Although cryoablation of tumor tissue is known to induce systemic antitumor immune responses, the underlying mechanisms remain poorly understood, and therapeutic efficacy is often unpredictable. During cryoablation, the cryoprobe creates a temperature gradient, resulting in a central zone of lethal tissue destruction and a periphery characterized by sub-lethal injury. In this peripheral zone, cells undergo programmed cell death triggered by thermal shock and ischemia. A widely cited, yet debated, hypothesis suggests that these apoptotic events in the tumor periphery may suppress the antitumor immune response. The purpose of this study was to experimentally test this hypothesis. We utilized the murine breast cancer cell line (4T1-Luc) and a panel of human cell lines (MCF-7, BT-20, BT-474, MDA-MB-231, MDA-MB-453, HBL-100). Following validation via flow cytometry using Annexin V-AF488, Rhodamine 123, TMRE, PI, and 7-AAD, we established −7 °C and −80 °C as temperature conditions that predominantly induce apoptosis and necrosis, respectively. These conditions were used to prepare cryo-treated 4T1-Luc cells for the vaccination of syngeneic mice. Ten days post-immunization, the efficacy of the treatment was evaluated in subcutaneous solid tumor and lung metastasis models by monitoring tumor growth, quantifying tumor-specific antibodies, performing histological analysis of tumor and lung tissues, and quantifying lung metastases via PCR and luciferase assays. Contrary to the initial hypothesis, our findings demonstrate that tumor cells undergoing sub-lethal cryoablation do not acquire immunosuppressive characteristics. Instead, they promote systemic antitumor immunity. Although antitumor immunity induced by apoptotic and necrotic tumor cells was comparable in a subcutaneous tumor model, vaccination with necrotic tumor cells proved more effective than vaccination with apoptotic cells in controlling lung metastasis formation.

BackgroundThis study investigates the potential of extracellular vesicles (EVs) in skin regeneration and rejuvenation. EVs, nanoscale vesicles released by various cell types, play a crucial role in intercellular communication.ObjectiveTo reaffirm the pivotal role of blood-derived exosomes in intercellular communication and their potential for skin tissue regeneration, leveraging existing research, including human data, to advocate for exosomes as a viable cell-free therapy for skin health.MethodsThe study employs a novel isolation technique combining PEG and Dextran with ultracentrifugation to extract EVs from plasma. Characterization techniques, including NTA, AFM, Cryo-TEM, and FC, confirm the successful isolation and characterization of EVs.ResultsThe study demonstrates positive effects of blood-derived EVs on fibroblast proliferation, collagen, and elastin production in murine and human models. Despite advancements, challenges persist in obtaining consistent EVs quality and concentration. The findings support the clinical relevance of EVs in skin health and suggest potential applications for skin rejuvenation. Future research directions and study limitations are also discussed, contributing to the evolving understanding of EVs-based therapies.

