Detection of circulating miRNAs : comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients

Funding Information: This study was supported by the Norwegian Financial Mechanism 2009–2014 under Project Contract No NFI/R/2014/045. The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Publisher Copyright: © 2017 The Author(s).Background: Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer. They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes. However, it is still debated if EVs contain biologically meaningful amounts of miRNAs and may provide a better source of miRNA biomarkers than whole plasma. The aim of this study was to systematically compare the diagnostic potential of prostate cancer-associated miRNAs in whole plasma and in plasma EVs. Methods: RNA was isolated from whole plasma and plasma EV samples from a well characterised cohort of 50 patient with prostate cancer (PC) and 22 patients with benign prostatic hyperplasia (BPH). Nine miRNAs known to have a diagnostic potential for PC in cell-free blood were quantified by RT-qPCR and the relative quantities were compared between patients with PC and BPH and between PC patients with Gleason score ≥ 8 and ≤6. Results: Only a small fraction of the total cell-free miRNA was recovered from the plasma EVs, however the EV-incorporated and whole plasma cell-free miRNA profiles were clearly different. Four of the miRNAs analysed showed a diagnostic potential in our patient cohort. MiR-375 could differentiate between PC and BPH patients when analysed in the whole plasma, while miR-200c-3p and miR-21-5p performed better when analysed in plasma EVs. EV-incorporated but not whole plasma Let-7a-5p level could distinguish PC patients with Gleason score ≥ 8 vs ≤6. Conclusions: This study demonstrates that for some miRNA biomarkers EVs provide a more consistent source of RNA than whole plasma, while other miRNAs show better diagnostic performance when tested in the whole plasma.publishersversionPeer reviewe

Resolution of Teenage Conflicts un School

Cilvēka dzīve nav iedomājama bez konfliktiem un problēmām. Indivīda vai grupas darbu vienmēr pavada pretrunas, bet pretrunu augstāko saasinājuma punktu veido konflikta situācija. Pētījuma mērķis bija atklāt skolu konfliktu cēloņus un to risināšanas prasmes pusaudžu vecumā. Pusaudžu skolu konfliktu cēloņi, to risināšanas taktika, prasmes tika pētītas “x”komercnovirziena vidusskolā ķīmijas un klašu audzināšanas stundās. Pētījums veikts skolā, kurā bez vispārizglītojošiem priekšmetiem māca arī komerczinību priekšmetus. Pētījumā izmantoja pētījumu metožu kompleksu: intervijas, anketēšanu, pārrunas un novērošanu. Darbā apkopotas teorētiskas atziņas par konflikta jēdzienu, būtību, cēloņiem un risināšanas iespējām. Praktiskajā daļā atklāti konflikta cēloņi skolā un to risināšanas prasmes. Pusaudži savu daudzveidīgo būtību īstenoja kopīgiem spēkiem, pastiprināti realizējot arī saskarsmes procesu, uz kā balstās konfliktu risināšanas prasmes. Procesā ārkārtīgi liela loma ir skolotājam, viņa saskarsmei ar pusaudžiem un darba metodei.We cannot imagine our life without conflicts and problems. There are contradictions in individual or a group work, but the conflict situation causes the highest aggravation point of the contradictions. The goal of the research was to find out the reasons of conflicts at schools and to discover the problem solvation skills in adolescence. The reasons of conflicts among adolescents, the way of solvation, skills were investigated in “x” commercial secondary school during the chemistry and class lessons. The research AS done at school where there are taught not only general education but also commercial subjects. During the research several methods were used: interviews, questionnaires, discussions and observation. Theoretical cognitions about the notion of conflict were summarized as well as notions about causes, basis and possibilities of solvation. The adolescents realised their varied activities together being intensively involved in communicative process that is the basis of solvation. The teacher and his interaction with adolescents plays a big role in this process. 15 literal sources were used in work

Vairogdziedzera audzēju veidošanās molekulāro mehānismu izpēte un biomarķieru identificēšana

