Cutaneous melanoma is an increasingly common skin cancer characterized by aggressive metastatic growth and poor prognosis. The mechanisms behind melanoma progression are not fully understood, but emerging evidence suggests that a group of newly discovered small regulatory RNAs, named microRNAs (miRNAs), plays an important role. miRNAs are ~ 22 nucleotide single strand non-coding RNAs that post-transcriptionally regulate gene expression by binding to target messenger RNAs (mRNAs), leading to mRNA degradation and translation inhibition. Abnormal expression of miRNAs has been observed in human malignancies and is associated with tumorigenesis. The main goals of this thesis are to investigate miRNA dysregulation in melanoma and to identify potential miRNAs involved in melanoma pathogenesis. Initially, the expression of 470 miRNAs was profiled in 8 metastatic melanoma and 8 benign nevus tissue samples. We discovered unique miRNA expression profiles and identified differentially expressed miRNAs in melanomas as compared to nevi. miR-193b was one of the most significantly downregulated miRNAs in melanoma, and its function and regulatory targets were unknown. Subsequently, in vitro functional studies revealed that ectopic expression of miR-193b in melanoma cells drastically repressed cell proliferation and migration. Although it does not directly induce apoptosis in melanoma cells, miR-193b does sensitize these cells to ABT-737-mediated cell death. In concert with functional studies, gene expression analysis and in silico target prediction were performed to globally screen for mRNA targets of miR-193b. We identified eighteen genes as candidates in that they were downregulated by miR-193b and contained predicted miR-193b binding sites. Based on their known biological functions, three genes were particularly interesting: cyclin D1 (CCND1), myeloid cell leukemia sequence 1 (Mcl-1), and stathmin 1 (STMN1). CCND1 and Mcl-1 are two well-known melanoma oncogenes, and we validated their role in cell proliferation and apoptosis respectively. Furthermore, using similar approach, we were the first to identify STMN1 as a novel melanoma oncogene. We demonstrated that CCND1, Mcl-1, and STMN1 were directly regulated by miR-193b. During melanoma progression, reduced expression of miR-193b may promote cell proliferation, migration and survival. Taken together, this thesis describes the dysregulation of miRNAs in melanoma and demonstrates that miR-193b functions as a tumor suppressor. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2012-05-31 15:27:01.707

Techn. Universiẗat, Diss., 2005--München.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os tumores de mama são caracterizados pela sua alta heterogeneidade. O câncer de mama é uma doença complexa, que possui o seu desenvolvimento fortemente influenciado por fatores ambientais, combinada a uma progressiva acumulação de mutações genéticas e desregulação epigenética de vias críticas. Alterações nos padrões de expressão gênica podem ser resultado de uma desregulação no controle de eventos epigenéticos, assim como, na regulação pós-transcricional pelo mecanismo de RNA de interferência endógeno via microRNA (miRNA). Estes eventos são capazes de levar à iniciação, à promoção e à manutenção da carcinogênese, como também ter implicações no desenvolvimento da resistência à terapia Os miRNAs formam uma classe de RNAs não codificantes, que durante os últimos anos surgiram como um dos principais reguladores da expressão gênica, através da sua capacidade de regular negativamente a atividade de RNAs mensageiros (RNAms) portadores de uma seqüencia parcialmente complementar. A importância da regulação mediada por miRNAs foi observada pela capacidade destas moléculas em regular uma vasta gama de processos biológicos incluindo a proliferação celular, diferenciação e a apoptose. Para avaliar a expressão de miRNAs durante a progressão tumoral, utilizamos como modelo experimental a série 21T que compreende 5 linhagens celulares originárias da mesma paciente diagnosticada com um tumor primário de mama do tipo ErbB2 e uma posterior metástase pulmonar. Essa série é composta pela linhagem obtida a partir do tecido normal 16N, pelas linhagens correspondentes ao carcinoma primário 21PT e 21NT e pelas linhagens obtidas um ano após o diagnóstico inicial, a partir da efusão pleural no sítio metastatico 21MT1 e 21MT2. O miRNAoma da série 21T revelou uma redução significativa nos níveis de miR-205 e nos níveis da proteina e-caderina e um enriquecimento do fator pró-metastático ZEB-1 nas células 21MT. Considerando a importância dos miRNAs na regulação da apoptose, e que a irradiação em diferentes espectros é comumente usada em procedimentos