Introduction. Absence of objective morphological signs of malignancy complicates the problem of preoperative differential diagnosis of nodular thyroid lesions with follicular structure. In ambiguous cases, selection of surgical treatment is justified but results of histological examination of operative material often show benign nature of the nodules. A possible solution for this problem is development of molecular diagnostic methods for this pathology. For example, the miR-THYROID test system is based on the differences in expression patterns of regulatory microRNAs (miRNAs) in follicular adenoma cells and follicular thyroid cancer cells. Aim. To develop and validate a set of reagents (miR-THYROID) for differential diagnosis of follicular adenoma and follicular thyroid cancer using reverse transcription with subsequent polymerase chain reaction (RT-PCR). Materials and methods. Selection of potential marker molecules (n = 53) is based on the results of previous study of histologically verified samples of follicular adenoma and follicular thyroid cancer using high-throughput sequencing. In this study, a test system based on RT reaction with two-tailed primer and quantitative PCR with TaqMan probe was developed. To evaluate analytical characteristics of this technology, synthetic analogues of miRNA molecules were used; for analysis of diagnostic characteristics of the method, cytology samples of follicular adenoma (n = 20) and follicular thyroid cancer (n = 20) were used. Results. Studies of analytical characteristics of RT-PCR system and concentration of potential marker molecules in cytology samples were performed which allowed to shorten the list of marker miRNAs from 53 to 7 (hsa-miR-15a-5p, -20-5p, -24-3p, -106b-5p, -143-3p, -146b-5p, -192-5p). Algorithm of RT-PCR result analysis was developed based on calculation of molecule concentration ratios relative to oppositely directed follicular cancer-associated changes in expression, combination of these results, and calculation of the miR-T diagnostic parameter. miR-THYROID test system based in RT-PCR technology allowed to differentiate follicular adenoma from follicular cancer with sensitivity 89.47 % and specificity 90 %, positive prognostic significance 89.47 %, negative prognostic significance 90 %, and accuracy 89.74 %. Conclusion. Test system miR-THYROID based on 7 microRNA molecules was validated on 40 samples (20 samples of follicular adenoma and 20 samples of follicular cancer) and demonstrated high diagnostic significance.

Background: Synthetic DNA aptamers are a class of molecules with potential applications in medicine, serving as molecular sensors or ligands for targeted drug delivery. Systematic evolution of ligands by exponential enrichment (SELEX) is a technology for selecting functional aptamers that was first reported three decades ago and has been actively developed since. SELEX involves multiple iterations of two fundamental steps: (i) target affinity-based partitioning of aptamers from a random library and (ii) amplification of selected aptamers by PCR, followed by isolation of single-stranded DNA (ssDNA). SELEX protocols have diversified considerably, with numerous variations possible for each step. This heterogeneity makes it challenging to identify optimal methods. Comparative analysis of different approaches for the major stages of SELEX is therefore of considerable practical importance. Methods: Four widely used methods for ssDNA generation were performed in parallel: (a) PCR followed by digestion of the antisense strand with exonuclease lambda, (b) PCR with an extended primer followed by size-dependent strand separation using denaturing PAGE, (c) asymmetric PCR, and (d) asymmetric PCR with a primer-blocker. Results: The specificity, efficiency, reproducibility, and duration of each method were compared. Conclusions: Asymmetric PCR with a primer-blocker yielded the most favorable results.

Введение. Дооперационная дифференциальная диагностика узловых образований щитовидной железы, имеющих фолликулярную структуру, является не тривиальной задачей в силу отсутствия объективных признаков злокачественности. Это стимулирует исследования с целью поиска молекулярных маркеров фолликулярного рака и разработку диагностических тест-систем. Малые регуляторные РНК — группа молекул, выполняющих разные биологические функции, но имеющих сходную биохимическую структуру. Количественный анализ разных представителей этой группы может быть проведен одновременно с помощью идентичной технологии. Это определяет возможность комплексной оценки биологии клеток в составе анализируемого биоптата и разработки новых диагностических критериев. Целью исследования был сравнительный анализ профиля экспрессии малых РНК в клетках фолликулярной карциномы и фолликулярной аденомы щитовидной железы. Материалы и методы. В исследование были включены образцы ткани фолликулярной карциномы (ФК, n = 12) и доброкачественной фолликулярной аденомы (ФА, n = 12) щитовидной железы, полученные после проведения тиреоидэктомии и гистологического исследования. Анализ профиля экспрессии коротких РНК был проведен методом глубокого секвенирования. Результаты. Экспрессия (концентрация) транспортных РНК (tRNA), малых ядерных РНК (snRNA) и большой группы неклассифицированных молекул (miscRNA) повышена в клетках фолликулярной карциномы щитовидной железы, по сравнению с клетками фолликулярной аденомы, но диагностический потенциал отдельных молекул относительно невысокий. Тотальные концентрации молекул микроРНК (miRNA) оказались сопоставимы в группах ФК и ФА, анализ «реципрокных пар» маркерных молекул позволяет дифференцировать ФК и ФА с высокой степенью вероятности (AUC: 0,94-0,98). Тотальное количество молекул пиРНК (piwiRNA) оказались несколько выше в образцах ФК, анализ «реципрокных пар» маркерных молекул позволяет уверенно дифференцировать ФК и ФА (AUC: 1,00). Выводы. Анализ концентрации маркерных молекул микроРНК (miRNA) и пиРНК (piwiRNA) в материале тонкоигольной аспирационной биопсии представляется перспективным методом дополнительной диагностики узловых образований щитовидной железы, имеющих фолликулярную структуру.

