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Next-generation sequencing analyzes EPC-drived exosome miRNA content and alterations of mRNA expression in exosomes treated HUVEC

ABSTRACT: Purpose: To dissect the mechanisms of EPC-drived exosome in promoting endothelial cells reendothelialization, next-generation sequencing was used to determine exosome miRNA content and alterations in mRNA expression in HUVEC. Methods: The miRNA of EPC-drived exosomes sequencing was carried out on the HiSeq 2500 Platform (NovelBio Corp. Laboratory, Shanghai). The data analysis was carried out with the QIAseq miRNA quantification platform using unique molecular index (UMI) counts according to the manufacturer’s instructions. The mRNA analysis of three independent samples of PBS or EPC exosomes treated HUVEC was performed using the the HiSeq 2500 Platform (NovelBio Corp. Laboratory, Shanghai) following the manufacturer’s instructions. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: The 20 most highly expressed microRNAs identified in EPC exosomes s are listed in Table 1. The genes predicted to be targeted by those microRNAs most highly expressed in EPC exosomes (top 20 microRNAs comprising ~75% of all reads, Table 1) are most significantly enriched in the functional category ‘blood vessel development’within the top 10 categories. Further, endothelial-specific miR-21-5P was the most highly expressed microRNA in EPC exosomes. Conclusions:EPC exosomes promote vascular endothelial repair by delivering functional miR-21-5p.

ORGANISM(S): Homo sapiens

PROVIDER: GSE124692 | GEO | 2022/01/03

REPOSITORIES: GEO

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Project description:Comparison of mRNA expression in human EPC vs. HUVEC vs. human monocytes. Cell-type specific gene expression under basal cell culture conditions (no stimulation). The hybridization was performed with three samples of EPC vs. three samples of HUVEC vs. 3 samples of CD14+ monocytes. â?¢ The origin of the biological sample:; Human endothelial progenitor cells (EPC): EPC were ex vivo cultivated from human peripheral blood-derived mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation from healthy human volunteers as previously described (Dimmeler et al., 2001). Pooled human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Verviers, Belgium). CD14+ monocytes were purified from PBMC by positive selection with anti-CD14-microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity assessed by FACS analysis was greater than 95%. â?¢ Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques:; EPC:; 8000000 PBMC/ml were plated on human fibronectin (Sigma, Taufkirchen, Germany) and maintained in endothelial basal medium (Cambrex) with EGM SingleQuots and 20% fetal calf serum (FCS). After 3 days, nonadherent cells were removed and adherent cells were incubated in fresh medium for 24 h before starting experiments. HUVEC:; HUVEC were cultured in endothelial basal medium (Cambrex) supplemented with hydrocortisone, bovine brain extract, gentamicin, amphotericin B, epidermal growth factor, and 10% FCS until the third passage according to the manufacturerâ??s recommendations. CD14+ monocytes: No culture. â?¢ Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol. Total RNA was extracted from EPC, HUVEC, and CD14+ monocytes using the RNeasy cleanup system (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Quantity and quality of total RNA was analyzed by the 2100 Bioanalyzer system (Agilent Technologies, Waldbronn, Germany) and agarose gel electrophoresis. â?¢ Labeling protocol(s):; The detailed protocol for the sample preparation and microarray processing is available from Affymetrix (Santa Clara, CA). Briefly, 10 Âµg of purified total RNA was reverse transcribed by Superscript II reverse transcriptase (Life Technologies, Grand Island, NY) using T7-(dT)24 primer containing a T7 RNA polymerase promoter. After synthesis of the second complementary DNA (cDNA) strand, this product was used in an in vitro transcription reaction to generate biotinylated complementary RNA (cRNA). â?¢ The protocol and conditions used during hybridization, blocking and washing:; Fifteen micrograms of fragmented, biotinylated cRNA were hybridized to a HG-U95Av2 microarray (Affymetrix Inc.) for 16 hours at 45Â° C with constant rotation at 60 rpm. This high-density oligonucleotide array targets 9,670 human genes as selected from the National Center for Biotechnology Information (NCBI) Gene Bank database with a total of 12,000 oligonucleotide sets. Each microarray was used to assay a single sample. After hybridization, the microarray was washed and stained on an Affymetrix fluidics station and scanned with an argon-ion confocal laser, with a 488 nm emission and detection at 570 nm. â?¢ GeneChip image analysis was performed using the Microarray Analysis Suite 5.0 (Affymetrix, Inc.). Expression data were analyzed utilizing the GeneSpringâ?¢ software version 4.2 (Silicon Genetics Inc., San Carlos, CA).

