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Cell-Free Nucleic Acids in the Molecular Pathogenesis of Diseases Development

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: closed (31 August 2020) | Viewed by 46346

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Special Issue Editor

Prof. Dr. Bálint Nagy

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Department of Human Genetics, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary
Interests: comparison of nucleic acid isolation methods; cell-free nucleic acid isolation; gene expression measurement; clinical application of liquid biopsy; diagnosis of genetic diseases; cell-free gDNA; mtDNA; miRNA and lncRNA studies in oncological and cardiovascular diseases; exosome isolation methods; nucleic acid measurements from exosomes
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Dear Colleagues,

Cell-free nucleic acids (DNA, mitochondrial DNA, microRNA, long non-coding RNA, circular RNA), which are released by cells to different body fluids via active transport or by apoptosis or necrosis in both normal and pathological conditions, have been the focus of recent research. They are considered to be biomarkers for detection and molecular classification of samples obtained from patients having cancer, stroke, myocardial infarction, diabetes, sepsis, etc. Liquid biopsy has recently become a very popular sampling procedure for such purposes. The main advantage of this method is the possibility of easy and repetitive obtaining of samples. Cell-free nucleic acid can be isolated from bloody fluids in a short time. Their use in diagnostic or screening processes is already accepted. They also provide the possibility to follow up patients’ treatments. Liquid biopsy is applicable during the diagnosis of oncological, cardiovascular, neurological, and infectious diseases, among others. Gene expression studies could provide valuable information to researchers and clinicians. Currently, it seems promising to isolate exosomes from liquid biopsies and understand their role in the development of pathological conditions. We can collect valuable information on cell–cell–tissue communication. Identification of new extra- and intracellular signaling nucleic acid molecules and finding out their pathways could help in the diagnosis and treatment of patients.

Cell-free DNA is already widely used in the prenatal detection of genetic diseases. Recently, oncological diseases have been the focus of research, and the newest area of research is cardiovascular and neurological diseases using regulator RNA molecules.

Therefore, authors are invited to submit original research and review articles that address the progress and current standing of cell-free nucleic acids to understand the molecular pathology of diseases and develop biomarkers for screening and follow up.

Topics include, but are not limited to, the following:

Gene expression studies of diseases using cell-free nucleic acids;
Gene mutation studies;
Application of cell-free nucleic acids such as biomarkers in different types of diseases;
Techniques for isolation, detection, analysis, and identification of signaling nucleic acid molecules, pathways, and networks;
Standardization of related protocols.

Prof. Bálint Nagy
Guest Editor

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Keywords

cell-free nucleic acids
liquid biopsy
biomarkers
non-invasive
exosomes

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Published Papers (8 papers)

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Research

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20 pages, 5685 KiB

Open AccessArticle

Analysis of Circulating miRNA Profile in Plasma Samples of Glioblastoma Patients

by Dóra Géczi, Bálint Nagy, Melinda Szilágyi, András Penyige, Álmos Klekner, Adrienn Jenei, József Virga and Zsuzsanna Birkó

Int. J. Mol. Sci. 2021, 22(10), 5058; https://doi.org/10.3390/ijms22105058 - 11 May 2021

Cited by 9 | Viewed by 2912

Abstract

(1) Background: Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis. Treatment options are limited, clinicians lack efficient prognostic and predictive markers. Circulating miRNAs—besides being important regulators of cancer development—may have potential as diagnostic biomarkers of GBM. (2) Methods: In this study, profiling of 798 human miRNAs was performed on blood plasma samples from 6 healthy individuals and 6 patients with GBM, using a NanoString nCounter Analysis System. To validate our results, five miRNAs (hsa-miR-433-3p, hsa-miR-362-3p, hsa-miR-195-5p, hsa-miR-133a-3p, and hsa-miR-29a-3p) were randomly chosen for RT-qPCR detection. (3) Results: In all, 53 miRNAs were significantly differentially expressed in plasma samples of GBM patients when data were filtered for FC 1 and FDR 0.1. Target genes of the top 39 differentially expressed miRNAs were identified, and we carried out functional annotation and pathway enrichment analysis of target genes via GO and KEGG-based tools. General and cortex-specific protein–protein interaction networks were constructed from the target genes of top miRNAs to assess their functional connections. (4) Conclusions: We demonstrated that plasma microRNA profiles are promising diagnostic and prognostic molecular biomarkers that may find an actual application in the clinical practice of GBM, although more studies are needed to validate our results. Full article

(This article belongs to the Special Issue Cell-Free Nucleic Acids in the Molecular Pathogenesis of Diseases Development)