Purpose of the study. Identification of potential miRNA markers in material of focal pancreatic lesions. Materials and methods . Samples of focal pancreatic lesions after histological evaluation were enrolled in the study including chronic pancreatitis (ChP) (n = 23), low-grade pancreatic intraepithelial neoplasia /PanIN‑1/2 (n = 19), high-grade pancreatic intraepithelial neoplasia /PanIN‑3 (n = 8), and invasive pancreatic ductal adenocarcinoma PDAC (n = 26). Workflow of research included the profiling of cancer-associated miRNA in pooled samples, the selection of potential marker miRNAs, the assessment of selected miRNAs expression in total collection of specimens, the identification of differentially expressed miRNAs, and the approbation of new algorithm of data interpretation via ratio of “reciprocal miRNA pair”. Consequent reactions of revers transcription and quantitative teal-time PCR were used. Results . The expression levels of miR‑216a and miR‑217 were decreased in the following order: PanIN‑1/2 > PanIN‑3 > PDAC. Moreover, miR‑375 was up-regulated while miR‑143 was down-regulated in the PDAC. Differential diagnostics of PDAC versus focal chronic pancreatitis might be performed with high accuracy (AUC > 0.95) by assessment panel of four molecules: miR‑216a, miR‑217, miR‑1246 and Let‑7a. Conclusion . The assessment of microRNAs in pancreatic lesions is a promising approach for the differential diagnosis of PDAC, but this technology requires further validation with an increase in the number of samples.

Aim: Neo-adjuvant chemotherapy is a common approach for the complex treatment of breast cancer (BC) and paclitaxel (PTX) is frequently included in the therapeutic regimen. However, the effect of PTX-based treatment is hard to predict precisely based on routinely used markers. As microRNAs are considered a new promising class of biomarkers, the link between miRNA expression and PTX resistance of BC cells needs to be well investigated. This study aimed at the identification of miRNAs associated with responses of BC cells to PTX. Methods: Intrinsic PTX sensitivity and miRNA profiling were assayed in five BC cell lines to identify candidate miRNAs. Selected miRNA (n. 15) expressions were analyzed by real-time-quantitative polymerase chain reaction (RT-qPCR) in BC tissue samples (n. 31) obtained from a diagnostic biopsy. Results were analyzed in the context of the effect of two cycles of PTX and the effect of the completed scheme of neoadjuvant therapy. The study's design facilitated the evaluation of the effect of PTX on cells and the identification of features of the microRNA expression profiles associated exclusively with sensitivity to this drug. Results: miR-186 and miR-7 expression in BC tissues was higher in patients with better outcomes of PTX-based neoadjuvant therapy. Conclusion: High expressions of miR-186 and miR-7 are associated with good response to PTX, whereas their low expressions may be associated with resistance to PTX in BC, indicating the possibility of developing innovative test systems for the prediction of the PTX response, which can be used before the start of neo-adjuvant chemotherapy for BC.

