Development of microRNA expression in exosomes derived from lncRNA HOTTIP overexpressed FaDu cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE163565 | GEO | 2022/02/26
REPOSITORIES: GEO
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Project description:The aim of this study is to discover loss of specific miRNA loci in Wilms' tumors using array CGH. Custom arrays were designed based on the Agilent 2x105K Human Whole Genome Genomic microarray with Agilent’s eArray program (https://earray.chem.agilent.com/earray/), with additional probes that cover all miRNA regions (200 bps before, within and after each miRNA from miRBase v13, with each probe in triplicates to enhance the reliability). All custom-designed probes were designed in UCSC hg18. Probes from Agilent’s database were lifted-over from hg19 to hg18 (LiftOver tool: http://genome.ucsc.edu).
2012-03-31 | GSE28397 | GEO
Project description:MicroRNAs (miRNAs) may modulate more than 60% of human coding genes and act as negative regulators, while long non-coding RNAs (lncRNAs) regulate gene expression on multiple levels by interacting with chromatin, functional proteins, and RNAs such as mRNAs and microRNAs. However, the crosstalk between lncRNA HOTTIP and miRNAs in leukemogenesis remains elusive. Using combined integrated analyses of global miRNA expression profiling and state-of-the-art genomic analyses of chromatin such as ChIRP seq., (genome wide HOTTIP binding analysis), ChIP-seq., and ATAC-seq., we found that miRNA genes are directly controlled by HOTTIP. Specifically, the HOX cluster miRNAs (miR-196a, miR-196b, miR-10a and miR-10b), located cis & trans, were most dramatically regulated and significantly decreased in HOTTIP–/– AML cells. HOTTIP bound to the miR- 196b promoter, and HOTTIP deletion reduced chromatin accessibility and enrichment of active histone modifications at HOX cluster associated miRNAs in AML cells, while reactivation of HOTTIP restored miR gene expression and chromatin accessibility in the CTCF-boundary-attenuated AML cells. Inactivation of HOTTIP or miR-196b promotes apoptosis by altering the chromatin signature at the FAS promoter and increasing FAS expression. Transplantation of miR-196b knockdown MOLM13 cells in NSG mice increased overall survival compared to wild-type cells. Thus, HOTTIP remodels the chromatin architecture around miRNAs to promote their transcription, consequently repressing tumor suppressors and promoting leukemogenesis.
2022-09-14 | GSE182601 | GEO
Project description:Exosomes are small membraneous vesicles secreted into body fluids by tumors. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Further exploration into the molecular profiling of exosomes may increase our understanding of their roles in melanoma progression in vivo, and may have potential application in biomarker studies. In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. Our results indicate that melanoma-derived exosomes have unique gene expression signatures and miRNA profiles that may have important functions in melanoma metastasis and progression. Total RNA from cells and exosomes were isolated using mirVana total RNA isolation kit according to the manufacturer’s guidelines. RNA was quantified using Nanodrop ND-1000. The integrity of these total RNAs was assessed using Agilent 2100 Bioanalyzer. Total high-quality RNA was labelled. The miRNA array profiling was performed by using the Affymetrix GeneChip miRNA Array 1.0. Two different RNA preparations from two cell lines and their exosomes were used, except that only one RNA preparation was used for HEMa-LP exosome miRNA array. Due to the limited number of passages (approximately 10), adequate exosomal RNA and proteins from HEMa-LP cells for multiple analyses was not available.