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19 pages, 3365 KiB

Open AccessArticle

The Cell-Free Expression of MiR200 Family Members Correlates with Estrogen Sensitivity in Human Epithelial Ovarian Cells

by Éva Márton, Alexandra Varga, Lajos Széles, Lóránd Göczi, András Penyige, Bálint Nagy and Melinda Szilágyi

Int. J. Mol. Sci. 2020, 21(24), 9725; https://doi.org/10.3390/ijms21249725 - 20 Dec 2020

Cited by 9 | Viewed by 2861

Abstract

Exposure to physiological estrogens or xenoestrogens (e.g., zearalenone or bisphenol A) increases the risk for cancer. However, little information is available on their significance in ovarian cancer. We present a comprehensive study on the effect of estradiol, zearalenone and bisphenol A on the phenotype, mRNA, intracellular and cell-free miRNA expression of human epithelial ovarian cell lines. Estrogens induced a comparable effect on the rate of cell proliferation and migration as well as on the expression of estrogen-responsive genes (GREB1, CA12, DEPTOR, RBBP8) in the estrogen receptor α (ERα)-expressing PEO1 cell line, which was not observable in the absence of this receptor (in A2780 cells). The basal intracellular and cell-free expression of miR200s and miR203a was higher in PEO1, which was accompanied with low ZEB1 and high E-cadherin expression. These miRNAs showed a rapid but intermittent upregulation in response to estrogens that was diminished by an ERα-specific antagonist. The role of ERα in the regulation of the MIR200B-MIR200A-MIR429 locus was further supported by publicly available ChIP-seq data. MiRNA expression of cell lysates correlated well with cell-free miRNA expression. We conclude that cell-free miR200s might be promising biomarkers to assess estrogen sensitivity of ovarian cells. Full article

(This article belongs to the Special Issue Cell-Free Nucleic Acids in the Molecular Pathogenesis of Diseases Development)

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16 pages, 1378 KiB

Open AccessArticle

Pathway Analysis of Selected Circulating miRNAs in Plasma of Breast Cancer Patients: A Preliminary Study

by Veronika Holubekova, Zuzana Kolkova, Marian Grendar, Dusan Brany, Dana Dvorska, Igor Stastny, Marianna Jagelkova, Katarina Zelinova, Marek Samec, Alena Liskova, Zuzana Laucekova, Erik Kudela, Martina Bobrovska, Michal Kalman, Pavol Zubor and Zuzana Dankova

Int. J. Mol. Sci. 2020, 21(19), 7288; https://doi.org/10.3390/ijms21197288 - 2 Oct 2020

Cited by 17 | Viewed by 3206

Abstract

MicroRNAs in the circulation of breast cancer (BC) patients have great potential for the early diagnosis, treatment and monitoring of breast cancer. The aim of this preliminary study was to obtain the expression profile of selected miRNAs in the plasma of BC patients that could discriminate BC patients from healthy volunteers and may be useful in early detection of BC. Significantly deregulated miRNAs were evaluated by pathway analysis with the prediction of potential miRNA targets. The study enrolled plasma samples from 65 BC patients and 34 healthy volunteers. Selected miRNAs were screened in pilot testing by the real-time PCR (qPCR) method, and the most appropriate reference genes were selected for normalisation by the geNorm algorithm. In the final testing, we detected miR-99a, miR-130a, miR-484 and miR-1260a (p < 0.05) as significantly up-regulated in the plasma of BC patients. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that all significantly deregulated miRNAs are involved in the Hippo and Transforming Growth Factor-beta (TGF-beta) signalling pathways. Our study confirmed a different profile of selected circulating miRNAs in the plasma of BC patients with an emphasis on some critical points in the analysis process. Full article

(This article belongs to the Special Issue Cell-Free Nucleic Acids in the Molecular Pathogenesis of Diseases Development)

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20 pages, 2110 KiB

Open AccessArticle

Elevated Pro-Inflammatory Cell-Free MicroRNA Levels in Cerebrospinal Fluid of Premature Infants after Intraventricular Hemorrhage

by Zsolt Fejes, Judit Erdei, Marianna Pócsi, Jun Takai, Viktória Jeney, Andrea Nagy, Alíz Varga, Attila Bácsi, László Bognár, László Novák, János Kappelmayer and Béla Nagy, Jr.