Cervical cancer is one of the most common gynecological malignancies and it is preventable through the yearly diagnosis and management of pre-cancerous cervical disease. The profile of miRNA expression in cervical epithelium cells is altered with cervical dysplasia development and further progression. The NOVAprep-miR-CERVIX is a new approach for the assessment of cervical dysplasia through the analysis of six marker miRNAs. This study aims to evaluate theperformance and diagnostic potency of the new method. Cytological smears from 226 women (NILM, n.114; HSIL, n.112) were included in the study. A VPH test was performed with RealBest DNAHPV HR screen Kit, six marker miRNAs (miR-21,-29b,-145,-451a,-1246,-1290) were assayed using NOVAprep-miRCERVIX kit. Obtained data were analyzed using the Delta Ct method and random forest machine learning algorithm. The results of the quantitative analysis of six microRNAs were expressed as a miR-CERVIX parameter, which ranged from 0 to 1, where "0" corresponded to the healthy cervical epithelium, while "1" corresponded to high-grade squamous intraepithelial dysplasia. The average value of miR-CERVIX differed in groups of NILM and HSIL samples (0.34 vs. 0.72; p < 0.000005). An estimation of miR-CERVIX allowed for the differentiation between healthy and pre-cancerous samples with sensitivity of 0.79 and specificity of 0.79, as well as to confirm HSIL with specificity of 0.98. Interestingly, the HSIL group included HPV(+) and HPV(-) samples, which were statistically significantly different in terms of miR-CERVIX value. Analysis of CC-associated miRNAs in material of cervical smear might serve as an additional method for the evaluation of cervical dysplasia severity.

BACKGROUND:The development of new non-invasive markers for prostate cancer (PC) diagnosis, prognosis, and management is an important issue that needs to be addressed to decrease PC mortality. Small extracellular vesicles (SEVs) secreted by prostate gland or prostate cancer cells into the plasma are considered next-generation diagnostic tools because their chemical composition might reflect the PC development. The population of plasma vesicles is extremely heterogeneous. The study aimed to explore a new approach for prostate-derived SEV isolation followed by vesicular miRNA analysis.METHODS:We used superparamagnetic particles functionalized by five types of DNA-aptamers binding the surface markers of prostate cells. Specificity of binding was assayed by AuNP-aptasensor. Prostate-derived SEVs were isolated from the plasma of 36 PC patients and 18 healthy donors and used for the assessment of twelve PC-associated miRNAs. The amplification ratio (amp-ratio) value was obtained for all pairs of miRNAs, and the diagnostic significance of these parameters was evaluated.RESULTS:The multi-ligand binding approach doubled the efficiency of prostate-derived SEVs' isolation and made it possible to purify a sufficient amount of vesicular RNA. The neighbor clusterization, using three pairs of microRNAs (miR-205/miR-375, miR-26b/miR375, and miR-20a/miR-375), allowed us to distinguish PC patients and donors with sensitivity-94%, specificity-76%, and accuracy-87%. Moreover, the amp-ratios of other miRNAs pairs reflected such parameters as plasma PSA level, prostate volume, and Gleason score of PC.CONCLUSIONS:Multi-ligand isolation of prostate-derived vesicles followed by vesicular miRNA analysis is a promising method for PC diagnosis and monitoring.