de diagnóstico como mamografia e na radioterapia, avaliamos a expressão de miRNAs após irradiação de alta e baixa energia e do tratamento doxorrubicina. Para os ensaios foram utilizados as linhagens não tumorais MCF-10A e HB-2 e as linhagens de carcinoma da mama MCF-7 e T-47D. Observou-se que raios-X de baixa energia são capazes de promover quebras na molécula do DNA e apoptose assim como, alterar sensivelmente miRNAs envolvidos nessas vias como o let-7a, miR-34a e miR-29b. No que diz respeito à resposta a danos genotóxicos, uma regulação positiva sobre a expressão de miR-29b, o qual em condições normais é regulado negativamente foi observada uma regulação positiva sobre miR-29b expressão após todos os tratamentos em células tumorais. Nossos resultados indicam que miR-29b é um possível biomarcador de estresse genotóxico e que miR-205 pode participar no potencial metastático das células 21T. / Breast tumors are characterized by their high heterogeneity. It is a complex disease, which has its development strongly influenced by environmental factors, combined with a progressive accumulation of genetic mutations and epigenetic dysregulation of critical pathways. Changes in gene expression patterns may be a result of a deregulation in epigenetic events as well as in post-transcriptional regulation driven by RNA interference endogenously represented by microRNA (miRNA) these mechanisms are capable to promote the initiation, maintenance and progression of carcinogenesis; they are also implicated on the development of therapy resistance. miRNAs form a class of non-coding RNAs which have emerged in recent years as one of the major regulators of gene expression through its capacity to silence messenger RNAs (mRNAs) containing a partially complementary sequence. The importance of regulation mediated by miRNAs was observed on their ability to regulate a wide range of biological processes including cell proliferation, differentiation and apoptosis.To gain insights into the mechanisms involved in breast cancer initiation and progression conducted a miRNA global expression on 21T series that are an in vitro model of breast cancer progression comprising cell lines derived from the same patient which include a normal epithelia (16N), primary in situ ductal carcinoma (21PT and 21NT) and cells derived from pleural effusion of lung metastasis (21MT-1 and 21MT-2). Considering the importance of miRNAs in the regulation of apoptosis, and that irradiation in different spectra is commonly used in diagnostic procedures as mammography and on radiotherapy, we evaluate the miRNA expression after cell low and high energy irradiation and doxorubicin treatment to determine whether miRNAs are useful biomarkers to detect cell response after DNA damage. The experiments were done on the non-tumoral cell lines MCF-10A and HB-2 and on the breast carcinoma derived cell lines MCF-7 and T-47D. We observed that of low energy X-rays is able to promote DNA strand breaks and apoptosis and to slightly change the expression of miRNAs involved on this pathway such as let-7a, miR-34a and miR-29b. Regarding DNA stress response pathways an upregulation on miR-29b expression, that in normal conditions is downregulated in tumor cell lines could be observed after all treatments. The microRNAome of 21T series revealed a significant downregulation of miR-205, an enrichment of the prometastatic factor ZEB-1, potential target for miR-205 and the consequent reduction of ecadherin levels in 21MT cells checked by western blot. Our results indicate that miR-29b is biomarkers of genotoxic stress and that miR-205can participate on the metastatic potential of 21T cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os tumores de mama são caracterizados pela sua alta heterogeneidade. O câncer de mama é uma doença complexa, que possui o seu desenvolvimento fortemente influenciado por fatores ambientais, combinada a uma progressiva acumulação de mutações genéticas e desregulação epigenética de vias críticas. Alterações nos padrões de expressão gênica podem ser resultado de uma desregulação no controle de eventos epigenéticos, assim como, na regulação pós-transcricional pelo mecanismo de RNA de interferência endógeno via microRNA (miRNA). Estes eventos são capazes de levar à iniciação, à promoção e à manutenção da carcinogênese, como também ter implicações no desenvolvimento da resistência à terapia Os miRNAs formam uma classe de RNAs não codificantes, que durante os últimos anos surgiram como um dos principais reguladores da expressão gênica, através da sua capacidade de regular negativamente a atividade de RNAs mensageiros (RNAms) portadores de uma seqüencia parcialmente complementar. A importância da