NOVAprep-miR-Cervix: New Method for Evaluation of Cervical Dysplasia Severity Based on Analysis of Six miRNAs.

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-miR-CERVIX 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.

Reciprocal Dysregulation of MiR-146b and MiR-451 Contributes in Malignant Phenotype of Follicular Thyroid Tumor.

Over the last few years, incidental thyroid nodules are being diagnosed with increasing frequency with the use of highly sensitive imaging techniques. The ultrasound thyroid gland examination, followed by the fine-needle aspiration cytology is the standard diagnostic approach. However, in cases of the follicular nature of nodules, cytological diagnosis is not enough. Analysis of miRNAs in the biopsy presents a promising approach. Increasing our knowledge of miRNA's role in follicular carcinogenesis, and development of the appropriate the miRNA analytical technologies are required to implement miRNA-based tests in clinical practice. We used material from follicular thyroid nodes (n.84), grouped in accordance with their invasive properties. The invasion-associated miRNAs expression alterations were assayed. Expression data were confirmed by highly sensitive two-tailed RT-qPCR. Reciprocally dysregulated miRNAs pair concentration ratios were explored as a diagnostic parameter using receiver operation curve (ROC) analysis. A new bioinformatics method (MiRImpact) was applied to evaluate the biological significance of the observed expression alterations. Coupled experimental and computational approaches identified reciprocal dysregulation of miR-146b and miR-451 as important attributes of follicular cell malignant transformation and follicular thyroid cancer progression. Thus, evaluation of combined dysregulation of miRNAs relevant to invasion and metastasis can help to distinguish truly malignant follicular thyroid cancer from indolent follicular adenoma.

Plasma exosomes stimulate breast cancer metastasis through surface interactions and activation of FAK signaling.

PURPOSE: The interaction between malignant cells and surrounding healthy tissues is a critical factor in the metastatic progression of breast cancer (BC). Extracellular vesicles, especially exosomes, are known to be involved in inter-cellular communication during cancer progression. In the study presented herein, we aimed to evaluate the role of circulating plasma exosomes in the metastatic dissemination of BC and to investigate the underlying molecular mechanisms of this phenomenon. METHODS: Exosomes isolated from plasma of healthy female donors were applied in various concentrations into the medium of MDA-MB-231 and MCF-7 cell lines. Motility and invasive properties of BC cells were examined by random migration and Transwell invasion assays, and the effect of plasma exosomes on the metastatic dissemination of BC cells was demonstrated in an in vivo zebrafish model. To reveal the molecular mechanism of interaction between plasma exosomes and BC cells, a comparison between un-treated and enzymatically modified exosomes was performed, followed by mass spectrometry, gene ontology, and pathway analysis. RESULTS: Plasma exosomes stimulated the adhesive properties, two-dimensional random migration, and transwell invasion of BC cells in vitro as well as their in vivo metastatic dissemination in a dose-dependent manner. This stimulatory effect was mediated by interactions of surface exosome proteins with BC cells and consequent activation of focal adhesion kinase (FAK) signaling in the tumor cells. CONCLUSIONS: Plasma exosomes have a potency to stimulate the metastasis-promoting properties of BC cells. This pro-metastatic property of normal plasma exosomes may have impact on the course of the disease and on its prognosis.

Combined quantitation of HMGA2 mRNA, microRNAs, and mitochondrial-DNA content enables the identification and typing of thyroid tumors in fine-needle aspiration smears.

BACKGROUND: Analysis of molecular markers in addition to cytological analysis of fine-needle aspiration (FNA) samples is a promising way to improve the preoperative diagnosis of thyroid nodules. Nonetheless, in clinical practice, applications of existing diagnostic solutions based on the detection of somatic mutations or analysis of gene expression are limited by their high cost and difficulties with clinical interpretation. The aim of our work was to develop an algorithm for the differential diagnosis of thyroid nodules on the basis of a small set of molecular markers analyzed by real-time PCR. METHODS: A total of 494 preoperative FNA samples of thyroid goiters and tumors from 232 patients with known histological reports were analyzed: goiter, 105 samples (50 patients); follicular adenoma, 101 (48); follicular carcinoma, 43 (28); Hürthle cell carcinoma, 25 (11); papillary carcinoma, 121 (56); follicular variant of papillary carcinoma, 80 (32); and medullary carcinoma, 19 (12). Total nucleic acids extracted from dried FNA smears were analyzed for five somatic point mutations and two translocations typical of thyroid tumors as well as for relative concentrations of HMGA2 mRNA and 13 microRNAs and the ratio of mitochondrial to nuclear DNA by real-time PCR. A decision tree-based algorithm was built to discriminate benign and malignant tumors and to type the thyroid cancer. Leave-p-out cross-validation with five partitions was performed to estimate prediction quality. A comparison of two independent samples by quantitative traits was carried out via the Mann-Whitney U test. RESULTS: A minimum set of markers was selected (levels of HMGA2 mRNA and miR-375, - 221, and -146b in combination with the mitochondrial-to-nuclear DNA ratio) and yielded highly accurate discrimination (sensitivity = 0.97; positive predictive value = 0.98) between goiters with benign tumors and malignant tumors and accurate typing of papillary, medullary, and Hürthle cell carcinomas. The results support an alternative classification of follicular tumors, which differs from the histological one. CONCLUSIONS: The study shows the feasibility of the preoperative differential diagnosis of thyroid nodules using a panel of several molecular markers by a simple PCR-based method. Combining markers of different types increases the accuracy of classification.

