Tatyana E. Skvortsova

Isolation and Comparative Study of Cell-Free Nucleic Acids from Human Urine

Abstract:  Cell‐free nucleic acids (NA) from human urine were investigated. Concentrations of DNA and RNA in the urine of healthy people were independent of gender and were in the range of 6 ng/mL to 50 ng/mL and 24 ng/mL to 140 ng/mL, respectively. DNA fragments of 150–400 bp represent the main part of cell‐free DNA, along with DNA fragments up to 1,300 bp, which were found in male urine, and DNA fragments up to 19 kbp, which were found in female urine. Analysis of circulating DNA, isolated from blood of breast cancer patients and cell‐free DNA isolated from their urine by methylation‐specific PCR, demonstrates that the presence of methylated promoters of RASSF1A and RARβ2 genes in plasma was accompanied by the detection of the same methylated markers in urine. The data obtained demonstrate applicability of cell‐free urine DNA in cancer diagnostics.

Comparative Study of Extracellular Vesicles from the Urine of Healthy Individuals and Prostate Cancer Patients.

Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90–95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.

Concentrations of circulating RNA from healthy donors and cancer patients estimated by different methods.

Abstract:  Circulating RNA (cirRNA) was isolated from plasma and cell surface‐bound fractions of blood of healthy women and breast cancer patients. RNA samples were DNase treated and quantified by a SYBR Green II assay. Concentrations of RNA sequences of GAPDH, Ki‐67 mRNA, and 18S rRNA were measured by real‐time quantitative PCR (RT‐qPCR) after reverse transcription with random hexamer primers. The obtained data spread over three orders of magnitude for GAPDH and Ki‐67 mRNA signals and two orders of magnitude for the copy number of 18S rRNA in blood fractions in both groups. In blood of healthy donors, no correlation was found between the copy number of GAPDH, Ki‐67 mRNA, and 18S rRNA and RNA concentrations measured by the SYBR Green II assay. Within the group of breast cancer patients, the concentration GAPDH and Ki‐67 mRNA correlated with the concentration of total RNA only in the cell surface‐bound fraction; whereas the concentration of 18S rRNA correlated with total RNA in both, the cell surface‐bound fraction and blood. The copy number of Ki‐67 mRNA correlated with copy numbers of GAPDH mRNA in all fractions of cirRNA of healthy donors and breast cancer patients. A correlation between copy numbers of Ki‐67 mRNA and 18S rRNA was found only in cell surface‐bound fraction of breast cancer patients. The data described here demonstrate the necessity of searching for more suitable RNA markers in order to estimate total cirRNA concentrations by RT‐qPCR, although mRNA of GAPDH could be used for normalization of the level of cancer‐specific mRNA among patients.

Extracellular DNA in breast cancer: Cell-surface-bound, tumor-derived extracellular DNA in blood of patients with breast cancer and nonmalignant tumors.

Abstract: A methylation‐specific polymerase chain reaction technique was used to investigate aberrant promoter methylation of RASSF1A and HIC‐1 genes in circulating extracellular DNA (exDNA) from the blood of breast cancer and fibroadenoma patients. Methylated DNA could be detected in the exDNA eluted from the surface of erythrocytes and leukocytes, even in the samples where no methylated DNA could be detected in plasma. The data obtained demonstrate that cell surface bound exDNA provides a valuable source of material for early noninvasive cancer diagnostics and monitoring.

Investigation of Tumor‐Derived Extracellular DNA in Blood of Cancer Patients by Methylation‐Specific PCR

The frequency of APC, RASSF1A, RARβ, CDH1 and CDH13 gene promoter methylation in samples of DNA isolated from breast and lung patient plasma was studied in order to develop the noninvasive tumor‐specific DNA detection method. Methylation of at least one of genes was detected in extracellular DNA from most of the cancer blood specimens. The results obtained indicate that promoter hypermethylation of a number of marker genes represents a promising serum marker for early breast and lung cancer detection.

Binding and penetration of methylated DNA into primary and transformed human cells.

Tumor progression was shown to be accompanied by the circulation of aberrantly methylated DNA both in the plasma and at the surface of blood cells. As the methylated DNA of tumor suppression genes in blood is a carrier of potentially harmful genetic information, we have studied the stability of methylated DNA fragments in the extracellular space, their binding to the cell surface, and their penetration into cells. It has been shown that the methylated fragments of the RARβ2 gene are more stable and penetrate into the primary and transformed cells at least twice as efficiently as the unmethylated fragments. The data obtained demonstrate the prevalence of methylated DNA in intracellular traffic compared to the same sequences of unmethylated DNA and, thus, a high transformation potential of methylated DNA.

