The presence of extracellular microRNAs in the media of cultured Drosophila cells

While regulatory RNA pathways, such as RNAi, have commonly been described at an intracellular level, studies investigating extracellular RNA species in insects are lacking. In the present study, we demonstrate the presence of extracellular microRNAs (miRNAs) in the cell-free conditioned media of two Drosophila cell lines. More specifically, by means of quantitative real-time PCR (qRT-PCR), we analysed the presence of twelve miRNAs in extracellular vesicles (EVs) and in extracellular Argonaute-1 containing immunoprecipitates, obtained from the cell-free conditioned media of S2 and Cl. 8 cell cultures. Next-generation RNA-sequencing data confirmed our qRT-PCR results and provided evidence for selective miRNA secretion in EVs. To our knowledge, this is the first time that miRNAs have been identified in the extracellular medium of cultured cells derived from insects, the most speciose group of animals

A microsatellite repeat in PCA3 long non-coding RNA is associated with prostate cancer risk and aggressiveness.

Short tandem repeats (STRs) are repetitive sequences of a polymorphic stretch of two to six nucleotides. We hypothesized that STRs are associated with prostate cancer development and/or progression. We undertook RNA sequencing analysis of prostate tumors and adjacent non-malignant cells to identify polymorphic STRs that are readily expressed in these cells. Most of the expressed STRs in the clinical samples mapped to intronic and intergenic DNA. Our analysis indicated that three of these STRs (TAAA-ACTG2, TTTTG-TRIB1, and TG-PCA3) are polymorphic and differentially expressed in prostate tumors compared to adjacent non-malignant cells. TG-PCA3 STR expression was repressed by the anti-androgen drug enzalutamide in prostate cancer cells. Genetic analysis of prostate cancer patients and healthy controls (N > 2,000) showed a significant association of the most common 11 repeat allele of TG-PCA3 STR with prostate cancer risk (OR = 1.49; 95% CI 1.11-1.99; P = 0.008). A significant association was also observed with aggressive disease (OR = 2.00; 95% CI 1.06-3.76; P = 0.031) and high mortality rates (HR = 3.0; 95% CI 1.03-8.77; P = 0.045). We propose that TG-PCA3 STR has both diagnostic and prognostic potential for prostate cancer. We provided a proof of concept to be applied to other RNA sequencing datasets to identify disease-associated STRs for future clinical exploratory studies

Common miRNA patterns of Alzheimer’s disease and Parkinson’s disease and their putative impact on commensal gut microbiota

With the rise of Next-Generation-Sequencing (NGS) methods, Micro-RNAs (miRNAs) have achieved an important position in the research landscape and have been found to present valuable diagnostic tools in various diseases such as multiple sclerosis or lung cancer. There is also emerging evidence that miRNAs play an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD) or Parkinson’s disease (PD). Apparently, these diseases come along with changes in miRNA expression patterns which led to attempts from researchers to use these small RNA species from several body fluids for a better diagnosis and in order to observe disease progression. Additionally, it became evident that microbial commensals might play an important role for pathology development and were shown to have a significantly different composition in patients suffering from neurodegeneration compared with healthy controls. As it could recently be shown that secreted miRNAs are able to enter microbial organisms, it is conceivable that the host’s miRNA might affect the gut microbial ecosystem. As such, miRNAs may inherit a central role in shaping the “diseased microbiome” and thereby mutually act on the characteristics of these neurodegenerative diseases. We have therefore 1) compiled a list of miRNAs known to be associated with AD and/or PD, 2) performed an in silico target screen for binding sites of these miRNA on human gut metagenome sequences and 3) evaluated the hit list for interesting matches potentially relevant to the etiology of AD and or PD. The examination of protein identifiers connected to bacterial secretion system, lipopolysaccharide biosynthesis and biofilm formation revealed an overlap of 37 bacterial proteins that were targeted by human miRNAs. The identified links of miRNAs to the biological processes of bacteria connected to AD and PD have yet to be validated via in vivo experiments. However, our results show a promising new approach for understanding aspects of these neurodegenerative diseases in light of the regulation of the microbiome

Computational analysis identifies a sponge interaction network between long non-coding RNAs and messenger RNAs in human breast cancer

