sdRNAs) in cells are limited primarily to their involvement in microRNA pathways. However, similar molecules have been observed in Saccharomyces cerevisiae, which is an organism lacking miRNA machinery. Here we examined the subcellular localization of sdRNAs in yeast. Our findings reveal that both sdRNAs and their precursors, snoRNAs, are present in the cytoplasm at levels dependent upon stress conditions. Moreover, both sdRNAs and snoRNAs may interact with translating ribosomes in a stress-dependent manner. Likely consequential to their ribosome association and protein synthesis suppression features, yeast sdRNAs may exert inhibitory activity on translation. Observed levels of sdRNAs and snoRNAs in the cytoplasm and their apparent presence in the ribosomal fractions suggest independent regulation of these molecules by yet unknown factors.

Abstract P165: Vascular Endothelial Exosomes Propogate Inflammation in Vascular Smooth Muscle Cells Through Up-Regulation of HMGB1

Vascular homeostasis is predicated on the integral interaction between a myriad of cell types. Principle to homeostasis is the interaction between the endothelial cells (ECs) of the intima and the vascular smooth muscle cells (VSMCs) of the media. Disruption of the tightly controlled communication between these cell types can lead to development of pathophysiology. In the past decade, much study has been given to a novel means of intercellular communication in exosomes. Exosomes are nanovesicles derived from endosomal compartments capable of carrying functional proteins, small RNAs and lipids into extracellular space. Initially characterized in the field of immunology, little is known on exosomes within vascular biology. Therefore, we present a model through which EC exosomes initiate inflammatory signaling and remodeling within VSMC. Primary rat aortic EC and VSMC exosomes isolated in serum-free conditions displayed characteristic size, shape and protein content of exosomes as analyzed through nanoparticle tracking analysis, electron microscopy, and western blot. Proteomics identified differential expression between EC and VSMC exosomes suggesting altered cargo between the two populations. Incubation of EC exosomes with VSMC identified a dose- and time-dependent increase in the expression of vascular cell adhesion molecule 1 (VCAM1) in VSMC (2.6 fold, p<0.01) and functional increase in leukocyte adhesion (p<0.05), that was a one-way mechanism of EC-VSMC. EC exosomes increased the prevalence of senescence cells (1.62 fold, p<0.05) in VSMC with increases in cellular volume, suggesting a pro-inflammatory remodeling. Unbiased mass spectroscopy on VSMC incubated with EC exosomes derived with/without TNF stimulation identified pro-inflammatory mediator HMGB1 as significantly up-regulated in both conditions. Interestingly, use of HMGB1 inhibitor Glycyrrhizin was able to abolish increase in VCAM1 (p<0.05) and increases in THP-1 adhesion (p<0.05). Identification of HMGB1 was also present in unbiased proteomics performed on EC exosomes themselves. Taken together, this suggests that EC exosomes derived from nutrient-stressed conditions induce inflammatory remodeling and signaling in VSMC through an HMGB1-dependent mechanism.

Differential expression of miRNAs and their targets in wax-deficient rapeseed

The cuticle of a plant, composed of cutin and wax, is the outermost hydrophobic layer covering the epidermis of all its aerial organs, protecting it from many abiotic and biotic stresses. The biosynthesis and regulation pathways of wax components have been well studied, whereas there are fewer reports on the small RNA-involved post-transcriptional regulation of wax biosynthesis in plants, particularly in Brassica napus. Previously, we conducted a study on a glossy mutant of rapeseed, and we assumed that there was a dominant repressor to inhibit the expression of wax-related genes. To verify this hypothesis and investigate the function of small RNAs in wax biosynthesis in B. napus, we constructed four small RNA libraries from the stem epidermis of wax-deficient mutant and wild-type plants for sequencing. Subsequently, 43,840,451 clean reads were generated and 24 nt sequences represented the dominant percentage. In total, 300 unique known miRNAs were identified and eight of them showed differential expression. In addition, the expression levels of six novel miRNAs were altered. Surprisingly, we found that four up-regulated miRNAs in the wax-deficient plants, bna-miR408b-5p, bna-miR165b-5p, bna-miR160a-3p, and bna-miR398-5p, were all complementary strands of their corresponding mature strands. Stem-loop qRT-PCR verified that the expression of bna-miR165a-5p was increased in the mutant stems, while its putative target, BnaA06g40560D (CYP96A2), was down-regulated. In addition, the expression of bna-miR827a was detected to be down-regulated in glossy mutant. 5′ RACE experimental data showed that bna-miR827a cleaves three NITROGEN LIMITATION ADAPTATION (NLA) genes (BnaC08g45940D, BnaA10g01450D and BnaC05g01480D). The down-regulation of bna-miR827a resulted in decreased cleavage on its targets, and led to the up-regulation of its targets, especially BnaA10g01450D gene. These results showed that bna-miR165a-5p might participate in wax biosynthesis process by regulating its putative target BnaA06g40560D (CYP96A2). The expression levels of a phosphate (Pi)-related miRNA, bna-miR827a, and its target genes were affected in wax-deficient rapeseeds. These results will promote the study of post-transcriptional regulation mechanisms of wax biosynthesis in B. napus and provide new directions for further research.