regulação mediada por miRNAs foi observada pela capacidade destas moléculas em regular uma vasta gama de processos biológicos incluindo a proliferação celular, diferenciação e a apoptose. Para avaliar a expressão de miRNAs durante a progressão tumoral, utilizamos como modelo experimental a série 21T que compreende 5 linhagens celulares originárias da mesma paciente diagnosticada com um tumor primário de mama do tipo ErbB2 e uma posterior metástase pulmonar. Essa série é composta pela linhagem obtida a partir do tecido normal 16N, pelas linhagens correspondentes ao carcinoma primário 21PT e 21NT e pelas linhagens obtidas um ano após o diagnóstico inicial, a partir da efusão pleural no sítio metastatico 21MT1 e 21MT2. O miRNAoma da série 21T revelou uma redução significativa nos níveis de miR-205 e nos níveis da proteina e-caderina e um enriquecimento do fator pró-metastático ZEB-1 nas células 21MT. Considerando a importância dos miRNAs na regulação da apoptose, e que a irradiação em diferentes espectros é comumente usada em procedimentos de diagnóstico como mamografia e na radioterapia, avaliamos a expressão de miRNAs após irradiação de alta e baixa energia e do tratamento doxorrubicina. Para os ensaios foram utilizados as linhagens não tumorais MCF-10A e HB-2 e as linhagens de carcinoma da mama MCF-7 e T-47D. Observou-se que raios-X de baixa energia são capazes de promover quebras na molécula do DNA e apoptose assim como, alterar sensivelmente miRNAs envolvidos nessas vias como o let-7a, miR-34a e miR-29b. No que diz respeito à resposta a danos genotóxicos, uma regulação positiva sobre a expressão de miR-29b, o qual em condições normais é regulado negativamente foi observada uma regulação positiva sobre miR-29b expressão após todos os tratamentos em células tumorais. Nossos resultados indicam que miR-29b é um possível biomarcador de estresse genotóxico e que miR-205 pode participar no potencial metastático das células 21T. / Breast tumors are characterized by their high heterogeneity. It is a complex disease, which has its development strongly influenced by environmental factors, combined with a progressive accumulation of genetic mutations and epigenetic dysregulation of critical pathways. Changes in gene expression patterns may be a result of a deregulation in epigenetic events as well as in post-transcriptional regulation driven by RNA interference endogenously represented by microRNA (miRNA) these mechanisms are capable to promote the initiation, maintenance and progression of carcinogenesis; they are also implicated on the development of therapy resistance. miRNAs form a class of non-coding RNAs which have emerged in recent years as one of the major regulators of gene expression through its capacity to silence messenger RNAs (mRNAs) containing a partially complementary sequence. The importance of regulation mediated by miRNAs was observed on their ability to regulate a wide range of biological processes including cell proliferation, differentiation and apoptosis.To gain insights into the mechanisms involved in breast cancer initiation and progression conducted a miRNA global expression on 21T series that are an in vitro model of breast cancer progression comprising cell lines derived from the same patient which include a normal epithelia (16N), primary in situ ductal carcinoma (21PT and 21NT) and cells derived from pleural effusion of lung metastasis (21MT-1 and 21MT-2). Considering the importance of miRNAs in the regulation of apoptosis, and that irradiation in different spectra is commonly used in diagnostic procedures as mammography and on radiotherapy, we evaluate the miRNA expression after cell low and high energy irradiation and doxorubicin treatment to determine whether miRNAs are useful biomarkers to detect cell response after DNA damage. The experiments were done on the non-tumoral cell lines MCF-10A and HB-2 and on the breast carcinoma derived cell lines MCF-7 and T-47D. We observed that of low energy X-rays is able to promote DNA strand breaks and apoptosis and to slightly change the expression of miRNAs involved on this pathway such as let-7a, miR-34a and miR-29b. Regarding DNA stress response pathways an upregulation on miR-29b expression, that in normal conditions is downregulated in tumor cell lines could be observed after all treatments. The microRNAome of 21T series revealed a significant downregulation of miR-205, an enrichment of the prometastatic factor ZEB-1, potential target for miR-205 and the consequent reduction of ecadherin levels in 21MT cells checked by western blot. Our results indicate that miR-29b is biomarkers of genotoxic stress and that miR-205can participate on the metastatic potential of 21T cells.