Lectin-induced agglutination method of urinary exosomes isolation followed by mi-RNA analysis: Application for prostate cancer diagnostic.

BACKGROUND: Prostate cancer is the most common cancer in men. Prostate-specific antigen has, however, insufficient diagnostic specificity. Novel complementary diagnostic approaches are greatly needed. MiRNAs are small regulatory RNAs which play an important role in tumorogenesis and are being investigated as a cancer biomarker. In addition to their intracellular regulatory functions, miRNAs are secreted into the extracellular space and can be found in various body fluids, including urine. The stability of extracellular miRNAs is defined by association with proteins, lipoprotein particles, and membrane vesicles. Among the known forms of miRNA packaging, tumour-derived exosome-enclosed miRNAs is thought to reflect the vital activity of cancer cells. The assessment of the exosomal fraction of urinary miRNA may present a new and highly specific method for prostate cancer diagnostics; however, this is challenged by the absence of reliable and inexpensive methods for isolation of exosomes. METHODS: Prostate cancer (PC) cell lines and urine samples collected from 35 PC patients and 35 healthy donors were used in the study. Lectins, phytohemagglutinin, and concanavalin A were used to induce agglutination of exosomes. The efficiency of isolation process was evaluated by AFM and DLS assays. The protein content of isolated exosomes was analysed by western blotting. Exosomal RNA was assayed by automated electrophoresis and expression level of selected miRNAs was evaluated by RT-qPCR. The diagnostic potency of the urinary exosomal miRNA assessment was estimated by the ROC method. RESULTS: The formation of multi-vesicular agglutinates in urine can be induced by incubation with lectin at a final concentration of 2 mg/ml. These agglutinates contain urinary exosomes and may be pelleted by centrifugation with a relatively low G-force. The analysis of PC-related miRNA in urinary exosomes revealed significant up-regulation of miR-574-3p, miR-141-5p, and miR-21-5p associated with PC. CONCLUSIONS: Lectin-induced aggregation is a low-cost and easily performed method for isolation of exosomes from urine. Isolated exosomes can be further analysed in terms of miRNA content. The miRNA profile of urinary exosomes reflects development of prostate cancer and may present a promising diagnostic tool.

Plasma exosomal miR-21 and miR-181a differentiates follicular from papillary thyroid cancer.

Thyroid cancer (TC) is the most common endocrine malignancy and its incidence has increased over the last few decades. As has been revealed by a number of studies, TC tissue's micro-RNA (miRNA) profile may reflect histological features and the clinical behavior of tumor. However, alteration of the miRNA profile of plasma exosomes associated with TC development has to date not been explored. We isolated exosomes from plasma and assayed their characteristics using laser diffraction particle size analysis, atomic force microscopy, and western blotting. Next, we profiled cancer-associated miRNAs in plasma exosomes obtained from papillary TC patients, before and after surgical removal of the tumor. The diagnostic value of selected miRNAs was evaluated in a large cohort of patients displaying different statuses of thyroid nodule disease. MiRNA assessment was performed by RT-qPCR. In total, 60 patients with different types of thyroid nodal pathology were included in the study. Our results revealed that the development of papillary TC is associated with specific changes in exosomal miRNA profiles; this phenomenon can be used for differential diagnostics. MiRNA-31 was found to be over-represented in the plasma exosomes of patients with papillary TC vs. benign tumors, while miRNA-21 helped to distinguish between benign tumors and follicular TC. MiRNA-21 and MiRNA-181a-5p were found to be expressed reciprocally in the exosomes of patients with papillary and follicular TC, and their comparative assessment may help to distinguish between these types of TC with 100 % sensitivity and 77 % specificity.