Long Non-Coding RNAs in Kidney Disease.

Non-coding RNA species contribute more than 90% of all transcripts and have gained increasing attention in the last decade. One of the most recent members of this group are long non-coding RNAs (lncRNAs) which are characterized by a length of more than 200 nucleotides and a lack of coding potential. However, in contrast to this simple definition, lncRNAs are heterogenous regarding their molecular function—including the modulation of small RNA and protein function, guidance of epigenetic modifications and a role as enhancer RNAs. Furthermore, they show a highly tissue-specific expression pattern. These aspects already point towards an important role in cellular biology and imply lncRNAs as players in development, health and disease. This view has been confirmed by numerous publications from different fields in the last years and has raised the question as to whether lncRNAs may be future therapeutic targets in human disease. Here, we provide a concise overview of the current knowledge on lncRNAs in both glomerular and tubulointerstitial kidney disease.

Small RNA Functions as a Trafficking Effector in Plant Immunity.

Small RNAs represent a class of small but powerful agents that regulate development and abiotic and biotic stress responses during plant adaptation to a constantly challenging environment. Previous findings have revealed the important roles of small RNAs in diverse cellular processes. The recent discovery of bidirectional trafficking of small RNAs between different kingdoms has raised many interesting questions. The subsequent demonstration of exosome-mediated small RNA export provided a possible tool for further investigating how plants use small RNAs as a weapon during the arms race between plant hosts and pathogens. This review will focus on discussing the roles of small RNAs in plant immunity in terms of three aspects: the biogenesis of extracellular small RNAs and the transportation and trafficking small RNA-mediated gene silencing in pathogens.

Intercellular delivery of small RNAs in plant gametes.

Small RNAs are 20-24 nucleotides in length. In plants, small RNAs are classified into microRNAs (miRNAs) and small interfering RNAs (siRNAs), based on their biogenesis and molecular features. In contrast to the extensive knowledge of the roles of small RNAs in sporophytic tissues, the distribution and function of small RNAs in gametophytic cells have been less well studied. However, with the improvement of single-cell sorting and RNA sequencing technologies, the distribution of small RNAs, especially siRNAs, between sperm cells and the vegetative cell, as well as the function of sperm-delivered small RNAs during early seed development have been elucidated. This review summarizes work from the past 5years regarding small RNAs in male gametes, emphasizing the intercellular communication and biological significance of small RNAs in Arabidopsis.

Analysis of Expression Pattern of snoRNAs in Different Cancer Types with Machine Learning Algorithms.

Small nucleolar RNAs (snoRNAs) are a new type of functional small RNAs involved in the chemical modifications of rRNAs, tRNAs, and small nuclear RNAs. It is reported that they play important roles in tumorigenesis via various regulatory modes. snoRNAs can both participate in the regulation of methylation and pseudouridylation and regulate the expression pattern of their host genes. This research investigated the expression pattern of snoRNAs in eight major cancer types in TCGA via several machine learning algorithms. The expression levels of snoRNAs were first analyzed by a powerful feature selection method, Monte Carlo feature selection (MCFS). A feature list and some informative features were accessed. Then, the incremental feature selection (IFS) was applied to the feature list to extract optimal features/snoRNAs, which can make the support vector machine (SVM) yield best performance. The discriminative snoRNAs included HBII-52-14, HBII-336, SNORD123, HBII-85-29, HBII-420, U3, HBI-43, SNORD116, SNORA73B, SCARNA4, HBII-85-20, etc., on which the SVM can provide a Matthew’s correlation coefficient (MCC) of 0.881 for predicting these eight cancer types. On the other hand, the informative features were fed into the Johnson reducer and repeated incremental pruning to produce error reduction (RIPPER) algorithms to generate classification rules, which can clearly show different snoRNAs expression patterns in different cancer types. The analysis results indicated that extracted discriminative snoRNAs can be important for identifying cancer samples in different types and the expression pattern of snoRNAs in different cancer types can be partly uncovered by quantitative recognition rules.