Introduction/Background Cervical cancer (CC) is one of the most common types of cancer and the fourth leading cause of cancer-related deaths in women. Cervical carcinogenesis is multistep process of the cervical dysplasia development and progression. Correct diagnostic and effective therapy of cervical dysplasia presents an important approach to reduce CC morbidity and mortality. MicroRNAs in cervical epithelium cells are considered as promising diagnostic markers of cervical dysplasia. MicroRNA-based diagnostic test-systems can provide a helpful addition to traditional diagnostic techniques. Methodology NOVAprep-miR-CERVIX is a new test-system based on RT-qPCR analysis of six miRNAs (miR-21–5p; miR-29b-3p; miR-145–5p; miR-451a-5p; miR-1246–5p and miR-1290–3p) in material of cervical smear. Test-system includes quality of material control and control of enzymatic reaction efficacy. Machine learning based of random forest algorithm was applied for RT-qPCR results evaluation. Cervical smear samples were obtained from 226 women: 114 samples of normal epithelium and 112 samples of cervical epithelium with high-grad intraepithelial lesion (HSIL) or carcinoma in situ (CIS) as a result of cytological evaluation. Moreover, any of HSIL/CIS diagnosis was confirmed histologically. Results The 38 samples from 216 (17.8%) did not pass quality controls and were excluded from analysis. NOVAprep-miR-CERVIX Index (miR-CERVIX-I varied from 0 to 1) was calculated on the base of results of six miRNA analysis for remaining 178 samples. Difference in miR-CERVIX-I was statistically significant in two groups of samples formed on the base of cytological/histological diagnosis (figure 1). Normal condition of cervical epithelium (miR-CERVIX-I < 0.49) was diagnosed with sensitivity 79.2%, specificity 80.46%. HSIL was diagnosed with sensitivity 70.83%, specificity 97.22% (miR-CERVIX-I > 0.78). Moreover, intermediate value of miR-CERVIX-I (between 0.5 and 0.77) is supposed to reflect condition of low-grade intraepithelial dysplasia. Conclusion NOVAprep-miR-CERVIX can be applied for cervical dysplasia diagnostic and management as a test system complimentary to standard methods.

Background. Heat stress (HS) induces the cellular secretion of heat shock proteins (HSP ) and extracellular nanovesicles (ENVs). The biological link between these phenomena is poorly understood. In the case of colorectal cancer (CRC) cells, the secretion of HSP s and ENV may be involved in the clinical response to intraperitoneal therapy of peritoneal carcinomatosis.Material and Methods. Established colon cancer cell lines COLO 320, HCT 116, HT29 and DLD 1 were used. ENVs were isolated from culture media by differential ultra-centrifugation and analyzed by dynamic light scattering, nanoparticle tracking analysis, atomic force microscopy and flow cytometry. Super-paramagnetic particles (SPMP ) covered by antibodies to the membrane form of Hsp70 were used for isolation and quantification of Hsp70(+) ENVs. Vesicular microRNA was assayed by RT-qPC R.Results. HS induces the secretion of ENVs by CRC cells, the resistance to HS correlates with the activity of HS-induced ENVs secretion. HS induces the secretion of a specific population of ENVs enriched by membrane form Hsp70 (mHsp70). The microRNA content of mHsp70(+) ENVs has qualitative and quantitative features. The concentration of miR-126-3p, -181-5p, -155-5p, -223 is increased in mHSP 70(+) ENVs secreted by three CRC cell lines.Conclusion. HS induces the secretion of mHSP 70(+) ENVs by CRC cells. This phenomenon may be involved in a clinical response to intraperitoneal chemo-hyperthermic perfusion therapy of peritoneal carcinomatosis.

A liquid biopsy based on circulating small extracellular vesicles (SEVs) has not yet been used in routine clinical practice due to the lack of reliable analytic technologies. Recent studies have demonstrated the great diagnostic potential of nanozyme-based systems for the detection of SEV markers. Here, we hypothesize that CD30-positive Hodgkin and Reed–Sternberg (HRS) cells secrete CD30 + SEVs; therefore, the relative amount of circulating CD30 + SEVs might reflect classical forms of Hodgkin lymphoma (cHL) activity and can be measured by using a nanozyme-based technique. A AuNP aptasensor analytics system was created using aurum nanoparticles (AuNPs) with peroxidase activity. Sensing was mediated by competing properties of DNA aptamers to attach onto surface of AuNPs inhibiting their enzymatic activity and to bind specific markers on SEVs surface. An enzymatic activity of AuNPs was evaluated through the color reaction. The study included characterization of the components of the analytic system and its functionality using transmission and scanning electron microscopy, nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and spectrophotometry. AuNP aptasensor analytics were optimized to quantify plasma CD30 + SEVs. The developed method allowed us to differentiate healthy donors and cHL patients. The results of the CD30 + SEV quantification in the plasma of cHL patients were compared with the results of disease activity assessment by positron emission tomography/computed tomography (PET-CT) scanning, revealing a strong positive correlation. Moreover, two cycles of chemotherapy resulted in a statistically significant decrease in CD30 + SEVs in the plasma of cHL patients. The proposed AuNP aptasensor system presents a promising new approach for monitoring cHL patients and can be modified for the diagnostic testing of other diseases.

