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Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions.

ABSTRACT: Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000g and 160,000g, and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC) patients. It was determined that 16,000g blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000g pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs' fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches.

SUBMITTER: Savelyeva AV

PROVIDER: S-EPMC5239830 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions.

Savelyeva Anna V AV Kuligina Elena V EV Bariakin Dmitry N DN Kozlov Vadim V VV Ryabchikova Elena I EI Richter Vladimir A VA Semenov Dmitry V DV

BioMed research international 20170103

Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000<i>g</i> and 160,000<i>g< ...[more]

PMID: 28127559

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Project description:BackgroundCircular RNA (circRNA) is a type of closed circular noncoding RNA (ncRNA), mostly formed by back-splicing or alternative splicing of pre-messenger RNA (mRNA). The aim of this study was to explore the expression profile of circRNA in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS) and discover potential molecular markers of AS.MethodsThe circRNA microarray technology was used to detect the expression of circRNAs in the peripheral blood of 6 patients with AS and 6 healthy controls (HC). To screen the differentially expressed circRNAs by fold change (FC) and P value, these differentially expressed circRNAs were analyzed by bioinformatics. In 60 cases of AS and 30 cases of HC, 4 circRNAs were subjected to real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and their correlation with various clinical indicators was analyzed. Finally, the receiver operating characteristic (ROC) curve was used to analyze their potential as AS diagnostic markers.ResultsThe microarray results showed that there were 1369 significantly differently expressed (P < 0.05, FC > 1.5) circRNAs between the AS and HC groups (675 upregulated and 694 downregulated). The results of bioinformatics analysis suggested that they were mainly involved in "enzyme binding," "adenosine ribonucleotide binding," "MAPK signaling pathway", etc. The RT-qPCR results showed that the expressions of hsa_circRNA_001544 (U = 486.5, P < 0.05) and hsa_circRNA_102532 (U = 645, P < 0.05) were significantly different between the AS group and the HC group. The AS group was further divided into two subgroups: active AS (ASA) and stable AS (ASS). After analysis, it was found that compared with the HC group, hsa_circRNA_001544 was significantly increased in both ASA (U = 214, P < 0.05) and ASS groups (U = 273, P < 0.05), while hsa_circRNA_008961 (U = 250, P < 0.05) and hsa_circRNA_102532 (U = 295, P < 0.05) were only significantly increased in the ASA group. Furthermore, hsa_circRNA_012732 was significantly different between the ASA and ASS groups (U = 194, P < 0.05), and there was no statistical significance among the remaining groups. Correlation analysis results showed that hsa_circRNA_012732 was negatively correlated with Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), high-sensitivity C-reactive protein (hsCRP), and globulin (GLOB) and positively correlated with lymphocyte count (LY), mean corpusular volume, and albumin (ALB), and hsa_circRNA_008961 was negatively correlated with platelet (PLT) count. ROC curve analysis showed that hsa_circRNA_001544 (95% CI = 0.610-0.831, P < 0.05) and hsa_circRNA_102532 (95% CI = 0.521-0.762, P < 0.05) were statistically significant, and their area under curve (AUC) values were 0.720 and 0.642, respectively.ConclusionsThere are differentially expressed circRNAs in PBMCs of AS patients, and they may be involved in the occurrence and development of AS. Among these differentially expressed circRNAs, hsa_circRNA_012732 has the potential to become an indicator of disease activity, and hsa_circRNA_001544 has the potential to become a molecular marker for AS diagnosis.

Project description:ObjectiveCirculating miRNAs are found in bodily fluids including plasma and can serve as biomarkers for diseases. The aim of this study was to provide the first insight into the landscape of circulating miRNAs in close proximity to the adrenocorticotropic hormone (ACTH) secreting PitNET. To achieve this objective next-generation sequencing of miRNAs in plasma from bilateral inferior petrosal sinus sampling (BIPSS) - a gold standard in diagnosing ACTH-secreting PitNETs was carried out and selected miRNA candidates were further tested by RT-qPCR in independent patient cohorts.MethodsSinistral (left) and dextral (right) BIPSS blood samples of the patient were collected in three time points: before the administration of corticotropin-releasing hormone, 5 and 15 minutes after stimulation. In differential expression analysis, sinistral plasma was compared with dextral. The selected miRNA candidates were tested in plasma by RT-qPCR in two patient groups: 1) in five ACTH secreting PitNET patients with plasma samples taken before and 24 hours after surgery, 2) in 12 ACTH secreting PitNET patients vs. 9 non-functioning PitNET patients.ResultsBIPSS concluded that the highest amount of ACTH was released in the sinistral side at the 5th minute mark indicating a presence of a tumor. The highest amount of differentially expressed miRNAs was observed 5 minutes after stimulation (20 upregulated, 14 downregulated). At the 5th minute mark in sinistral plasma, two miRNAs were identified: hsa-miR-7-5p and hsa-miR-375-3p that were highly upregulated compared to other BIPSS samples and peripheral plasma samples. Further testing by qPCR revealed significant reduction of miR-7-5p in plasma 24 hours after surgery and upregulation in plasma of ACTH secreting PitNET patients compared to non-functioning PitNET patients (P =0.0013).ConclusionsBy stimulating the ACTH secreting PitNET with CRH a rapid increase of two miRNAs (hsa-mir-7-5p, hsa-mir-375-3p) and ACTH can be observed in sinistral inferior petrosal (tumor side). A decrease of miR-7-5p in plasma after surgery and upregulation in plasma of ACTH secreting PitNET patients was discovered implying that further studies of this miRNA as diagnostic marker is needed.

Dataset Information

Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions.

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Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions.

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