MicroRNA-guided gene expression in prostate cancer: Literature and database overview.

Insight into the aberrant expression of microRNAs (miRNAs) and the genes that they regulate during the progression of cancer in general and prostate cancer (PCa) in particular is one of the most important issues in current molecular biomedicine and allows for the discovery of therapeutic or diagnostic miRNA targets. The present study aimed to analyze the available data regarding the direct or indirect effects of miRNAs on the expression of the mRNAs involved in carcinogenesis and to enable updating and optimizing the selection of the corresponding targets. The present review focuses on the data related to the genes with miRNA-dependent expression during the development of PCa. The data used in this review have been extracted from research papers and the databases STRING, PANTHER and TargetScan, with a special focus on the genes directly associated with cell transformation and the maintenance of the transformed genotype, as well as tumor invasion and spread. The search for miRNA markers of PCa and therapeutically active molecules should rely on bioinformatics resources, such as data from recent experimental studies, as well as meta-analysis and cross-analysis of the data on the state of the tumor, patient status, histological/immunohistological data and data on mRNA-miRNA coexpression.

Searching for the Novel Specific Predictors of Prostate Cancer in Urine: The Analysis of 84 miRNA Expression.

The aim of this study was to investigate miRNA profiles of clarified urine supernatant and combined urine vesicle fractions of healthy donors and patients with benign prostatic hyperplasia and prostate cancer (PCa). The comparative analysis of miRNA expression was conducted with a custom miRCURY LNA miRNA qPCR panel. Significant combinations of miRNA pairs were selected by the RandomForest-based feature selection algorithm Boruta; the difference of the medians between the groups and a 95% confidence interval was built using the bootstrap approach. The Asymptotic Wilcoxon-Mann-Whitney Test was performed for miRNA combinations to compare different groups of donors. Benjamini-Hochberg correction was used to adjust the statistical significance for multiple comparisons. The most diagnostically significant miRNAs pairs were miR-107-miR-26b.5p and miR-375.3p-miR-26b.5p in the urine supernatant fraction that discriminated the group of healthy patients and PCa patients, as well as miR-31.5p-miR-16.5p, miR-31.5p-miR-200b, miR-31.5p-miR-30e.3p and miR-31.5p-miR-660.5p in the fraction extracellular vesicles that were different between healthy men and benign prostate hyperplasia patients. Such statistical criteria as the occurrence of individual significant miRNA pairs in the total number of comparisons, median ΔCt difference, and confidence interval can be useful tools for determining reliable markers of PCa.

Protocol for miRNA isolation from biofluids.

MicroRNAs (miRNAs) have been identified as promising biomarkers in cancer and other diseases. Packaging of miRNAs into vesicles and complexes with proteins ensures their stability in biological fluids but also complicates their isolation. Conventional protocols used to isolate cell-free RNA are generally successful in overcoming these difficulties; however, they are costly, labor-intensive, or heavily reliant on the use of hazardous chemicals. Here we describe a protocol that is suitable for isolating miRNAs from biofluids, including blood plasma and urine. The protocol is based on precipitation of proteins, denaturation of miRNA-containing complexes with octanoic acid and guanidine isothiocyanate, and subsequent purification of miRNA on spin columns. The efficacy of miRNA extraction by phenol-chloroform extraction, miRCURY RNA isolation kit--biofluids (Exiqon), and the proposed protocol was compared by quantitative reverse-transcription PCR of miR-16 and miR-126. The proposed protocol was slightly more effective for isolating miRNA from plasma and significantly superior to the other two methods for miRNA isolation from urine. Spectrophotometry and SDS-PAGE data suggest that the disparity in performance between miRCURY Biofluids and the proposed protocol can be attributed to differences in precipitation mechanisms, as confirmed by the retention of different proteins in the supernatant.

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.

miRNAs and radiotherapy response in prostate cancer.