Introduction. Testicular germ cell tumor is a relatively rare disease. Its high social significance is due to the fact that this pathology occurs in young patients. The standard schemes of polychemotherapy determine the potential possibility of effective treatment for most of the patients even with an advanced disease. Several circulating markers (alpha-fetoprotein, human chorionic gonadotropin and lactate dehydrogenase) are being used for therapy monitoring, but the low diagnostic specificity of these molecules determines the need to develop new approaches. Over the past years, circulating microRNA, for instance miR-371a-3p, appeared to be promising marker for testicular germ cell tumor monitoring. However, to develop and to implement in practice the microRNA-based diagnostic technologies, it’s necessarily to understand the features of the microRNA expression alterations specific for different histological types of testicular germ cell tumor.The study objective – to evaluate changes in the expression of several potential marker microRNA molecules (miR-302/ miR-367, miR-371/miR-373) in testicular germ cell tumor samples of various histological types.Materials and methods. Testicular germ cell tumor samples (n = 61), including seminomas, embryonic carcinomas, post-pubertal teratomas, yolk sac tumors, chorioncarcinomas, and corresponding normal tissue samples (n = 61) were included in the study. The analysis of selected miRNA expression was performed by reverse transcription and polymerase chain reaction.Results. We identified the changes in the expression profile of the miR-302/miR-367 cluster typical for semines, embryonic carcinomas, post-pubertal teratomas, yolk sac tumors and chorioncarcinomas, as well as changes in the expression profile of the miR-371/miR-373 cluster, universal for all histotypes except chorioncarcinomas. Inhibition of miR-10b and miR-145 expression in semines, embryonic carcinomas, and post-pubertal teratomas was demonstrated.Conclusion. Activation of miR-302b, miR-302d, miR-371a expression and inhibition of miR-10b, miR-145 expression in the tissue of the most common variants of testicular germ cell tumor is a characteristic feature of these tumors. The detected changes are significant and can lead to corresponding changes in the profile of circulating microRNAs.

Vesicular miRNA has emerged as a promising marker for various types of cancer, including prostate cancer (PC). In the advanced stage of PC, the cancer-cell-derived small extracellular vesicles (SEVs) may constitute a significant portion of circulating vesicles and may mediate a detectable change in the plasma vesicular miRNA profile. However, SEVs secreted by small tumor in the prostate gland constitute a tiny fraction of circulating vesicles and cause undetectable miRNA pattern changes. Thus, the isolation and miRNA profiling of a specific prostate-derived fraction of SEVs can improve the diagnostic potency of the methods based on vesicular miRNA analysis. Prostate-specific membrane antigen (PSMA) was selected as a marker of prostate-derived SEVs. Super-paramagnetic beads (SPMBs) were functionalized by PSMA-binding DNA aptamer (PSMA–Apt) via a click reaction. The efficacy of SPMB–PSMA–Apt complex formation and PSMA(+)SEVs capture were assayed by flow cytometry. miRNA was isolated from the total population of SEVs and PSMA(+)SEVs of PC patients (n = 55) and healthy donors (n = 30). Four PC-related miRNAs (miR-145, miR-451a, miR-143, and miR-221) were assayed by RT-PCR. The click chemistry allowed fixing DNA aptamers onto the surface of SPMB with an efficacy of up to 89.9%. The developed method more effectively isolates PSMA(+)SEVs than relevant antibody-based technology. The analysis of PC-related miRNA in the fraction of PSMA(+)SEVs was more sensitive and revealed distinct diagnostic potency (AUC: miR-145, 0.76; miR-221, 0.7; miR-451a, 0.65; and miR-141, 0.64) than analysis of the total SEV population. Thus, isolation of prostate-specific SEVs followed by analysis of vesicular miRNA might be a promising PC diagnosis method.