2012-10-10 | E-GEOD-35387 | biostudies-arrayexpress
Project description:Currently, micro RNAs (miRNAs) constitute a promising models for cell-to-cell communication since they are transferred between cells to execute essential roles in many processes. Their structure and size, in addition to various transport mechanisms, allow them to remain stable within various biological fluids, surviving in extremely adverse conditions, including low pH, boiling, and freezing. The presence of miRNAs in reproductive fluids, such as follicular, uterine and seminal fluid, indicate potential roles in the reproductive system. These extracellular miRNAs can be transported by lipoproteins (both HDL and LDL) or other proteins, including Argonaute2 (AGO2) and nucleophosmin1 (NPM1). Another transport system is mediated by extracellular vesicles (EVs), such as apoptotic bodies, microvesicles (MVs) and/or exosome-like vesicles. EVs protect miRNAs from degradation and contribute to their stability within biological fluids. Furthermore, EVs can transport a wide range of components packaged in a selective way. For instance, Squadrito et al. (2014), used macrophages and endothelial cells to demonstrate that the sorting of miRNAs into EVs for heterotrophic cell communication is altered by both, the presence of target transcripts and the self-presence of the respectively miRNA. In addition, the signature of miRNAs found in the exosomes significantly differed for those detected in the parent cells. To date, mechanisms controlling the specific loading of miRNAs into exosomes remain unclear. Indeed, several mechanisms may govern exosome sorting of specific subsets of miRNAs. MiRNA sorting appears to be influenced by different pathways and molecules in different cell types and tissues, and miRNAs contain well defined motifs (i.e., EXOmotifs), that direct the miRNA allocation into exosomes before delivery into recipient cells. A recent study showed that this RNA sequence can be recognized by the sumoylated form of the heterogeneous ribonucleoprotein A2B1 (hnRNPA2B1). Moreover, a terminal 30 nucleotide addition in miRNAs affects their selective sorting in B cells. Another hypothesis suggests that RNAs are selectively sorted depending on the differential affinity of RNA motifs towards the raft-like region of the cytoplasmic surface of microvesicular body (MVB) limiting membranes. Current data indicate that cells can communicate with each other through the transfer of miRNA-loaded exosomes. For example, monocyte-derived exosomes deliver miR-150 to endothelial cells and enhance endothelial cell migration by reducing c-myb expression. The miRNA content of exosomes plays a critical role in such cell-to-cell communication and determines the fate of recipient cells. Thus, exosomes derived from the bone marrow mesenchymal stromal cells of myeloma patients promote tumor growth depending on the content of miR-15a in exosomes. Our group has described a novel cell-to-cell communication mechanism involving the delivery of endometrial miRNAs from the maternal endometrium to the trophectoderm cells of preimplantation embryos. Specifically, in B6C3 derived mouse embryos, we found EV-associated and free miR-30d to cause overexpression of genes involved in embryonic adhesion processes, including Itb3, Itga7 and Cdh5. Furthermore, supplementing murine embryos with miR-30d significantly improved embryo adhesion, suggesting that external miRNAs may have a functional role as transcriptomic modifiers of preimplantation embryos. Based on profiling of miRNAs in endometrial fluid, maternally-derived miRNAs are present within EVs in the uterine microenvironment. The internalization of maternally-derived exosomes has been visualized, but the mechanism by which miR-30d becomes incorporated into exosomes remains unknown. The present study aimed to elucidate the underlying mechanism of hsa-miR-30d transfer from human endometrial epithelial cells (hEECs) to the interior of exosomes and eventually to early-stage blastocysts, using a mir-30d knockout murine mode.
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Project description:To determine whether hypoxia augments the activity of exosomes by modulating miRNAs, we use miRNA array profile to find the differential expression of miRNAs of exosomes in different culture conditions (normoxia, hypoxia, hypoxia supplemented with GW4869 which is an inhibitor of exosomes generation).