BACKGROUND: Gaining insight into microRNAs (miRNAs) and genes that regulate the therapeutic response of cancer diseases in general and prostate cancer (PCa) in particular is an important issue in current molecular biomedicine and allows the discovery of predictive miRNA targets. OBJECTIVES: The aim of this study was to analyze the available data on the influence of radiotherapy (RT) on miRNA expression and on miRNA involved in radiotherapy response in PCa. MATERIALS AND METHODS: The data used in this review were extracted from research papers and the DIANA, STRING, and other databases with a special focus on the mechanisms of radiotherapy PCa response and the miRNA involved and associated genes. RESULTS AND DISCUSSION: A search for miRNA prognostic and therapeutic effectiveness markers should rely on both the data of recent experimental studies on the influence of RT on miRNA expression and miRNAs involved in regulation of radiosensitivity in PCa and on bioinformatics resources. miRNA panels and genes targeted by them and involved in radioresponse regulation highlighted by meta-analysis and cross-analysis of the data in the present review have. CONCLUSION: Selected miRNA and gene panel has good potential as prognostic and radiotherapy effectiveness markers for PCa and, moreover, as radiotherapy effectiveness markers in other types of cancer, as the proposed model is not specific to PCa, which opens up opportunities for the development of a universal diagnostic system (or several intersecting systems) for oncology radiotherapy in general.

miRNAs and androgen deprivation therapy for prostate cancer.

Androgen deprivation therapy (ADT) is mainly used for the treatment of advanced, metastatic or recurrent prostate cancer (PCa). However, patients progress to ADT resistance and castration-resistant prostate cancer (CRPC) with a poor prognosis. Reliable validated markers of ADT resistance with proven clinical utility are necessary for timely correction of the therapy as well as for improvement of patient quality of life. MiRNAs involved in the ADT response and CRPC development via multiple mechanisms may act as biomarkers for patient outcomes. Available data on miRNAs associated with the ADT response (resistance and sensitivity) are summarized and analyzed in the manuscript, including analyses using bioinformatics resources. Molecular targets of miRNAs, as well as reciprocal relations between miRNAs and their targets, were studied using different databases. Special attention was dedicated to the mechanisms of ADT resistance and CRPC development, including testosterone, PI3K-AKT, VEGF pathways and associated genes. Several different approaches can be used to search for miRNAs associated with the ADT response, each of which focuses on the associated set of miRNAs - potential markers of ADT. The intersection of these approaches and combined analysis allowed us to select the most promising miRNA markers of the ADT response. Meta-analysis of the current data indicated that the selected 5 miRNAs (miRNAs - 125b, miR-21, miR-23b, miR-27b and miR-221) and 14 genes are involved in the regulation of key processes of CRPC development and represent the most promising predictors of the ADT response, further demonstrating their potential in combination therapy for advanced PCa.

Isolation of Extracellular Vesicles from Biological Fluids via the Aggregation-Precipitation Approach for Downstream miRNAs Detection.

Extracellular vesicles (EVs) have high potential as sources of biomarkers for non-invasive diagnostics. Thus, a simple and productive method of EV isolation is demanded for certain scientific and medical applications of EVs. Here we aim to develop a simple and effective method of EV isolation from different biofluids, suitable for both scientific, and clinical analyses of miRNAs transported by EVs. The proposed aggregation-precipitation method is based on the aggregation of EVs using dextran blue and the subsequent precipitation of EVs using 1.5% polyethylene glycol solutions. The developed method allows the effective isolation of EVs from plasma and urine. As shown using TEM, dynamic light scattering, and miRNA analyses, this method is not inferior to ultracentrifugation-based EV isolation in terms of its efficacy, lack of inhibitors for polymerase reactions and applicable for both healthy donors and cancer patients. This method is fast, simple, does not need complicated equipment, can be adapted for different biofluids, and has a low cost. The aggregation-precipitation method of EV isolation accessible and suitable for both research and clinical laboratories. This method has the potential to increase the diagnostic and prognostic utilization of EVs and miRNA-based diagnostics of urogenital pathologies.

The Influence of Radical Prostatectomy on the Expression of Cell-Free MiRNA.