MiR-873a-5p Targets A20 to Facilitate Morphine Tolerance in Mice.

Long-term morphine administration leads to tolerance and a gradual reduction in analgesic potency. Noncoding microRNAs (miRNAs) modulate gene expression in a posttranscriptional manner, and their dysregulation causes various diseases. Emerging evidence suggests that miRNAs play a regulatory role in the development of morphine tolerance. In the present study, we hypothesized that miR-873a-5p is a key functional small RNA that participates in the development and maintenance of morphine tolerance through the regulation of A20 (tumor necrosis factor α-induced protein 3, TNFAIP3) in mice. We measured the percentage of maximum possible effect (MPE %) to evaluate the analgesic effect of morphine. The expression of miR-873a-5p and its target gene A20 were determined after the morphine-tolerant model was successfully established. Intrathecal injection with lentivirus to intervene in the expression of A20 and the miR-873a-5p antagomir was used to explore the role of miR-873a-5p in the development of morphine tolerance. Chronic morphine administration significantly increased the expression of miR-873a-5p, which was inversely correlated with decreased A20 expression in the spinal cord of morphine-tolerant mice. Downregulation of miR-873a-5p in the spinal cord attenuated and partly reversed the development of morphine tolerance accompanied by overexpression of A20. Similarly, A20 was upregulated by a recombinant lentivirus vector, which attenuated and reversed the pathology of morphine tolerance by inhibiting the activation of nuclear factor (NF)-κB. Collectively, our results indicated that miR-873a-5p targets A20 in the spinal cord to facilitate the development of morphine tolerance in mice. Downregulating the expression of miR-873a-5p may be a potential strategy to ameliorate morphine tolerance.

Synergistic and Global Effect of C‐terminus of Hfq in Small RNA Regulation

Hfq, a homohexameric RNA chaperone, is known for stabilizing small noncoding RNAs (sRNAs) and facilitating riboregulation by promoting sRNA base pairing with target mRNAs. The proteins encoded by these target mRNAs are often involved in virulence and stress responses. Hfq has a conserved N‐terminal region sufficient for most of its function and a poorly conserved disordered C‐terminus (CT). The length of the C‐terminus varies among bacteria; it is 37 aa in E. coli. Since the role of C‐terminus of Hfq has been poorly understood we have investigated its role in vivo in small RNA stability and function. Mutant derivatives of Hfq were expressed from the chromosomal locus, allowing in vivo comparisons with the wild‐type (WT) Hfq, expressed under similar conditions and at physiological levels. While deletion of the CT (Hfq65) had modest phenotypes, combinations of this deletion with a weak mutation (K31A) in the distal face of the protein was fully defective for function in different assays. This strong synergistic phenotype of CT deletion with K31A allowed us to further dissect the CT. Loss of the region between 65–72aa, the best conserved part of the CT, was critical for the synergistic defect. These results suggest that the initial region of the CT supports the function of the distal face, where many mRNAs and a subset of sRNAs are known to bind. To understand the global effect of CT on interacting RNAs, we compared RNA seq data of Hfq immunoprecipitation for WT and Hfq65. Overall, Hfq binding RNA patterns were not much affected. However, consistent with previous findings, and with the cooperation of the CT with the distal face of Hfq, Hfq65 was most defective in the accumulation of the sRNAs (Class II) that bind the proximal and distal faces of Hfq. Some class I sRNAs, which bind the proximal and rim face of Hfq, were also modestly depleted in Hfq65, suggesting the possibility of a broader role for the CT. The mechanistic aspects of CT collaboration with the Hfq core are under investigation. Our results suggest that the C‐terminus collaborates with the Hfq distal face, reinforcing the specificity and strength of RNA binding to Hfq.Support or Funding InformationThis research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Small extrachromosomal circular DNAs, microDNA, produce short regulatory RNAs that suppress gene expression independent of canonical promoters.