Resistance of breast cancer cells to paclitaxel is associated with low expressions of miRNA-186 and miRNA-7.

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.

Diagnosis of Prostate Cancer through the Multi-Ligand Binding of Prostate-Derived Extracellular Vesicles and miRNA Analysis.

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.

Potential of Plant Exosome Vesicles from Grapefruit (Citrus × paradisi) and Tomato (Solanum lycopersicum) Juices as Functional Ingredients and Targeted Drug Delivery Vehicles.

Plant-derived extracellular vesicles (PEVs) have gained attention as promising bioactive nutraceutical molecules; their presence in common fruit juices has increased their significance because human interaction is inevitable. The goal of this study was to evaluate the potential of PEVs derived from grapefruit and tomato juices as functional ingredients, antioxidant compounds, and delivery vehicles. PEVs were isolated using differential ultracentrifugation and were found to be similar in size and morphology to mammalian exosomes. The yield of grapefruit exosome-like vesicles (GEVs) was higher than that of tomato exosome-like vesicles (TEVs), despite the latter having larger vesicle sizes. Furthermore, the antioxidant activity of GEVs and TEVs was found to be low in comparison to their juice sources, indicating a limited contribution of PEVs to the juice. GEVs showed a higher efficiency in being loaded with the heat shock protein 70 (HSP70) than TEVs, as well as a higher efficiency than TEV and PEV-free HSP70 in delivering HSP70 to glioma cells. Overall, our results revealed that GEVs present a higher potential as functional ingredients present in juice and that they exert the potential to deliver functional molecules to human cells. Although PEVs showed low antioxidant activity, their role in oxidative response in cells should be further addressed.

AuNP Aptasensor for Hodgkin Lymphoma Monitoring.

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.

CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry.

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.

Evaluation of Colon-Specific Plasma Nanovesicles as New Markers of Colorectal Cancer.

PURPOSE: Developing new and efficient approaches for the early diagnosis of colorectal cancer (CRC) is an important issue. Circulating extracellular nanovesicles (ENVs) present a promising class of cancer markers. Cells of well-differentiated adenocarcinomas retain the molecular characteristics of colon epithelial cells, and the ENVs secreted by these cells may have colon-specific surface markers. We hypothesize that an increase in the number of ENVs carrying colon-specific markers could serve as a diagnostic criterion for colorectal cancer. EXPERIMENTAL DESIGN: Potential colon-specific markers were selected based on tissue-specific expression profile and cell surface membrane localization data. Plasma was collected from CRC patients (n = 48) and healthy donors (n = 50). The total population of ENVs was isolated with a two-phase polymer system. ENVs derived from colon epithelium cells were isolated using immune-beads with antibodies to colon-specific markers prior to labelling with antibodies against exosomal tetraspanins (CD63 and CD9) and quantification by flow cytometry. RESULTS: The number of ENVs positive for single colon cancer markers was found to be significantly higher in the plasma of CRC patients compared with healthy donors. The efficacy of detection depends on the method of ENV labelling. The diagnostic efficacy was estimated by ROC analysis (the AUC varied between 0.71 and 0.79). The multiplexed isolation of colon-derived ENVs using immune-beads decorated with antibodies against five markers allowed for a further increase in the diagnostic potency of the method (AUC = 0.82). CONCLUSIONS: ENVs derived from colon epithelium may serve as markers of differentiated CRC (adenocarcinomas). The composition of ligands used for capturing colon-derived ENVs and their method of labelling are critical for the efficacy of this proposed diagnostic approach.

A New Approach for Prostate Cancer Diagnosis by miRNA Profiling of Prostate-Derived Plasma Small Extracellular Vesicles.

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.

Formation and Evaluation of a Two-Phase Polymer System in Human Plasma as a Method for Extracellular Nanovesicle Isolation.

The aim of the study was to explore the polyethylene glycol-dextran two-phase polymer system formed in human plasma to isolate the exosome-enriched fraction of plasma extracellular nanovesicles (ENVs). Systematic analysis was performed to determine the optimal combination of the polymer mixture parameters (molecular mass and concentration) that resulted in phase separation. The separated phases were analyzed by nanoparticle tracking analysis and Raman spectroscopy. The isolated vesicles were characterized by atomic force microscopy and dot blotting. In conclusion, the protein and microRNA contents of the isolated ENVs were assayed by flow cytometry and by reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR), respectively. The presented results revealed the applicability of a new method for plasma ENV isolation and further analysis with a diagnostic purpose.