Int. J. Mol. Sci. 2020, 21(18), 6870; https://doi.org/10.3390/ijms21186870 - 19 Sep 2020

Cited by 14 | Viewed by 2906

Abstract

Intraventricular hemorrhage (IVH) represents a high risk of neonatal mortality and later neurodevelopmental impairment in prematurity. IVH is accompanied with inflammation, hemolysis, and extracellular hemoglobin (Hb) oxidation. However, microRNA (miRNA) expression in cerebrospinal fluid (CSF) of preterm infants with IVH has been unknown. Therefore, in the present study, candidate pro-inflammatory cell-free miRNAs were analyzed in CSF samples from 47 preterm infants with grade III or IV IVH vs. clinical controls (n = 14). miRNAs were quantified by RT-qPCR, normalized to “spike-in” cel-miR-39. Oxidized Hb and total heme levels were determined by spectrophotometry as well as IL-8, VCAM-1, ICAM-1, and E-selectin concentrations by ELISA. To reveal the origin of the investigated miRNAs, controlled hemolysis experiments were performed in vitro; in addition, human choroid plexus epithelial cell (HCPEpiC) cultures were treated with metHb, ferrylHb, heme, or TNF-α to replicate IVH-triggered cellular conditions. Levels of miR-223, miR-155, miR-181b, and miR-126 as well as Hb metabolites along with IL-8 were elevated in CSF after the onset of IVH vs. controls. Significant correlations were observed among the miRNAs, oxidized Hb forms, and the soluble adhesion molecules. During the post-IVH follow-up, attenuated expression of miRNAs and protein biomarkers in CSF was observed upon elimination of Hb metabolites. These miRNAs remained unaffected by a series of artificially induced hemolysis, which excluded red blood cells as their origin, while stimulation of HCPEpiCs with oxidized Hb fractions and heme resulted in increased extracellular miRNA levels in the cell culture supernatant. Overall, the hemorrhage-induced CSF miRNAs reflected inflammatory conditions as potential biomarkers in preterm IVH. Full article

(This article belongs to the Special Issue Cell-Free Nucleic Acids in the Molecular Pathogenesis of Diseases Development)

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Review

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43 pages, 2780 KiB

Open AccessReview

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

by Zuzana Pös, Ondrej Pös, Jakub Styk, Angelika Mocova, Lucia Strieskova, Jaroslav Budis, Ludevit Kadasi, Jan Radvanszky and Tomas Szemes

Int. J. Mol. Sci. 2020, 21(22), 8634; https://doi.org/10.3390/ijms21228634 - 16 Nov 2020

Cited by 35 | Viewed by 5553

Abstract

Analyzes of cell-free nucleic acids (cfNAs) have shown huge potential in many biomedical applications, gradually entering several fields of research and everyday clinical care. Many biological properties of cfNAs can be informative to gain deeper insights into the function of the organism, such as their different types (DNA, RNAs) and subtypes (gDNA, mtDNA, bacterial DNA, miRNAs, etc.), forms (naked or vesicle bound NAs), fragmentation profiles, sequence composition, epigenetic modifications, and many others. On the other hand, the workflows of their analyzes comprise many important steps, from sample collection, storage and transportation, through extraction and laboratory analysis, up to bioinformatic analyzes and statistical evaluations, where each of these steps has the potential to affect the outcome and informational value of the performed analyzes. There are, however, no universal or standard protocols on how to exactly proceed when analyzing different cfNAs for different applications, at least according to our best knowledge. We decided therefore to prepare an overview of the available literature and products commercialized for cfNAs processing, in an attempt to summarize the benefits and limitations of the currently available approaches, devices, consumables, and protocols, together with various factors influencing the workflow, its processes, and outcomes. Full article

(This article belongs to the Special Issue Cell-Free Nucleic Acids in the Molecular Pathogenesis of Diseases Development)

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24 pages, 1879 KiB

Open AccessReview

Putative Origins of Cell-Free DNA in Humans: A Review of Active and Passive Nucleic Acid Release Mechanisms

by Stefan Grabuschnig, Abel Jacobus Bronkhorst, Stefan Holdenrieder, Ingund Rosales Rodriguez, Klaus Peter Schliep, Daniel Schwendenwein, Vida Ungerer and Christoph Wilhelm Sensen

Int. J. Mol. Sci. 2020, 21(21), 8062; https://doi.org/10.3390/ijms21218062 - 29 Oct 2020

Cited by 114 | Viewed by 10448

Abstract

Through various pathways of cell death, degradation, and regulated extrusion, partial or complete genomes of various origins (e.g., host cells, fetal cells, and infiltrating viruses and microbes) are continuously shed into human body fluids in the form of segmented cell-free DNA (cfDNA) molecules. While the genetic complexity of total cfDNA is vast, the development of progressively efficient extraction, high-throughput sequencing, characterization via bioinformatics procedures, and detection have resulted in increasingly accurate partitioning and profiling of cfDNA subtypes. Not surprisingly, cfDNA analysis is emerging as a powerful clinical tool in many branches of medicine. In addition, the low invasiveness of longitudinal cfDNA sampling provides unprecedented access to study temporal genomic changes in a variety of contexts. However, the genetic diversity of cfDNA is also a great source of ambiguity and poses significant experimental and analytical challenges. For example, the cfDNA population in the bloodstream is heterogeneous and also fluctuates dynamically, differs between individuals, and exhibits numerous overlapping features despite often originating from different sources and processes. Therefore, a deeper understanding of the determining variables that impact the properties of cfDNA is crucial, however, thus far, is largely lacking. In this work we review recent and historical research on active vs. passive release mechanisms and estimate the significance and extent of their contribution to the composition of cfDNA. Full article