Background: Non-coding RNAs (ncRNAs) are emerging as key regulators of many cellular processes in both physiological and pathological states. Moreover, the constant discovery of new non-coding RNA species suggests that the study of their complex functions is still in its very early stages. This variegated class of RNA species encompasses the well-known microRNAs (miRNAs) and the most recently acknowledged long non-coding RNAs (lncRNAs). Interestingly, in the last couple of years, a few studies have shown that some lncRNAs can act as miRNA sponges, i.e. as competing endogenous RNAs (ceRNAs), able to reduce the amount of miRNAs available to target messenger RNAs (mRNAs).Results: We propose a computational approach to explore the ability of lncRNAs to act as ceRNAs by protecting mRNAs from miRNA repression. A seed match analysis was performed to validate the underlying regression model. We built normal and cancer networks of miRNA-mediated sponge interactions (MMI-networks) using breast cancer expression data provided by The Cancer Genome Atlas.Conclusions: Our study highlights a marked rewiring in the ceRNA program between normal and pathological breast tissue, documented by its " on/off" switch from normal to cancer, and vice-versa. This mutually exclusive activation confers an interesting character to ceRNAs as potential oncosuppressive, or oncogenic, protagonists in cancer. At the heart of this phenomenon is the lncRNA PVT1, as illustrated by both the width of its antagonist mRNAs in normal-MMI-network, and the relevance of the latter in breast cancer. Interestingly, PVT1 revealed a net binding preference towards the mir-200 family as the bone of contention with its rival mRNAs. © 2014 Paci et al.; licensee BioMed Central Ltd

Cis-Acting Polymorphisms Affect Complex Traits through Modifications of MicroRNA Regulation Pathways

Genome-wide association studies (GWAS) have become an effective tool to map genes and regions contributing to multifactorial human diseases and traits. A comparably small number of variants identified by GWAS are known to have a direct effect on protein structure whereas the majority of variants is thought to exert their moderate influences on the phenotype through regulatory changes in mRNA expression. MicroRNAs (miRNAs) have been identified as powerful posttranscriptional regulators of mRNAs. Binding to their target sites, which are mostly located within the 3′-untranslated region (3′-UTR) of mRNA transcripts, they modulate mRNA expression and stability. Until today almost all human mRNA transcripts are known to harbor at least one miRNA target site with an average of over 20 miRNA target sites per transcript. Among 5,101 GWAS-identified sentinel single nucleotide polymorphisms (SNPs) that correspond to 18,884 SNPs in linkage disequilibrium (LD) with the sentinels () we identified a significant overrepresentation of SNPs that affect the 3′-UTR of genes (OR = 2.33, 95% CI = 2.12–2.57, ). This effect was even stronger considering all SNPs in one LD bin a single signal (OR = 4.27, 95% CI = 3.84–4.74, ). Based on crosslinking immunoprecipitation data we identified four mechanisms affecting miRNA regulation by 3′-UTR mutations: (i) deletion or (ii) creation of miRNA recognition elements within validated RNA-induced silencing complex binding sites, (iii) alteration of 3′-UTR splicing leading to a loss of binding sites, and (iv) change of binding affinity due to modifications of 3′-UTR folding. We annotated 53 SNPs of a total of 288 trait-associated 3′-UTR SNPs as mediating at least one of these mechanisms. Using a qualitative systems biology approach, we demonstrate how our findings can be used to support biological interpretation of GWAS results as well as to provide new experimentally testable hypotheses

MicroRNA-135a regulates NHE9 to inhibit proliferation and migration of glioblastoma cells