Detection of Cervical Lesions and Cancer in Air-Dried Cytologic Smears by Combined Analysis of mRNA and miRNA Expression Levels.

Cervical cancer screening is based on cytologic analysis and high-risk human papillomavirus (HR-HPV) testing, each having their drawbacks. Implementation of new biomarker-based methods may improve screening accuracy. Here, the levels of 25 microRNAs (miRNAs, miRs) and 12 mRNAs involved in cervical carcinogenesis in 327 air-dried Papanicolaou-stained cervical smears from patients with cervical precancerous lesions, cancer, or without the disease were estimated by real-time PCR. Using logistic regression analysis, small-scale miRNA-based, mRNA-based, and combined molecular classifiers were built based on paired ratios of miRNA or mRNA concentrations; their ability to detect high-grade cervical lesions and cancer was then compared. Combined mRNA-miRNA classifiers manifested a better combination of sensitivity and specificity than miRNA- and mRNA-based classifiers. The best classifier, combining miR-375, miR-20, miR-96, CDKN2A, TSP4, and ECM1, predicted high-grade lesions with diagnostic sensitivity of 89.0%, specificity of 84.2%, and a receiver-operating characteristic area under the curve of 0.913. Additionally, in a subsample of the same specimens, the levels of MIR124-2 and MAL promoter methylation, HR-HPV genotypes, and viral loads were analyzed. The relative high-grade lesion risk estimated by the classifier correlated with the frequency of MAL and MIR124-2 methylation but not with the HR-HPV genotype or viral load. The results support the feasibility of cellular biomarker-based methods for cervical screening and patient management.

PAMAM dendrimers for siRNA delivery: computational and experimental insights.

Short double-stranded RNAs, which are known as short interfering RNA (siRNA), can be used to specifically down-regulate the expression of the targeted gene in a process known as RNA interference (RNAi). However, the success of gene silencing applications based on the use of synthetic siRNA critically depends on efficient intracellular delivery. Polycationic branched macromolecules such as poly(amidoamine) (PAMAM) dendrimers show a strong binding affinity for RNA molecules and, hence, can provide an effective, reproducible, and relatively nontoxic method for transferring siRNAs into animal cells. Notwithstanding these perspectives, relatively few attempts have been made so far along these lines to study in detail the molecular mechanisms underlying the complexation process between PAMAMs and siRNAs. In this work we combine molecular simulation and experimental approaches to study the molecular requirements of the interaction of RNA-based therapeutics and PAMAM dendrimers of different generations. The dendrimers and their siRNA complexes were structurally characterized, and the free energy of binding between each dendrimer and a model siRNA was quantified by using the well-known MM/PBSA approach. DOSY NMR experiments confirmed the structural in silico prediction and yielded further information on both the complex structure and stoichiometry at low N/P ratio values. siRNA/PAMAM complex formation was monitored at different N/P ratios using gel retardation assays, and a simple model was proposed, which related the amount of siRNA complexed to the entropy variation upon complex formation obtained from the computer simulations.

Inhibition of Sp1-dependent transcription and antitumor activity of the new aureolic acid analogues mithramycin SDK and SK in human ovarian cancer xenografts.

OBJECTIVE: Increased activity of Sp family of transcription factors is a frequent and critical event in cancer development and progression. Genes governing tumor growth, invasion and angiogenesis are regulated by Sp factors, like Sp1, Sp3 or Sp4, and are frequently over-expressed in tumors. Targeting Sp factors has been explored as a therapeutic approach. Mithramycin (MTM) is a natural antibiotic that binds DNA and inhibit Sp1-dependent transcription. New analogues, named MTM-SDK and MTM-SK, were recently obtained by genetic engineering of the MTM biosynthetic pathway and have demonstrated improved transcriptional and antiproliferative activity in ovarian cancer cell lines in vitro. In the present study we evaluated the activity of the new compounds in human ovarian cancer xenografts. METHODS: Expression of Sp1 and target proteins in ovarian cancer specimens and tumor xenografts was assessed by immunohistochemistry. Drug-induced silencing of Sp1-regulated genes in cells and tumor xenograft samples was assessed by quantitative RT-PCR. Toxicity and antitumor activity of the compounds were investigated in healthy and tumor-bearing immunocompromised mice, respectively. RESULTS: Expression of Sp1 was frequently increased in human epithelial ovarian cancers. MTM-SDK and MTM-SK acted as potent inhibitors of Sp1-dependent transcription both in vitro and in tumor xenografts. Both compounds were well tolerated even after prolonged administration and delayed growth of ovarian tumor xenografts. MTM-SDK was particularly effective against orthotopic tumors leading to a significant increase of survival and delay of tumor progression. CONCLUSIONS: MTM-SDK and MTM-SK show relevant activity in vivo and represent interesting candidates for treatment of ovarian cancers.