Breast cancer diagnostics based on extracellular DNA and RNA circulating in blood

Extracellular DNA and RNA were extracted from blood plasma and cell surface-bound fractions of healthy women and patients with fibroadenoma and breast cancer. Frequency of methylation of RASSF1A, Cyclin D2, and RARβ2 genes was detected in the extracellular DNA using methylation-specific PCR. Methylation of at least one of these genes was found in plasma of 13% patients with nonmalignant breast fibroadenoma and in 60% of breast cancer patients. Employment cell-surface bound DNA as the substrate for PCR increased the detection frequency of gene methylation up to 87% in patients with fibroadenoma and 95% in breast cancer patients. In clinically healthy women the methylation markers have not been found in extracellular DNA. GAPDH, RASSF8, Ki-67 mRNAs, and 18S rRNA copies were quantified using RT-qPCR of extracellular RNA circulating in blood of patients with breast tumors and healthy controls. The major part of blood extracellular RNA is associated with cell surface. ROC analysis has shown that differences in concentrations 18S RNA, RASSF8, and Ki-67 mRNAs in blood plasma are highly sensitive and specific in discrimination of benign and malignant breast tumors. Thus, analysis of methylated forms of tumor suppressor genes in blood extracellular and quantification of specific extracellular RNA circulating in blood plasma may detect mammary gland tumors and discriminate malignant and benign neoplasms.

Plasma miR-19b and miR-183 as Potential Biomarkers of Lung Cancer

Lung cancer is a complex disease that often manifests at the point when treatment is not effective. Introduction of blood-based complementary diagnostics using molecular markers may enhance early detection of this disease and help reduce the burden of lung cancer. Here we evaluated the diagnostic potential of seven plasma miRNA biomarkers (miR-21, -19b, -126, -25, -205, -183, -125b) by quantitative reverse transcription PCR. Influence clinical and demographical characteristics, including age, tumor stage and cancer subtype on miRNA levels was investigated. Four miRNAs were significantly dysregulated (miR-19b, -21, -25, -183) in lung cancer patients. Combination of miR-19b and miR-183 provided detection of lung cancer with 94.7% sensitivity and 95.2% specificity (AUC = 0.990). Thus, miRNAs have shown the potential to discriminate histological subtypes of lung cancer and reliably distinguish lung cancer patients from healthy individuals.

Dynamic changes in circulating miRNA levels in response to antitumor therapy of lung cancer

ABSTRACT Purpose: Expression levels of cancer-associated microRNAs were reported to be altered in serum/plasma samples from lung cancer patients compared with healthy subjects. The purpose of this study was to estimate the value of five selected miRNAs plasma levels as markers of response to antitumor therapy in lung cancer patients. Materials and Methods: Expression levels of miR-19b, miR-126, miR-25, miR-205, and miR-125b have been evaluated by quantitative reverse transcription PCR versus control miR-16 in blood plasma samples from 23 lung cancer (LC) patients. Plasma samples were obtained from LC patients before treatment (untreated-UT), within 30 days after completing two courses of chemotherapy (postchemotherapy-PC) and 15 days after surgery (postoperative-PO). Results: Repeated Measures ANOVA demonstrated that miR-19b expression levels were decreased in PC and increased in PO samples. These changes were characterized by a significant quadratic trend (p = 0.03). Expression levels of miR-125b increased both after chemotherapy and again after surgery and demonstrated a significant linear trend (p = 0.03). The miR-125b/miR-19b ratio changed during the course of the antitumor treatment with a significant linear trend (p = 0.04). Individual analysis in the groups of patients with partial response to chemotherapy and patients with stable or progressive disease showed different trends for miR-19b, miR-125b, and miR-125b/miR-19b ratio between the groups. The Kaplan–Meier survival curves demonstrated an association of miR-125b/miR-19b ratio value with the survival time without the tumor relapse (p < 0.1). Conclusions: Dynamic change of trends for miR-19b and miR-125b expression levels and miR-125b/miR-19b ratio in the blood plasma have shown a potentiality to discriminate types of response to antitumor therapy in lung cancer patients. Further in-depth investigation is needed to establish a direct link the miRNAs expression levels in blood plasma with therapy response and patients survival.

A phenol-free method for isolation of microRNA from biological fluids.

MicroRNAs (miRNAs) found in biological fluids such as blood and urine have been identified as promising biomarkers for many human disorders, including cancer, cardiopathies, and neurodegenerative diseases. However, circulating miRNAs are either encapsulated into vesicles or found in complexes with proteins and lipoproteins and, thus, require a special approach to their isolation. Acid phenol-chloroform extraction can solve this problem, but it is a labor-intensive procedure that relies heavily on the use of hazardous chemicals. Here we describe a fast and simple phenol-free protocol for miRNA isolation from biofluids. MiRNA is extracted from complexes with biopolymers by a high concentration of guanidine isothiocyanate combined with water/organic composition of solvents. Purification is finished using silica-based spin columns. Comparison of miRNA isolation from blood plasma and urine using the single-phase method and acid phenol-chloroform extraction by means of radioisotope spike-ins and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) showed similar performance of the two methods.