MiRNAs of blood and urine have been shown to represent a convenient source of biomarkers for prostate cancer (PCa) diagnosis and assessment of the therapy effectiveness due to their high stability and representation and the low invasiveness of sample collection. Here, we studied the influence of radical prostatectomy (RP) on the expression of 12 cell-free miRNAs previously shown as potential markers of PCa (i.e., miR-19b, miR-22, miR-92a, miR-378, miR-425, miR-30e, miR-31, miR-125b, miR-200b, miR-205, miR-375 and miR-660). The relative expression of the miRNAs combined into 31 paired ratios was evaluated in the urine extracellular vesicles (EVs), clarified urine (CU) and blood plasma of healthy donors, pre- and post-RP samples of PCa patients. Nineteen miRNA ratios based on combinations of ten of the miRNAs (miR-19b, miR-30e, miR-31, miR-125b, miR-200b, miR-205, miR-375, miR-378, miR-425, and miR-660) were altered by RP. The comparative expression analysis of the cell-free miRNA ratios between healthy donors and PCa patients revealed miR-125b/miR-30e and miR-375/miR-30e as potential markers for evaluating therapeutic efficacy. MiR-378/miR-19b, miR-425/miR-19b, miR-200/miR-30e, miR-660/miR-30e, and miR-205/miR-30e had minor prognostic value but could be used to increase the steadiness of the diagnostic system. The urine EVs had the highest potential as a source of markers.

The Panel of 12 Cell-Free MicroRNAs as Potential Biomarkers in Prostate Neoplasms.

Prostate cancer is a global biological, medical, and social issue aggravated by the lack of reliable, highly specific, and sensitive non-invasive tests for diagnosis and staging of prostate cancer. One prospective source of biomarkers are the cell-free miRNAs present in various biological fluids. In the present study, we validated the diagnostic potential of cell-free miRNAs: miR-19b, miR-22, miR-92a, miR-378, miR-425, miR-30e, miR-31, miR-125b, miR-200b, miR-205, miR-375, and miR-660; we estimated the required sample size and the minimal miRNA set for a subsequent large-scale validation study. Relative expression of 12 miRNA combined in 31 ratios was investigated in three fractions of biological fluids (urine extracellular vesicles, clarified urine, and plasma) obtained from patients with prostate cancer (n = 10), benign prostate hyperplasia (n = 8), and healthy volunteers (n = 11). Eight of the miRNAs found in urine vesicles (miR-19b, miR-30e, miR-31, miR-92a, miR-125, miR-200, miR-205, and miR-660) showed great promise and when combined into six ratios (miR-125b/miR-30e, miR-200/miR-30e, miR-205/miR-30e, miR-31/miR-30e, miR-660/miR-30e, and miR-19b/miR-92a) could classify patients with prostate cancer, benign prostate hyperplasia, and healthy donors with 100% specificity, 100% sensitivity, and with a high degree of reliability for most donors.

Isolation and comparative study of cell-free nucleic acids from human urine.

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 RARbeta2 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.

Cell-surface-bound nucleic acids: Free and cell-surface-bound nucleic acids in blood of healthy donors and breast cancer patients.

Concentrations of extracellular DNA and RNA in the blood of healthy donors and patients with malignant and nonmalignant breast tumors were investigated. Cell-surface-bound extracellular DNA and RNA were detached by PBS-EDTA treatment or mild trypsin treatment of erythrocytes and leukocytes. In healthy donors, almost all extracellular nucleic acids (98%) are bound at the surface of blood cells. In the blood of cancer patients, extracellular nucleic acids were found in plasma and not at the cell surface. In patients with nonmalignant breast tumors, extracellular nucleic acids were found both at the surface of blood cells and in plasma. In healthy donors, the cell-surface-bound DNA is represented by 20-kbp DNA fragments and smaller fragments that varied in amounts in different fractions.

Release of nucleic acids by eukaryotic cells in tissue culture.

Extracellular nucleic acids in cultures of A431 and HeLa cells were investigated. The data obtained demonstrate the presence of high weight DNA and RNA in the extracellular medium. Temporal changes of extracellular nucleic acids levels in growth medium were investigated.

Investigation of tumor-derived extracellular DNA in blood of cancer patients by methylation-specific PCR.

The frequency of APC, RASSF1A, RARbeta, 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.

Extracellular circulating nucleic acids in human plasma in health and disease.

The concentration of extracellular DNA and RNA in blood plasma of healthy donors, trauma patients, patients with breast and lung cancer, nonmalignant breast tumors and nonmalignant lung diseases were estimated. Significant amounts of extracellular RNA were found in plasma of trauma patients. The concentration of DNA and RNA in plasma of trauma patients correlates with the extent of posttraumatic organ failure. Extracellular RNA was not found in the plasma of breast cancer patients and patients with nonmalignant breast tumors, whereas a very high concentration of extracellular RNA was found in patients with malignant and nonmalignant diseases of lung.