(This article belongs to the Special Issue Cell-Free Nucleic Acids in the Molecular Pathogenesis of Diseases Development)

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14 pages, 980 KiB

Open AccessReview

Novel Molecular Markers in Glioblastoma—Benefits of Liquid Biopsy

by Zsuzsanna Birkó, Bálint Nagy, Álmos Klekner and József Virga

Int. J. Mol. Sci. 2020, 21(20), 7522; https://doi.org/10.3390/ijms21207522 - 12 Oct 2020

Cited by 45 | Viewed by 6154

Abstract

Glioblastoma is a primary Central Nervous System (CNS) malignancy with poor survival. Treatment options are scarce and despite the extremely heterogeneous nature of the disease, clinicians lack prognostic and predictive markers to characterize patients with different outcomes. Certain immunohistochemistry, FISH, or PCR-based molecular markers, including isocitrate dehydrogenase1/2 (IDH1/2) mutations, epidermal growth factor receptor variant III (EGFRvIII) mutation, vascular endothelial growth factor overexpression (VEGF) overexpression, or (O6-Methylguanine-DNA methyltransferase promoter) MGMT promoter methylation status, are well-described; however, their clinical usefulness and accuracy is limited, and tumor tissue samples are always necessary. Liquid biopsy is a developing field of diagnostics and patient follow up in multiple types of cancer. Fragments of circulating nucleic acids are collected in various forms from different bodily fluids, including serum, urine, or cerebrospinal fluid in order to measure the quality and quantity of these markers. Multiple types of nucleic acids can be analyzed using liquid biopsy. Circulating cell-free DNA, mitochondrial DNA, or the more stable long and small non-coding RNAs, circular RNAs, or microRNAs can be identified and measured by novel PCR and next-generation sequencing-based methods. These markers can be used to detect the previously described alterations in a minimally invasive method. These markers can be used to differentiate patients with poor or better prognosis, or to identify patients who do not respond to therapy. Liquid biopsy can be used to detect recurrent disease, often earlier than using imaging modalities. Liquid biopsy is a rapidly developing field, and similarly to other types of cancer, measuring circulating tumor-derived nucleic acids from biological fluid samples could be the future of differential diagnostics, patient stratification, and follow up in the future in glioblastoma as well. Full article

(This article belongs to the Special Issue Cell-Free Nucleic Acids in the Molecular Pathogenesis of Diseases Development)

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20 pages, 988 KiB

Open AccessReview

Circulating Cell-Free Nucleic Acids: Main Characteristics and Clinical Application

by Melinda Szilágyi, Ondrej Pös, Éva Márton, Gergely Buglyó, Beáta Soltész, Judit Keserű, András Penyige, Tomas Szemes and Bálint Nagy

Int. J. Mol. Sci. 2020, 21(18), 6827; https://doi.org/10.3390/ijms21186827 - 17 Sep 2020

Cited by 125 | Viewed by 11612

Abstract

Liquid biopsy recently became a very promising diagnostic method that has several advantages over conventional invasive methods. Liquid biopsy may serve as a source of several important biomarkers including cell-free nucleic acids (cf-NAs). Cf-DNA is widely used in prenatal testing in order to characterize fetal genetic disorders. Analysis of cf-DNA may provide information about the mutation profile of tumor cells, while cell-free non-coding RNAs are promising biomarker candidates in the diagnosis and prognosis of cancer. Many of these markers have the potential to help clinicians in therapy selection and in the follow-up of patients. Thus, cf-NA-based diagnostics represent a new path in personalized medicine. Although several reviews are available in the field, most of them focus on a limited number of cf-NA types. In this review, we give an overview about all known cf-NAs including cf-DNA, cf-mtDNA and cell-free non-coding RNA (miRNA, lncRNA, circRNA, piRNA, YRNA, and vtRNA) by discussing their biogenesis, biological function and potential as biomarker candidates in liquid biopsy. We also outline possible future directions in the field. Full article

(This article belongs to the Special Issue Cell-Free Nucleic Acids in the Molecular Pathogenesis of Diseases Development)

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