Abstract Background Glioblastoma multiformae (GBM) is the most aggressive type of malignant brain tumor with complex molecular profile. Overexpression of Na+/H+ Exchanger isoform 9 (NHE9) promotes tumor progression and correlates positively with insensitivity to radiochemotherapy and poor prognosis. However, molecular mechanisms responsible for increase in NHE9 levels beyond a critical threshold have not been identified. Methods Bioinformatics analysis, luciferase reporter assays, real-time PCR and western blotting were conducted to examine the expression profiles and identify microRNAs (miRNA) that target NHE9. Cell proliferation and migration assays were conducted in U87 glioblastoma cells to determine the consequence of miRNA mediated targeting of NHE9. Endosomal pH measurements, immunofluorescence microscopy and surface biotinylation experiments were conducted to characterize the mechanistic basis of regulation. Results We show that microRNA 135a (miR-135a) targets NHE9 to downregulate its expression in U87 cells. MiR-135a levels are significantly lower in glioblastoma cells compared to normal brain tissue. Downregulation of NHE9 expression by miR-135a affects proliferative and migratory capacity of U87 cells. Selectively increasing NHE9 expression in these cells restored their ability to proliferate and migrate. We demonstrate that miR-135a takes a two-pronged approach affecting epidermal growth factor receptors (EGFRs) to suppress tumor cell growth and migration. EGFR activity is a potent stimulator of oncogenic signaling. While miR-135a targets EGFR transcripts to decrease the total number of receptors made, by targeting NHE9 it routes the few EGFRs made away from the plasma membrane to dampen oncogenic signaling. NHE9 is localized to sorting endosomes in glioblastoma cells where it alkalinizes the endosome lumen by leaking protons. Downregulation of NHE9 expression by miR-135a acidifies sorting endosomes limiting EGFR trafficking to the glioblastoma cell membrane. Conclusions We propose downregulation of miR-135a as a potential mechanism underlying the high NHE9 expression observed in subset of glioblastomas. Future studies should explore miR-135a as a potential therapeutic for glioblastomas with NHE9 overexpression.https://deepblue.lib.umich.edu/bitstream/2027.42/140393/1/12964_2017_Article_209.pd

SINTHESIS, MECHANISMS OF REGULATION AND FUNCTION OF microRNA

MiRNA razred su malih (~22-25 nukleotida), jednolančanih, evolucijski vrlo očuvanih, nekodirajućih molekula RNA koje imaju specifičnu ulogu u kontroli genske ekspresije na post-translacijskoj i transkripcijskoj razini. Kod životinja se miRNA sintetiziraju u dva koraka, djelovanjem dva enzima endoribonukleaza III (RNaza III) lokalizirana u jezgri i citoplazmi, dok se dorada miRNA u biljkama u cijelosti odvija u citoplazmi i provodi pomoću jedne RNaze III. Kao dio kompleksa miRISC, zrela miRNA prepoznaje ciljnu mRNA. Nakon prepoznavanja, ovisno o stupnju komplementarnosti, dolazi do egzonukleazne aktivnosti proteina Ago. Kod životinja je utvrđeno da miRNA reguliraju signalne putove te osnovne stanične procese poput proliferacije, diferencijacije, te da imaju važnu ulogu u neuralnom razvoju, imunitetu te održavanju homeostaze reguliranjem programirane stanične smrti, , itd. Biljne pak miRNA na specifičan način reguliraju razvoj biljnih organa poput listova i cvijeta, kao i vaskularni razvoj, homeostazu fitohormona i nutrijenata, a uključene su i u odgovor na stres do kojeg dolazi uslijed djelovanja biotičkih i abiotičkih čimbenika.MicroRNAs (miRNAs) are a class of small, noncoding RNAs, approximatey ~22-25 nucleotides long, single-standed, evolutionary conserved, which are known to play specific regulatory role on post-transcription and transcription level. In animals, miRNAs synthesizes occurs in two steps, processed by two exoribonucleases III (RNase III), which are localised in nucleus and cytoplasm, while plant miRNA processing is dependent on one RNase III, which is localised in cytoplasm. As a part of microRNA-induced silencing complex, miRNAs are able to recognise the target mRNAs. After recognition, depending on the degree of complementarity, the exonuclease (slicer) activity of Ago protein occurs. In animals, it was determined that miRNA regulates signal pathways and basic cellular processes such as proliferation, differentiation, and play an important role in neural development, immunity, and homeostatic maintenance by regulating programmed cell death, etc. Plants miRNAs specifically regulate the development of plant organs such as leaves and flowers, as well as vascular development, phytohormones and nutrition homeostasis, and are included in response to the stress caused by the action of biotic and abiotic factors