The quantification of the specific disease-associated populations of circulating extracellular membrane nanovesicles (ENVs) has opened up new opportunities for liquid biopsy in cancer and other chronic diseases. However, the sensitivity of such methods is mediated by an optimal combination of the isolation and labeling approaches, and is not yet sufficient for routine clinical application. The presented study aimed to develop, characterize, and explore a new approach to non-specific ENV staining, followed by size-exclusive chromatography (SEC), which allows us to increase the sensitivity of bead-assisted flow cytometry. Plasma from healthy donors was purified from large components, stained with lipophilic CM-Dil dye, and fractionated by means of SEC. The obtained fractions were analyzed in terms of particle size and concentration using NTA, as well as vesicular markers and plasma protein content via dot-blotting. We characterized the process of CM-Dil-stained plasma fractionation in detail and indicated the fractions with optimal characteristics. Finally, we explored the sensitivity of on-bead flow cytometry for the analysis of specific populations of plasma ENVs and demonstrated the advantages and limitations of the proposed technique.

Background . MicroRNAs (miRNAs) circulating in plasma are promising markers for the diagnosis of malignant tumors, including prostate cancer. However, the existing techniques used for their detection fail to ensure sufficient diagnostic accuracy. One of the possible ways to improve it is to isolate membrane nano-sized extracellular vesicles (nsEVs) secreted by prostate cells. Presumably, the analysis of miRNAs originating from this prostate-specific fraction of nsEVs more accurately reflects the process of prostate cancer development and has a greater diagnostic potential. Objective: to develop the method of miRNA isolation from the prostate-specific fraction of plasma nsEVs and to evaluate its performance characteristics. Materials and methods . Prostate-specific membrane antigen (PSMA) was used as a prostate-specific marker of nsEVs. The total population of plasma nsEVs was isolated using a two-phase polymer system. To isolate PSMA-positive (PSMA (+) ) nsEVs, we used superparamagnetic particles with PSMA-binding DNA aptamer immobilized on their surface. The efficacy of PSMA (+) nsEV isolation was assessed using flow cytometry and dot-blotting. RNA from nsEVs was isolated using proteolysis; miRNA analysis was performed using reverse transcription polymerase chain reaction. Plasma samples collected from patients with prostate cancer ( n = 33) and healthy donors (controls) ( n = 30) were used to evaluate the diagnostic parameters of the method. Results . We developed the method of PSMA (+) nsEV isolation from plasma and estimated its performance characteristics. We found that measurement of potential miRNA markers in PSMA (+) nsEVs was more effective than its measurement in the entire nsEV population and could distinguish between patients with prostate cancer and controls. Conclusion . The new technique of PSMA (+) nsEV isolation can be used for the development of novel diagnostic methods for the diagnosis of prostate cancer.

Introduction: Malignant transformation of follicular epithelium of the thyroid gland is associated with specific alterations of miRNA profile. Evaluation of miRNAs expression changes is being applied for primary or differential diagnostic of thyroid nodes. Anaplastic thyroid cancer (ATC) is relatively rare form of thyroid cancer with high malignant potency and rate of lethality. Investigation of miRNAs role in ATC might provide with soul for development of new diagnostic and therapeutic approaches. Goal: To analyze expression profile of miRNA in ATC and to identify miRNAs involved in pathogenesis of ATC. Material and Methods: Samples of ATC (n.20) and normal thyroid tissue (n. 22) were included in the study, expression levels of 85 cancer-associated miRNAs were analyzed by RT-PCR. Results: Expression of miR-375, miR-1246 and miR-21 is activated while expression of miR-Let7b, miR-125b and miR-181a is suppressed in cells of ATC. Conclusions: Further investigation of miRNA involvement into carcinogenesis of ATC is needed for development of new diagnostic and therapeutic approaches.