Elektroniskā versija nesatur pielikumusŠī doktora darba ietvaros tika identificēts multipleks biomerķieru modelis vairogdziedzera vēža pacientiem, kam nepieciešams apstiprinājums FNAB. Kā arī šajā darbā autors pēta TFF3, vienu no daudzsološākiem vairogdziedzera vēža biomarķieriem, funkcijas vairogdziedzera vēzī un demonstrē, ka tas var funkcionēt gan kā tumor suppresors, gan onkogēns atkarībā no šūnu konteksta. Šī darba ietvaros tika arī sistemātiski pētīts vēža asociēto autoantivielu repertuārs vairogdziedzera vēža pacientos. Kopumā šis darbs sniedz dziļāku izpratni par molekulārām izmaiņām, kas noved pie vairogdziedzera vēža attīstības un identificē potenciālos biomarķierus diagnostikas testa attīstīšanai un signālceļus terapijas attīstīšanai vairogdziedzera vēža pacientos.In this doctoral thesis, a multiplex biomarker model for thyroid cancer was identified and warrants further studies in FNAB to improve it. Also in this work, for the first time author investigated the functional role of TFF3 - one of the most promising thyroid cancer tissue biomarker known so far, and demonstrated that TFF3 can act as tumor suppresor or onco gene depending on cellular context. Furthermore, in this work for the first time author systematically studied the repertoire of cancer-associated autoantibodies in patients with thyroid cancer. Hence, this study provided a deeper insight into the molecular alterations leading to the development of thyroid cancer and revealed biomarkers for the diagnosis and putative therapeutic targets for the development of drugs against thyroid cancer

Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

Abstract. Background Macrophages are one of the most important players in the tumor microenvironment. The polarization status of tumor associated macrophages into a pro-inflammatory type M1 or anti-inflammatory type M2 may influence cancer progression and patient survival. Extracellular vesicles (EVs) are membrane-bound vesicles containing different biomolecules that are involved in cell to cell signal transfer. Accumulating evidence suggests that cancer-derived EVs are taken up by macrophages and modulate their phenotype and cytokine profile. However, the interactions of cancer-derived EVs with monocytes and macrophages at various differentiation and polarization states are poorly understood. In the current study, we have analyzed the uptake and functional effects of primary (SW480) and metastatic (SW620) isogenic colorectal cancer (CRC) cell line-derived EVs on monocytes (M), inactive macrophages (M0) and M1 and M2 polarized macrophages. Methods THP-1 monocytes were differentiated into M0 macrophages by addition of phorbol-12-myristate-13-acetate. Then M0 macrophages were further polarized into M1 and M2 macrophages in the presence of LPS, IFN- γ, IL-4, and IL-13 respectively. Internalization of SW480 and SW620-derived EVs was analyzed by flow cytometry and fluorescence microscopy. Changes in monocyte and macrophage immunophenotype and secretory profile upon EV exposure were analyzed by flow cytometry, quantitative PCR and Luminex assays. Results THP-1 monocytes and M0 macrophages efficiently take up SW480 and SW620-derived EVs, and our results indicate that dynamin-dependent endocytic pathways may be implicated. Interestingly, SW480 and SW620-derived EVs increased CD14 expression in M0 macrophages whereas SW480-derived EVs decreased HLA-DR expression in M1 and M2 polarized macrophages. Moreover, SW480-derived EVs significantly increased CXCL10 expression in monocytes and M0 macrophages. In contrast, SW620-derived EVs induced secretion of IL-6, CXCL10, IL-23 and IL-10 in M0 macrophages. However, addition of CRC cell line-derived EVs together with LPS, IFN- γ (M1) and IL-4, IL-13 (M2) stimuli during macrophage polarization had no additional effect on cytokine expression in M1 and M2 macrophages. Conclusion Our results suggest that CRC cell line-derived EVs are internalized and reprogram the immunophenotype and secretory profile in monocytes and inactive macrophages inducing mixed M1 and M2 cytokine response. Although CRC EVs decreased HLA-DR expression in M1, M2 polarized macrophages, their effect on the secretory profile of M1 and M2 polarized macrophages was negligible

Additional file 4: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

TNFα, IL-23, IL-6, IL-1 β, CXCL10, CCL22, IL-10 and MMP9 secretion profile at different monocyte-macrophage differentiation stages. The graphs represent average biomolecule concentrations SEM (n = 3). Statistical analysis carried out with one-way ANOVA test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and **** ≤ 0.0001 vs. untreated cell control of the respective monocyte-macrophage cell subset. (PDF 63 kb

Additional file 3: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

Effect of temperature on the SW480 EV uptake in THP-1 monocytes. Flow cytometry histograms showing Syto RNA Select fluorescence intensities of untreated (left) and Syto RNA Select-labeled SW480 EV-treated THP-1 monocytes following incubation at 4 °C (middle) and 37 °C (right). Histogram markers show the percentage of Syto RNA Select-positive cells. (PDF 53 kb

Additional file 2: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

SW480 and SW620-derived EV effect on monocyte (M) and macrophage (M0, M1, M2) viability. a OD values at 450 nm which are in direct proportion of viable cell counts. b SW480 and SW620 EV cytotoxicity on THP-1 monocytes and M0, M1 and M2 macrophages. The graphs represent mean ± SEM (n = 3). Statistical analysis carried out with the t-test. *p ≤ 0.05, **p ≤ 0.01 vs. untreated cell control of the respective monocyte-macrophage cell subset. (PDF 50 kb