Interest in extrachromosomal circular DNA (eccDNA) molecules has increased recently because of their widespread presence in normal cells across every species ranging from yeast to humans, their increased levels in cancer cells and their overlap with oncogenic and drug-resistant genes. However, the majority of eccDNA (microDNA) in mammalian tissues and cell lines are too small to carry protein coding genes. We have tested functional capabilities of microDNA by creating artificial microDNA molecules mimicking known microDNA sequences and have discovered that they express functional small regulatory RNA including microRNA and novel si-like RNA. MicroDNA are transcribed in vitro and in vivo independent of a canonical promoter sequence. MicroDNA that carry miRNA genes form transcripts that are processed by the endogenous RNA-interference pathway into mature miRNA molecules, which repress a luciferase reporter gene as well as endogenous mRNA targets of the miRNA. Further, microDNA that contain sequences of exons repress the endogenous gene from which the microDNA were derived through the formation of novel si-like RNA. We also show that endogenous microDNA associate with RNA polymerases subunits, POLR2H and POLR3F. Together, these results suggest that microDNA may modulate gene expression through the production of both known and novel regulatory small RNA.

MicroRNA-663 antagonizes apoptosis antagonizing transcription factor to induce apoptosis in epithelial cells.

MicroRNAs are small functional RNAs that modulate various biological processes in cells by interfering with gene translation. We have previously demonstrated that certain miRNAs play a crucial role in the innate immune responses of human oral epithelial cells to Porphyromonas gingivalis. While addressing the mechanisms of P. gingivalis induced apoptosis in these cells, we discovered that certain miRNAs are upregulated upon stimulation with live bacteria. These upregulated miRNAs include hsa-miR-584, hsa-miR-572, hsa-miR-210, hsa-miR-492, hsa-miR-623 and hsa-miR-663. Further analysis revealed an unexpected role for hsa-miR-663 (miR-663). To further evaluate miR-663 function, we overexpressed miR-663 in epithelial cells which resulted in cellular apoptosis. The bioinformatics analysis of the miR-663 target prediction, revealed a strong binding affinity to a 3' UTR region of Apoptosis Antagonizing Transcription Factor (AATF) mRNA. To demonstrate the binding of miR-663 to AATF mRNA, the putative miR-663 target site within the 3'-UTR region of AATF was cloned in luciferase vector and transfected to HEK293T cells. Luminescence data showed the downregulation of luciferase activity in cells that had the full length target region of the putative binding site, confirming that AATF is one of the targets for miR-663. This prompted us to further evaluate its role in a cancer cell line (MCF-7) to determine miR-663s' apoptotic function. The overexpression of miR-663 led to a significant increase in apoptosis of MCF-7 cells. Taken together, miR-663 may function as an 'apoptomiR' by inhibiting the anti-apoptotic gene AATF to induce apoptosis. These findings could have therapeutic implications for epithelial cell targeting in cancer therapy.

Small RNA Function in Plants: From Chromatin to the Next Generation.

Small RNA molecules can target a particular virus, gene, or transposable element (TE) with a high degree of specificity. Their ability to move from cell to cell and recognize targets in trans also allows building networks capable of regulating a large number of related targets at once. In the case of epigenetic silencing, small RNA may use the widespread distribution of TEs in eukaryotic genomes to coordinate many loci across developmental and generational time. Here, we discuss the intriguing role of plant small RNA in targeting transposons and repeats in pollen and seeds. Epigenetic reprogramming in the germline and early seed development provides a mechanism to control genome dosage, imprinted gene expression, and incompatible hybridizations via the "triploid block."

Expansion and Divergence of Argonaute Genes in the Oomycete Genus Phytophthora.

Modulation of gene expression through RNA interference is well conserved in eukaryotes and is involved in many cellular processes. In the oomycete Phytophthora, research on the small RNA machinery and function has started to reveal potential roles in the pathogen, but much is still unknown. We examined Argonaute (AGO) homologs within oomycete genome sequences, especially among Phytophthora species, to gain a clearer understanding of the evolution of this well-conserved protein family. We identified AGO homologs across many representative oomycete and stramenopile species, and annotated representative homologs in P. sojae. Furthermore, we demonstrate variable transcript levels of all identified AGO homologs in comparison to previously identified Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) homologs. Our phylogenetic analysis further refines the relationship of the AGO homologs in oomycetes and identifies a conserved tandem duplication of AGO homologs in a subset of Phytophthora species.