Made available in DSpace on 2018-08-01T21:36:53Z (GMT). No. of bitstreams: 1 tese_10765_Dissertação_final_Rayza.pdf: 2180620 bytes, checksum: 5e88e543a5adef7347618d2eef3cfb62 (MD5) Previous issue date: 2017-03-27 / A calibração multivariada associada à técnica de infravermelho é uma alternativa aos métodos tradicionais para determinação de parâmetros físico-químicos em petróleo. Entretanto, o modelo multivariado construído é aplicável somente para o instrumento no qual os espectros foram obtidos. A transferência de calibração de modelos multivariados é de fundamental importância para a indústria petrolífera, levando-se em conta que aumenta a aplicabilidade de modelos e permite estimar com rapidez e com poucos gastos diversas propriedades físico-químicas. Este trabalho foi dividido em duas seções: a primeira é dedicada à avaliação modelos de transferência entre dois instrumentos de infravermelho a região média (MIR) para modelos PLS (mínimos quadrados parciais) e OPLS (projeções ortogonais em estruturas latentes); e a segunda visa aplicar a transferência PDS (padronização direta por partes) a modelos PLS para predição de densidade API, TIAC (temperatura de início de aparecimento de cristais), NAT (número de acidez total) e NAN (número de acidez naftênica). Dentre os métodos quimiométricos SBC (correção de declive e viés), FR (recalibração total), DS (padronização direta) e PDS avaliados para previsão de densidade API, o modelo de transferência PDS aplicado ao modelo OPLS resultou na melhor capacidade preditiva (RMSEP de 1,48). A aplicação direta de PDS a um modelo PLS original conta com a vantagem de aproveitar modelos já consolidados, e os resultados da primeira seção indicam que isto é uma possibilidade. Na segunda seção, as propriedades API, TIAC, NAT e NAN foram modeladas e validadas por PLS para o instrumento primário. Espectros secundários, diferentes daqueles da primeira seção, passaram por uma interpolação polinomial a fim de igualar as variáveis às do instrumento primário, antes da aplicação da transferência PDS. Os espectros transferidos e processados por airPLS, técnica iterativa adaptativa, se tornaram indistiguíveis por PCA (análise por componentes principais) dos espectros primários. A técnica de PDS associada a modelos PLS mostrou-se promissora, especialmente sendo capaz de estimar com boa exatidão o valor da densidade API de amostras de petróleo classificadas como medianas.

La mucine MUC1 est une oncoprotéine transmembranaire dont la surexpression dans 90% des adénocarcinomes pancréatiques a été associée à un mauvais pronostic. MUC1 est impliquée dans la transduction des signaux intracellulaires et dans les interactions cellulaires permettant de conférer aux cellules tumorales des propriétés accrues en termes de prolifération et d’invasion cellulaire. De plus il a été montré un rôle important de MUC1 dans la chimiorésistance à la gemcitabine, et dans la transition épithélio-mésenchymateuse des cellules cancéreuses pancréatiques. De manière intéressante il a pu être montré que MUC1 pouvait être internalisée et localisée dans le compartiment nucléaire afin d’agir comme un co-activateur transcriptionnel permettant de moduler l’expression de nombreux gènes comme ceux de la voie Wnt/β-caténine, les cibles de Stat1/3 ou le cluster miR-200c/miR141. De plus, il a été démontré que MUC1 pouvait être régulé de façon épigénétique, par méthylation de son promoteur, par acétylation des histones et par les miARN.Notre objectif a été d’étudier l'inhibition de MUC1 par des miARN dérégulés dans le cancer pancréatique, afin de proposer une nouvelle stratégie thérapeutique innovante dans le but de ralentir la progression de ce cancer. Nous avons sélectionné des miARN pouvant cibler la mucine MUC1 aussi bien au niveau de son 3’UTR, de son 5’UTR ou de sa région codante par l'utilisation des bases de données miRanda, miRWalk et TargetScan. Afin d’affiner cette sélection, nous avons ensuite retenu seulement ceux étant dérégulés dans le cancer pancréatique en étudiant leur expression dans des lignées cellulaires cancéreuses pancréatiques humaines ainsi que dans un modèle murin transgénique de cancérogenèse pancréatique et dans les tissus de patients atteints d'adénocarcinome pancréatique.Nous avons dans un premier temps mis en évidence que parmi les miARN sélectionnés, la surexpression de miR-29a, miR-183, miR-200a, miR-330-5p, miR-876-3p et miR-939 entraînait une diminution de l'expression protéique de MUC1. En établissant le profil d'expression des miARN dans les trois modèles de cancer pancréatique dont nous disposions, nous avons pu mettre en évidence une dérégulation globale de miR-29a et miR-330-5p dans les lignées cellulaires cancéreuses pancréatiques