Project description:Comparison of mRNA expression in human EPC vs. HUVEC vs. human monocytes. Cell-type specific gene expression under basal cell culture conditions (no stimulation). The hybridization was performed with three samples of EPC vs. three samples of HUVEC vs. 3 samples of CD14+ monocytes. • The origin of the biological sample: Human endothelial progenitor cells (EPC): EPC were ex vivo cultivated from human peripheral blood-derived mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation from healthy human volunteers as previously described (Dimmeler et al., 2001). Pooled human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Verviers, Belgium). CD14+ monocytes were purified from PBMC by positive selection with anti-CD14-microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity assessed by FACS analysis was greater than 95%. • Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques: EPC: 8000000 PBMC/ml were plated on human fibronectin (Sigma, Taufkirchen, Germany) and maintained in endothelial basal medium (Cambrex) with EGM SingleQuots and 20% fetal calf serum (FCS). After 3 days, nonadherent cells were removed and adherent cells were incubated in fresh medium for 24 h before starting experiments. HUVEC: HUVEC were cultured in endothelial basal medium (Cambrex) supplemented with hydrocortisone, bovine brain extract, gentamicin, amphotericin B, epidermal growth factor, and 10% FCS until the third passage according to the manufacturer’s recommendations. CD14+ monocytes: No culture. • Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol. Total RNA was extracted from EPC, HUVEC, and CD14+ monocytes using the RNeasy cleanup system (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Quantity and quality of total RNA was analyzed by the 2100 Bioanalyzer system (Agilent Technologies, Waldbronn, Germany) and agarose gel electrophoresis. • Labeling protocol(s): The detailed protocol for the sample preparation and microarray processing is available from Affymetrix (Santa Clara, CA). Briefly, 10 µg of purified total RNA was reverse transcribed by Superscript II reverse transcriptase (Life Technologies, Grand Island, NY) using T7-(dT)24 primer containing a T7 RNA polymerase promoter. After synthesis of the second complementary DNA (cDNA) strand, this product was used in an in vitro transcription reaction to generate biotinylated complementary RNA (cRNA). • The protocol and conditions used during hybridization, blocking and washing: Fifteen micrograms of fragmented, biotinylated cRNA were hybridized to a HG-U95Av2 microarray (Affymetrix Inc.) for 16 hours at 45° C with constant rotation at 60 rpm. This high-density oligonucleotide array targets 9,670 human genes as selected from the National Center for Biotechnology Information (NCBI) Gene Bank database with a total of 12,000 oligonucleotide sets. Each microarray was used to assay a single sample. After hybridization, the microarray was washed and stained on an Affymetrix fluidics station and scanned with an argon-ion confocal laser, with a 488 nm emission and detection at 570 nm. • GeneChip image analysis was performed using the Microarray Analysis Suite 5.0 (Affymetrix, Inc.). Expression data were analyzed utilizing the GeneSpring™ software version 4.2 (Silicon Genetics Inc., San Carlos, CA). Keywords: parallel sample

Project description:Background: In recent times a subset of bone marrow (BM) derived cells; endothelial progenitor cells (EPCs), have generated tremendous interest, as these cells are suggested to home to sites of neovascularization and neoendothelialization and differentiate into mature endothelial cells (ECs), a process referred to as postnatal vasculogenesis. EPCs are being considered as a potential regenerative tool for treating various pathophysiological disease states including cardiovascular disorder and as a possible target to restrict vessel growth in tumour pathology. However, conflicting results have been reported in the field due to lack of EPC specific biomarkers, and the identification, characterization, and the precise role of EPCs in vascular biology still remains a subject of great debate. Therefore, the objective of this study was to use a bioinformatics approach to identify putative novel EPC specific biomarkers. Methods: This study reports a detailed gene expression profile of human umbilical cord blood (UCB) derived non-adherent CD133 + cells at different time points during in vitro differentiation (day 4 and day 7) which were compared with differentiated donor matched human umbilical vein endothelial cells (HUVEC). Results: EPC gene expression was profiled at both days 4 and 7, using affymetrix human 1.0ST exon arrays. Affymetrix gene expression profiling revealed significant expression changes in genes associated with stem/progenitor cell properties such as adhesion, signalling, molecular transport, cell structure organisation and growth. Conclusions: This is the first study utilizing gene expression profiling to examine non adherent CD133+ progenitor cells. Alterations in the gene expression reported in this study may be involved in the cellular processes characteristic of EPC development and differentiation. Gene expression profiling was performed on EPCs cultured for D4 and D7, on Affymetrix human 1.0ST exon array chips. Gene Spring (version GX11) was used to perform the gene expression analysis on three experimental groups; (a) D4 EPCs versus HUVEC; (b) D7 EPCs versus HUVEC and (c) D4 versus D7 EPCs.

Project description:Background: Docosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA’s anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA’s anticancer action. Results: Exosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA’s anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release. Conclusions: We conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA’s anticancer action, further supporting its use in cancer therapy. Examination of small RNA populations in MCF7 cells and exosomes after DHA treatment.

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Next-generation sequencing analyzes EPC-drived exosome miRNA content and alterations of mRNA expression in exosomes treated HUVEC

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