Background. Currently, there are no reliable markers for the prognosis of the low-grade squamous intraepithelial lesion (LSIL) of the cervical epithelium. Scientific literature provides with inconsistent recommendations regarding the management of the young patients with a cytological diagnosis of LSIL. The progression of cervical dysplasia and the development of cervical cancer are associated with characteristic alterations of the microRNA expression profile.Objective: to assess the prognostic value of microRNA in LSIL. Materials and methods. Samples (cytological smears) obtained from patients diagnosed with LSIL (n = 36), but with a different course of the disease over the next 6–12 months. Analysis of miRNA expression was carried out by the method of “two-tailed” reverse transcription and subsequent PCR.Results. The expression level of miR-126, miR-21, miR-1246, miR-182 was statistically significantly different in the compared groups, but the predictive value of the analysis of individual molecules was low (AUC <0.65). Calculation of the concentration ratios of the “reciprocal” pairs of microRNAs made it possible to obtain a more effective prognostic marker. ROC analysis of such ratios (miR-126/miR-182, miR-21/miR-182, miR-1246/miR-182) yielded AUC values: 0.82–0.89, sensitivity: 0.71–0.92; specificity: 0.86.Conclusions. Analysis of a panel of microRNA marker molecules in the material of the cervical epithelium and calculation of the concentration ratios of “reciprocal” pairs is a promising method for prognosis of LSIL course.

Introduction: Malignant transformation of follicular epithelium of the thyroid gland is associated with specific alterations of miRNA profile. Evaluation of miRNAs expression changes is being applied for primary or differential diagnostic of thyroid nodes. Anaplastic thyroid cancer (ATC) is relatively rare form of thyroid cancer with high malignant potency and rate of lethality. Investigation of miRNAs role in ATC might provide with soul for development of new diagnostic and therapeutic approaches. Goal: To analyze expression profile of miRNA in ATC and to identify miRNAs involved in pathogenesis of ATC. Material and Methods: Samples of ATC (n.20) and normal thyroid tissue (n. 22) were included in the study, expression levels of 85 cancer-associated miRNAs were analyzed by RT-PCR. Results: Expression of miR-375, miR-1246 and miR-21 is activated while expression of miR-Let7b, miR-125b and miR-181a is suppressed in cells of ATC. Conclusions: Further investigation of miRNA involvement into carcinogenesis of ATC is needed for development of new diagnostic and therapeutic approaches.

Background: The current approaches to distinguish follicular adenomas (FA) and follicular thyroid cancer (FTC) at the pre-operative stage have low predictive value. Liquid biopsy-based analysis of circulating extracellular vesicles (EVs) presents a promising diagnostic method. However, the extreme heterogeneity of plasma EV population hampers the development of new diagnostic tests. We hypothesize that the isolation of EVs with thyroid-specific surface molecules followed by miRNA analysis, may have improved diagnostic potency. Methods: The total population of EVs was isolated from the plasma of patients with FA (n = 30) and FTC (n = 30). Thyroid peroxidase (TPO)-positive EVs were isolated from the total populations using immune-beads. The miRNA from the TPO(+)EVs obtained from the plasma of FA and FTC patients was assayed by RT-PCR. The diagnostic potency of the selected miRNAs was estimated by the receiver operating characteristic (ROC) analysis. Results: TPO(+)EVs can be efficiently isolated by immunobeads. The analysis of Let-7 family members in TPO(+)EVs allows one to distinguish FA and FTC with high accuracy (area under curve defined by ROC = 0.77–0.84). Conclusion: The isolation of TPO(+)EVs, followed by RT-qPCR analysis of Let-7 family members, may present a helpful approach to manage follicular nodules in the thyroid gland.