humaines ainsi que chez les patients atteints d'adénocarcinome pancréatique, et une dérégulation plus spécifique pour les autres miARN. Nous avons alors pu mettre en évidence que parmi l'ensemble des miARN sélectionnés, seuls les miARN miR-29a et miR-330-5p avaient la capacité d’interagir avec l'ARNm de MUC1 au niveau de son 3’UTR. Nous avons donc entrepris dans un second temps d’étudier le rôle de miR-29a et miR-330-5p dans le cancer du pancréas. Pour cela, nous avons utilisé une stratégie transitoire de surexpression et d'inhibition des miARN et une stratégie stable en réalisant des lignées surexprimant les miARN ainsi qu’une lignée déficiente en MUC1. Nous avons pu mettre en évidence que la surexpression de miR-29a et miR-330-5p, permettait de ralentir la prolifération cellulaire, la migration, l'invasion cellulaire, la croissance tumorale et augmentait la chimiosensibilité des cellules cancéreuses pancréatiques à la gemcitabine. En conclusion, l'ensemble de ces données a permis de mettre en évidence un ensemble de miARN dérégulés dans le cancer du pancréas ayant la capacité de réguler négativement l'expression protéique de la mucine MUC1. Nous avons également montré que miR-29a et miR-330-5p étaient les seuls à réguler directement l'expression de MUC1 et qu’ils agissaient comme des suppresseurs de tumeurs en altérant les propriétés biologiques des cellules cancéreuses pancréatiques, in vitro et in vivo. Ces données nous permettent de proposer ces deux miARN comme une nouvelle piste thérapeutique potentielle pour le traitement de ce cancer. / The mucin MUC1 is a transmembrane oncoprotein overexpressed in 90% of pancreatic adenocarcinoma and associated with a poor prognosis. MUC1 is involved in cell signaling and cell interaction to enhanced tumor cell properties like cell proliferation and invasion. Furthermore it has been shown an important role of MUC1 in chemoresistance to gemcitabine, the basic treatment of pancreatic cancer, and in the epithelial-mesenchymal transition of pancreatic cancer cells. Interestingly it has been shown that MUC1 could be internalized and localized in the nuclear compartment to act as a transcriptional coactivator to modulate the expression of many genes such as the Wnt/β-catenin, the targets of Stat1/3 or the miR-200c/miR141 cluster. Furthermore, it has been shown that MUC1 is regulated by epigenetics: by methylation of the promoter, histone acetylation and by miRNAs in breast and ovarian cancer.Our aim was to study the inhibition of MUC1 by miRNAs deregulated in pancreatic cancer, to propose a new innovative therapeutic strategy to slow down progression of this cancer.We selected miRNAs targeting the mucin MUC1, in its 3\\\'UTR, its 5\\\'UTR or its coding region by using databases such as Miranda, miRWalk and TargetScan. To refine this selection, we then selected only those being deregulated in pancreatic cancer by studying their expression in human pancreatic cancer cell lines, tissues from patients with pancreatic adenocarcinoma and transgenic mouse model of early pancreatic carcinogenesis.We initially demonstrated that among the selected miRNAs, overexpression of miR-29a, miR-183, miR-200a, miR-330-5p, miR-and miR-939 876-3p led to a decrease of MUC1 protein expression. By establishing the miRNA expression profile in the three models of pancreatic cancer that we had, we were able to demonstrate an overall deregulation of miR-29a and miR-330-5p in human pancreatic cancer cell lines and in patients with a pancreatic adenocarcinoma, and a more specifically deregulation for the other miRNAs.We were then able to show that among the selected miRNAs, only miRNAs miR-29a and miR-330-5p had the ability to interact with MUC1 mRNA on its 3\\\'UTR. We therefore undertook to study the role of miR-29a and miR-330-5p in pancreatic cancer. For this, we used a transient strategy to overexpress or inhibit miRNAs and stable cell lines overexpressing the miRNA as well as a deficient cell line for MUC1. We were able to show that overexpression of miR-29a and miR-330-5p slowed down cell proliferation, migration, cell invasion, tumor growth and increased chemosensitivity of pancreatic cancer cells to gemcitabine.In conclusion, all these data allowed us to identify a set of deregulated miRNAs in pancreatic cancer which have the ability to decrease the mucin MUC1 protein expression level. We also showed that miR-29a and miR-330-5p were the only ones that can regulate the expression of MUC1 directly and act as tumor suppressors by altering the biological properties of pancreatic cancer cells in vitro and in vivo. These data allow us to propose these two miRNAs as a new potential therapeutic approach for the treatment of this cancer.

