The Search for and Functional Analysis of Genetic Variants in microRNA-Binding Sites using Massively Parallel Reporter Assay.

A large-scale search for the genetic variants with a bias in the representation of alleles in transcriptome data (AE SNPs) and the binding sites in microRNA 3'-UTRs was performed and their functional significance was assessed using massively parallel reporter assay (MPRA). Of the 629,559 associated "SNP-gene" pairs (eQTLs) discovered in the human liver tissue according to the GTEx Analysis V8 data, 4394 polymorphic positions in the 3'-UTRs of the genes, which represent the eQTLs for these genes were selected. The TargetScanHuman 7.0 algorithm and PolymiRTS database were searched for the potential microRNA-binding sites. Of the predicted microRNA sites affected by eQTL-SNPs, we selected 51 sites with the best evidence of functionality according to Ago2-CLIP-seq, CLEAR-CLIP, and eCLIP-seq for RNA-binding proteins. For MPRA, a library of the plasmids carrying the main and alternative alleles for each AE SNP (in total, 102 constructs) was created. Allele-specific expression for 6 SNPs was detected by transfection of the HepG2 cell line with the constructed plasmid library and sequencing of target DNA and RNA sequences using the Illumina (MiSeq) platform.

MIRA analysis of RARß2 gene methylation in DNA circulating in the blood in lung cancer.

Analysis of DNA epigenetic mutations in the blood circulating DNA is a prospective trend for creation of noninvasive methods for the diagnosis and treatment efficiency monitoring in cancer. The methylation status of target genes in circulating DNA was evaluated by methods based on preliminary bisulfite conversion of DNA. We used a different approach based on selection of hypermethylated sequences of circulating DNA by means of DNA-methyl-binding protein (methylated CpG island recovery assay, MIRA). Methylation was evaluated for RARß2 tumor suppression gene in circulating DNA in lung cancer and a trend was detected to higher methylation of this gene in the patients in comparison with healthy donors.

Data analysis algorithm for the development of extracellular miRNA-based diagnostic systems for prostate cancer.

Urine of prostate cancer (PCa) carries miRNAs originated from prostate cancer cells as a part of both nucleoprotein complexes and cell-secreted extracellular vesicles. The analysis of such miRNA-markers in urine can be a convenient option for PCa screening. The aims of this study were to reveal miRNA-markers of PCa in urine and design a robust and precise diagnostic test, based on miRNA expression analysis. The expression analysis of the 84 miRNAs in paired urine extracellular vesicles (EVs) and cell free urine supernatant samples from healthy donors, patients with benign and malignant prostate tumours was done using miRCURY LNA miRNA qPCR Panels (Exiqon, Denmark). Sets of miRNAs differentially expressed between the donor groups were found in urine EVs and urine supernatant. Diagnostically significant miRNAs were selected and algorithm of data analysis, based on expression data on 24-miRNA in urine and obtained using 17 analytical systems, was designed. The developed algorithm of data analysis describes a series of steps necessary to define cut-off values and sequentially analyze miRNA expression data according to the cut-offs to facilitate classification of subjects in case/control groups and allows to detect PCa patients with 97.5% accuracy.

[Representation analysis of miRNA from clarified urine and urine microvesicles in prostate malignancies and non-malignant neoplasms]. / Analiz predstavlennosti mikroRNK v mikrovezikulakh mochi i beskletochnoi moche pri zabolevaniiakh predstatel'noi zhelezy.

Urine of prostate cancer patients contains tumor-specific biopolymers, including protein- and microvesiclesassociated miRNAs that can potentially be used as oncomarkers. Previously we have characterized urine extracellular vesicles and demonstrated diagnostic potential of their miRNA cargo. In this study, we have performed a comparative analysis of the expression of 84 miRNA in paired samples of urine microvesicles and clarified urine from healthy men, patients with benign hyperplasia and cancer of the prostate using miRCURY LNA miRNA qPCR Panels. Subsets of miRNAs with differences in expression between the fractions of the urine were found in all three groups. Two groups of miRNA were identified based on the patterns of their differential expression. They regulate several key signaling pathways associated with prostate cancer development.

Cell surface oligonucleotide-binding proteins of human squamous carcinoma A431 cells.

Affinity modified with Flu-DAP-p(N)16degU oligonucleotide-binding proteins were isolated by affinity chromatography using Ultrogel A2-anti fluorescein antibodies. After separation by SDS-PAGE the proteins with molecular masses about 68 kDa were MS/MS sequenced and identified as keratin K1, keratin K10, keratin K2e and albumin.

Extracellular DNA in culture of primary and transformed cells, infected and not infected with mycoplasma.

The composition and kinetics of accumulation of extracellular DNA in cultures of primary human endotheliocytes, cervical adenocarcinoma, and mycoplasma-infected cervical adenocarcinoma cells were studied. The content of DNA bound to cell surface did not change during culturing. The concentration of extracellular DNA in culture medium increased during the lag phase and at the beginning of the exponential growth phase, which probably attests to active secretion of DNA by cells. Spontaneous extracellular DNA synthesis was observed only in cell culture infected with mycoplasma.

Plasma content of extracellular nucleic acids in donors and patients with mammary tumors.

The concentrations of extracellular DNA and RNA were measured in the plasma of donors and patients with fibroadenoma and breast cancer. The content of extracellular DNA surpassed the normal in 80% plasma samples from patients with mammary tumors. Extracellular RNA was detected in 30% plasma samples from donors and patients with breast tumors. No correlations were found between plasma concentration of extracellular DNA and size and stage of tumor growth. Hence, measurement of extracellular DNA in the plasma of patients can be used only as an accessory test for tumor diagnosis.

Interaction of keratin K1 with nucleic acids on the cell surface.

The interaction of surface proteins from A431 cells and cellular extracts with nucleic acids was investigated using affinity modification with 32P-labeled reactive oligonucleotide derivatives. Proteins with molecular weights of 68, 46, 38, and 28 kD as well as several low molecular weight proteins capable of binding to nucleic acids were found on the surface of intact cells. It was demonstrated that a protein with molecular weight of 68 kD is exposed at the cell surface, since the treatment of cells with trypsin results in the cleavage of this protein. Disruption of the integrity of the cell membrane (scrapping, treatment with trypsin, or permeabilization of the cell membrane with streptolysin O or saponin) disrupts the interaction of the reactive oligonucleotides with the cell surface proteins. Affinity modification of the cytosolic and membrane-cytosolic cell fractions with labeled oligonucleotides results in the modification of a large number of proteins, where proteins with molecular weights of 68, 46, 38, and 28 kD can be found as minor components. Surface oligonucleotide-binding proteins with molecular weight of ~68 kD were isolated by affinity chromatography after the modification of intact A431 cells with a reactive oligonucleotide derivative. The isolated surface oligonucleotide-binding proteins from A431 cells were sequenced, and one of the proteins was identified as keratin K1.