Regulation of DNA Double-Strand Break Repair by Non-Coding RNAs.

DNA double-strand breaks (DSBs) are deleterious lesions that are generated in response to ionizing radiation or replication fork collapse that can lead to genomic instability and cancer. Eukaryotes have evolved two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ) to repair DSBs. Whereas the roles of protein-DNA interactions in HR and NHEJ have been fairly well defined, the functions of small and long non-coding RNAs and RNA-DNA hybrids in the DNA damage response is just beginning to be elucidated. This review summarizes recent discoveries on the identification of non-coding RNAs and RNA-mediated regulation of DSB repair.

Transfer RNA-derived fragments and tRNA halves: biogenesis, biological functions and their roles in diseases.

The number of studies on non-coding RNAs has increased substantially in recent years owing to their importance in gene regulation. However, the biological functions of small RNAs from abundant species of housekeeping non-coding RNAs (rRNA, tRNA, etc.) remain a highly studied topic. tRNA-derived small RNAs (tsRNAs) refer to the specific cleavage of tRNAs by specific nucleases [e.g., Dicer and angiogenin (ANG)] in particular cells or tissues or under certain conditions such as stress and hypoxia. tsRNAs are a type of non-coding small RNA that are widely found in the prokaryotic and eukaryotic transcriptomes and are generated from mature tRNAs or precursor tRNAs at different sites. There are two main types of tsRNAs, tRNA-derived fragments (tRFs) and tRNA halves. tRFs are 14-30 nucleotides (nt) long and mainly consist of three subclasses: tRF-5, tRF-3, and tRF-1. tRNA halves, which are 31-40 nt long, are generated by specific cleavage in the anticodon loops of mature tRNAs. There are two types of tRNA halves, 5'-tRNA halves and 3'-tRNA halves. tsRNAs have multiple biological functions including acting as signaling molecules in stress responses and as regulators of gene expression. Additionally, they have been considered to be involved in RNA processing, cell proliferation, translation suppression, the modulation of DNA damage response, and neurodegeneration. More importantly, they are closely related to the occurrence of many human diseases such as tumors, infectious diseases, metabolic diseases, and neurological diseases. Moreover, tsRNAs have the potential to become new biomarkers for disease diagnosis. Continuous investigations will help us to understand their generation and regulatory mechanisms as well as the possible roles of tRFs and tRNA halves.

Abstract P144: Characterization of Vascular Endothelial and Smooth Muscle Exosomes and Their Role in Vascular SignalTransduction

Vascular homeostasis is predicated on the integral interaction between a myriad of cell types. Principle to homeostasis is the interaction between the endothelial cells (ECs) of the intima and the vascular smooth muscle cells (VSMCs) of the media. Disruption of the tightly controlled communication between these cell types can lead to development of pathophysiology. In the past decade, much study has been given to a novel means of intercellular communication in exosomes. Exosomes are nanovesicles derived from endosomal compartments capable of carrying functional proteins, small RNAs and lipids into extracellular space. Initially characterized in the field of immunology, little is known on exosomes within vascular biology. Therefore, we present an in vitro characterization of rat EC and VSMC exosomes and evidence of EC exosomes as initiators of signal transduction within VSMCs. Primary rat aortic EC and VSMC exosomes isolated in serum-free conditions displayed characteristic size, shape and protein content of exosomes as analyzed through nanoparticle tracking analysis, electron microscopy, and western blot. Interestingly, alpha smooth muscle actin was found to be cell-specific for VSMC exosomes. Proteomics identified differential expression between EC and VSMC exosomes suggesting altered cargo between the two populations. Incubation of EC exosomes with VSMC identified a dose- and time-dependent increase in the expression of vascular cell adhesion molecule 1 in VSMC with a maximum at 10 9 EC exosomes and 6 hours through immunoblot analysis. In addition, EC exosomes stimulated expression of early growth response protein 1 at 1-3 hours, suggesting activation of pro-inflammatory gene expression. EC exosomes stimulated a maximal rate of protein synthesis at 3 hours in VSMC (3.53 fold increase over control, p=0.016) determined through puromycin incorporation showing qualitative changes in cell function. Analysis of cell volume identified a hypertrophic response in VSMC after exosome incubation as well, displaying a role for exosomes in vascular hypertrophy. In conclusion, we present a broad characterization of exosomes in vascular biology and a functional crosstalk mechanism between RAEC and VSMC through exosome transfer.