Deoxyribonuclease activity and circulating DNA concentration in blood plasma of patients with prostate tumors.

The DNase activity and circulating DNA (cirDNA) concentration in blood plasma of healthy donors, patients with chronic prostatitis, and patients with prostate tumors were analyzed. The concentration of the cirDNA from plasma was determined by PicoGreen fluorescent assay. DNase activity in blood was measured using the immunoassay based on the cleavage of a hapten-labeled 974-bp DNA substrate. The mean cirDNA concentration in the plasma of healthy donors was low (21 +/- 4 ng/mL total blood) and was accompanied by high DNase activity (0.17 +/- 0.04 U/mL blood). The mean cirDNA concentration was 90 ng/mL blood (10-234 ng/mL) in the patients with nonmalignant prostate tumors and 115 ng/mL blood (13-339 ng/mL) in those with prostate cancer. The mean DNase activity in blood plasma of the patients with prostate tumors was 0.06 U/mL blood (0-0.12 U/mL). The results obtained demonstrate that increased concentrations of cirDNA in blood of the patients with prostate tumors is accompanied by a decreased DNase activity, confirming our previous data that a low DNase activity in blood plasma of cancer patients is one reason for a high cirDNA concentration.

Methylation-specific sequencing of GSTP1 gene promoter in circulating/extracellular DNA from blood and urine of healthy donors and prostate cancer patients.

Hypermethylated promoters of cancer-related genes represent convenient targets for early cancer diagnosis and monitoring based on circulating/extracellular DNA (cir/exDNA) from human blood and urine. The frequency of detection of methylated tumor suppressor genes in plasma or urine samples is usually lower than in the samples of tumor tissue because of a low concentration of target DNA and potential polymorphism of cirDNA methylation. Sequencing of the methylated cir/exDNA of tumor suppression genes provides information about methylation of the cirDNA originating from the tumor cells, which is necessary for optimization of cancer diagnosis. In this work, by sequencing chemically converted cir/exDNA, we have studied the cytosine methylation profile of GSTP1 gene promoter (1001-1302, X08058) in the pool of cir/exDNA from the blood and urine of healthy men, prostate cancer (PCa) patients, and patients with benign prostatic hyperplasia (BPH). We demonstrated that the data on cir/exDNA methylation could be obtained from sequencing of the cir/exDNA from blood and urine. The DNA isolated from blood plasma and the eluates of blood cells and urine of each patient were characterized by the same methylation profile of the GSTP1 gene. The profile of GSTP1 gene methylation in the extracellular DNA of PCa patients differs from the profiles characteristic of healthy donors and patients with BPH.

Cell-surface-bound circulating DNA as a prognostic factor in lung cancer.

Since the mortality of lung cancer patients remains very high, development of prognostic methods essential for efficient therapy is an immediate task. This study was designed to assess the value of circulating DNA (cirDNA) in blood as a prognostic marker in patients with non-small cell lung cancer. The average concentration of cirDNA in plasma was shown to be similar in healthy donors and lung cancer patients. However, the concentration of cell-surface-bound circulating DNA (csb-cirDNA) in lung cancer patients is significantly lower than that found in healthy donors (P < 0.0001) and correlates with a poor prognosis of tumor disease. Quantification of the cell-surface-bound DNA in blood of untreated patients allows persons with a poor prognosis of tumor disease to be detected with 94% sensitivity and 50% specificity.

Circulating DNA in the blood of gastric cancer patients.

The concentration of cell-free DNA and promoter methylation status of the MGMT, p15, and hMLH1 genes were analyzed by a fluorescence-based assay and methylation-specific PCR (MSP) in the blood of gastric cancer patients (n= 20) and healthy subjects (n= 22). Gastric cancer patients were characterized by an increased concentration of circulating DNA in the plasma; the amount of cell-surface-bound DNA was not decreased compared with controls and amounted to 80 +/- 15% of the total circulating DNA. MSP analysis of three genes in the cell-surface-bound DNA permits the detection of gastric cancer patients with a sensitivity of 75% and a specificity of 54%. Thus, the cell-surface-bound DNA is a convenient source of DNA for MSP analysis of cancer-specific markers. The data on the presence of methylated DNA in plasma combined with the analysis of other cancer-related changes in DNA can significantly contribute to cancer diagnostics.