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Project description:Development of microRNA expression in exosomes derived from lncRNA HOTTIP overexpressed FaDu cells
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Project description:MicroRNAs (miRNAs) are small non-coding RNAs that repress gene expression at the post transcriptional level via an antisense RNA-RNA interaction. Generally, miRNAs derived from snap frozen or fresh samples are used for array based profiling. Since tissues in most pathology departments are available only in formalin fixed and paraffin embedded state, we sought to evaluate the miRNA derived from formalin fixed and paraffin embedded (FFPE) samples for microarray analysis. In this study, miRNAs extracted from matched snap frozen and FFPE samples were profiled using the Agilent miRNA array platform. Each miRNA sample was hybridized to arrays containing probes interrogating 470 human miRNAs. A total of seven cases were compared in either duplicate or triplicate. Intrachip and interchip analyses demonstrated that the processes of miRNA extraction, labeling and hybridization from both frozen and FFPE samples are highly reproducible and add little variation to the results, as technical replicates showed high correlations (Kendall tau=0.722-0.853, Spearman rank correlation coefficient=0.891-0.954). Our results showed consistent high correlation between matched frozen and FFPE samples (Kendall tau=0.669-0.815, Spearman rank correlation coefficient=0.847-0.948), supporting the use of FFPE-derived miRNAs for profiling. Keywords: formalin-fixed and paraffin-embedded (FFPE) miRNA profiling
2008-11-01 | GSE12458 | GEO
Project description:Exosomes are small membraneous vesicles secreted into body fluids by tumors. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Further exploration into the molecular profiling of exosomes may increase our understanding of their roles in melanoma progression in vivo, and may have potential application in biomarker studies. In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. Our results indicate that melanoma-derived exosomes have unique gene expression signatures and miRNA profiles that may have important functions in melanoma metastasis and progression. Total RNA from cells and exosomes were isolated using mirVana total RNA isolation kit according to the manufacturer’s guidelines. RNA was quantified using Nanodrop ND-1000. The integrity of these total RNAs was assessed using Agilent 2100 Bioanalyzer. Total high-quality RNA was converted to cDNA, transcribed and labelled, and then hybridized to human HG-U133 plus 2 arrays (Affymetrix) then scanned according to the standard protocol recommended by Affymetrix. Two different RNA preparations from two cell lines and their exosomes were used.
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Project description:Most cancer-related deaths are caused by distant metastases, which are tumour cells that have escaped from a primary tumour and passed into the bloodstream to colonize a new organ. In this context, communication between tumour and stromal cells is essential. Indeed, tumor cells interact with cells in the tumor microenvironment and are able to modify them to their advantage. Both extracellular vesicles (EVs) and exosomes are heterogeneous populations of small vesicles present in the tumor microenvironment and in body fluids that have recently emerged as powerful mediators involved in this communication and their transport in fluids. Tumor cells release large quantities of exosomes containing tumor markers, which can then spread to distant locations.
The exosomes are of endosomal origin. They are composed of proteins, lipids, RNA and DNA, and they circulate in the bloodstream. They can be internalized by specific distant cells and thus deliver a functional message. It has recently been shown that tumor exosomes containing pro-metastatic factors form pre-metastatic niches, before the tumor cells actually arrive, while determining the metastatic organotropism of tumors. These properties are now opening up new avenues of research in tumor biomarkers. In recent years, several studies have highlighted different markers contained specifically in exosomes derived from cancer cells. Consequently, exosomes are considered as potential reservoirs of tumor biomarkers that could be clinically useful for the non-invasive diagnosis of cancer, with the advantage of being performed by liquid biopsy. The study of microRNA (miRNA) is of particular interest. Indeed, miRNAs are small non-coding RNAs (between 21 and 25 nucleotides) involved in the regulation of gene expression and which are frequently deregulated in cancer. Several studies underline that the variation of free miRNAs in the blood is correlated with the progression of the disease, particularly in colon cancer. However, the stability of free miRNAs is controversial. Therefore, exosomes represent a very advantageous means of transporting miRNAs in the blood, as they are able to protect miRNAs from degradation by RNAase.
The hypothesis of the project is that circulating exosomes derived from tumours contain markers including specific miRNAs that could be used as biomarkers of early prognosis (survival and progression), easily measured in blood samples from patients with colon cancer. But other molecules contained in exosomes could also be of interest.
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Project description:Exosomes are released by most cells to the extracellular environment, and are involved in cell-to-cell-communication. Exosomes contain specific repertoires of mRNAs, miRNAs and other non-coding RNAs that can be functionally transferred to recipient cells. However, the mechanisms that control the specific loading of RNA species into exosomes remain unknown. Here we describe short sequence motifs present in miRNAs that control their localization into exosomes. The protein hnRNPA2B1 specifically binds exosomal miRNAs through the recognition of these motifs and controls their loading into exosomes. Moreover, hnRNPA2B1 in exosomes is sumoylated, and sumoylation controls the binding of hnRNPA2B1 to miRNAs. The loading of miRNAs into exosomes can be modulated by mutagenesis of the identified motifs or changes in hnRNPA2B1 expression levels. These findings identify hnRNPA2B1 as a key player in miRNA sorting into exosomes and provide potential tools for the packaging of selected regulatory RNAs into exosomes and their use in biomedical applications.
2014-04-16 | PXD000350 | Pride