MiRNA let-7 from TPO(+) Extracellular Vesicles is a Potential Marker for a Differential Diagnosis of Follicular Thyroid Nodules.

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.

Analysis of Reciprocally Dysregulated miRNAs in Eutopic Endometrium Is a Promising Approach for Low Invasive Diagnostics of Adenomyosis.

Endometriosis is a chronic disease characterized by the growth of endometrial tissue outside of the uterine cavity. Endometriosis affects up to 10% of women of reproductive age and has great social impact. The diagnostics of endometriosis are based on clinical appearance, ultrasound, and magnetic resonance imaging (MRI); however, a diagnosis is frequently hampered by the absence of objective criteria. Adenomyosis (AM) is a particular type of endometriosis wherein the spread of the ectopic endometrial gland is limited by the uterine myometrium. Alteration of the microRNA expression profile in the eutopic endometrium can be associated with AM, and may be assayed for diagnostic purposes. In the presented study, we aimed to explore the diagnostic potency of this approach. Eutopic endometrium specimens were collected from patients (n = 33) and healthy women (n = 30). The microRNA expression was profiled to select individual microRNAs with AM-associated expression alterations. A new method of two-tailed RT-qPCR microRNA analysis was applied to assay potential markers. The expression ratios of reciprocally dysregulated microRNAs were calculated, and the diagnostic potency of these parameters was evaluated by receiver operation curve (ROC) analysis. Mir-10b, miR-200c and miR-191 were significantly dysregulated in the eutopic endometrium of AM patients. The expression ratio of reciprocally dysregulated microRNAs allowed us to diagnose AM with a range of sensitivity from 65% to 74%, and of specificity from 72% to 86%. The analysis of microRNAs from the eutopic endometrium might present a promising low-invasive method of AM diagnostics.

Extracellular vesicle (ECV)-modified polyethylenimine (PEI) complexes for enhanced siRNA delivery in vitro and in vivo.

Extracellular vesicles (ECVs) are secreted cell-derived membrane particles involved in intercellular signaling and cell-cell communication. By transporting various bio-macromolecules, ECVs and in particular exosomes are relevant in various (patho-) physiological processes. ECVs are also released by cancer cells and can confer pro-tumorigenic effects. Their target cell tropism, effects on proliferation rates, natural stability in blood and immunotolerance makes ECVs particularly interesting as delivery vehicles. Polyethylenimines (PEIs) are linear or branched polymers which are capable of forming non-covalent complexes with small RNA molecules including siRNAs or antimiRs, for their delivery in vitro and in vivo. This study explores for the first time the combination of PEI-based nanoparticles with naturally occurring ECVs from different cell lines, for the delivery of small RNAs. ECV-modified PEI/siRNA complexes are analyzed by electron microscopy vs. ECV or complex alone. On the functional side, we demonstrate increased knockdown efficacy and storage stability of PEI/siRNA complexes upon their modification with ECVs. This is paralleled by enhanced tumor cell-inhibition by ECV-modified PEI/siRNA complexes targeting Survivin. Pre-treatment with various inhibitors of cellular internalization reveals alterations in cellular uptake mechanisms and biological activities of PEI/siRNA complexes upon their ECV modification. Extending our studies towards PEI-complexed antimiRs against miR-155 or miR-1246, dose-dependent cellular and molecular effects are enhanced in ECV-modified complexes, based on the de-repression of direct miRNA target genes. Differences between ECVs from different cell lines are observed regarding their capacity of enhancing PEI/siRNA efficacies, independent of the target cell line for transfection. Finally, an in vivo therapy study in mice bearing s.c. PC3 prostate carcinoma xenografts reveals marked inhibition of tumor growth upon treatment with ECVPC3-modified PEI/siSurvivin complexes, based on profound target gene knockdown. We conclude that ECV-modification enhances the activity of PEI-based complexes, by altering pivotal physicochemical and biological nanoparticle properties.