SINTHESIS, MECHANISMS OF REGULATION AND FUNCTION OF microRNA

MiRNA razred su malih (~22-25 nukleotida), jednolančanih, evolucijski vrlo očuvanih, nekodirajućih molekula RNA koje imaju specifičnu ulogu u kontroli genske ekspresije na post-translacijskoj i transkripcijskoj razini. Kod životinja se miRNA sintetiziraju u dva koraka, djelovanjem dva enzima endoribonukleaza III (RNaza III) lokalizirana u jezgri i citoplazmi, dok se dorada miRNA u biljkama u cijelosti odvija u citoplazmi i provodi pomoću jedne RNaze III. Kao dio kompleksa miRISC, zrela miRNA prepoznaje ciljnu mRNA. Nakon prepoznavanja, ovisno o stupnju komplementarnosti, dolazi do egzonukleazne aktivnosti proteina Ago. Kod životinja je utvrđeno da miRNA reguliraju signalne putove te osnovne stanične procese poput proliferacije, diferencijacije, te da imaju važnu ulogu u neuralnom razvoju, imunitetu te održavanju homeostaze reguliranjem programirane stanične smrti, , itd. Biljne pak miRNA na specifičan način reguliraju razvoj biljnih organa poput listova i cvijeta, kao i vaskularni razvoj, homeostazu fitohormona i nutrijenata, a uključene su i u odgovor na stres do kojeg dolazi uslijed djelovanja biotičkih i abiotičkih čimbenika.MicroRNAs (miRNAs) are a class of small, noncoding RNAs, approximatey ~22-25 nucleotides long, single-standed, evolutionary conserved, which are known to play specific regulatory role on post-transcription and transcription level. In animals, miRNAs synthesizes occurs in two steps, processed by two exoribonucleases III (RNase III), which are localised in nucleus and cytoplasm, while plant miRNA processing is dependent on one RNase III, which is localised in cytoplasm. As a part of microRNA-induced silencing complex, miRNAs are able to recognise the target mRNAs. After recognition, depending on the degree of complementarity, the exonuclease (slicer) activity of Ago protein occurs. In animals, it was determined that miRNA regulates signal pathways and basic cellular processes such as proliferation, differentiation, and play an important role in neural development, immunity, and homeostatic maintenance by regulating programmed cell death, etc. Plants miRNAs specifically regulate the development of plant organs such as leaves and flowers, as well as vascular development, phytohormones and nutrition homeostasis, and are included in response to the stress caused by the action of biotic and abiotic factors

Common miRNA Patterns of Alzheimer’s Disease and Parkinson’s Disease and Their Putative Impact on Commensal Gut Microbiota

With the rise of Next-Generation-Sequencing (NGS) methods, Micro-RNAs (miRNAs) have achieved an important position in the research landscape and have been found to present valuable diagnostic tools in various diseases such as multiple sclerosis or lung cancer. There is also emerging evidence that miRNAs play an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD) or Parkinson’s disease (PD). Apparently, these diseases come along with changes in miRNA expression patterns which led to attempts from researchers to use these small RNA species from several body fluids for a better diagnosis and in order to observe disease progression. Additionally, it became evident that microbial commensals might play an important role for pathology development and were shown to have a significantly different composition in patients suffering from neurodegeneration compared with healthy controls. As it could recently be shown that secreted miRNAs are able to enter microbial organisms, it is conceivable that the host’s miRNA might affect the gut microbial ecosystem. As such, miRNAs may inherit a central role in shaping the “diseased microbiome” and thereby mutually act on the characteristics of these neurodegenerative diseases. We have therefore (1) compiled a list of miRNAs known to be associated with AD and/or PD, (2) performed an in silico target screen for binding sites of these miRNA on human gut metagenome sequences and (3) evaluated the hit list for interesting matches potentially relevant to the etiology of AD and or PD. The examination of protein identifiers connected to bacterial secretion system, lipopolysaccharide biosynthesis and biofilm formation revealed an overlap of 37 bacterial proteins that were targeted by human miRNAs. The identified links of miRNAs to the biological processes of bacteria connected to AD and PD have yet to be validated via in vivo experiments. However, our results show a promising new approach for understanding aspects of these neurodegenerative diseases in light of the regulation of the microbiome