Leveraging miRNA-Seq data and the 1000 Genomes imputed genotypes, we identified rs174561-C as a strong miRQTL for circulating miRNA-1908-5p (P=4.8x10-31) which has an inverse relationship with circulating LDL-C, fasting glucose and A1c. Here I investigated the molecular mechanism(s) linking miR1908-5p to cholesterol metabolism. First, by overexpression experiments in HuH-7 cells demonstrate that the presence of the C allele, associated with lower LDL-C levels, significantly increases miR-1908-5p by 2.15-fold relative to the T allele. Further experiments revealed that 72-hour cholesterol depletion increases miR-1908-5p expression (2.11-fold) whereas cholesterol loading decreases miR-1908-5p expression (0.69-fold). Differential miR-1908-5p expression was then used to profile genes involved in lipoprotein signaling and cholesterol metabolism using a PCR array to identify LDLR as a gene of interest. Although total RNA and protein expression of LDLR was unchanged in response to differential miR-1908-5p expression, the ratio of the mature form to the cleaved form of LDLR decreased following miR-1908-5p inhibition (0.85-fold) and conversely, increased with mimic treatment (1.63-fold). Cleavage of the mature LDLR is known to reduce cell surface affinity for LDL. These findings uncover a potential mechanism linking miR-1908-5p to lower LDL-cholesterol levels through reduced LDLR cleavage.

Indiana University-Purdue University Indianapolis (IUPUI) / Store-operated Ca2+ entry (SOCE) is involved in the maintenance of endoplasmic reticulum (ER) Ca2+ levels. The SOCE involves Stromal Interaction Molecule 1 (STIM1), distributed throughout the ER, and Orai1 channels, dispersed on the plasma membrane. SOCE is activated by the depletion of ER Ca2+ causing STIM1 to induce ER expansion and recruits Orai1 channels thus replenishing ER Ca2+. We reported downregulation of STIM1 in human islets from donors with type 2 diabetes (T2D) and in INS-1 β-cells treated with cytokines, and loss of STIM1 expression impairs β-cell SOCE, ER stress, and reduced insulin secretion. However, the regulatory mechanisms of STIM1 downregulation are unknown. To test this, actinomycin D and cycloheximide chase assay was performed to define whether IL-1β treatment impacted STIM1 mRNA or protein half-life. IL-1β had no impact on mRNA or protein decay. MicroRNAs (miRNAs), a class of small non-coding RNAs can regulate gene expression post-transcriptionally by binding to complementary regions in the 3’ untranslated region (UTR) of target mRNAs, affecting mRNA stability and translatability. The objective of this study was to establish miRNA regulation of STIM1 expression and altered SOCE. To identify potential miRNA candidates, RNA sequencing was done in human islets, treated with IL-1β and IFN-γ for 24 hrs. A total of 20 miRNAs were differentially expressed using a FC value of ≥ 1.5 and a p value of < 0.05. Of these, two miRNAs (miR-146a-5p and miR-4640-5p) were predicted by TargetScan to bind the 3’UTR of STIM1.To validate these findings, INS-1 β-cells, and human islets were treated with or without IL-1β. Only miR-146a-5p was upregulated in both systems. Consistent with inverse correlation, INS-1 β-cells transfected with miR-146a-5p mimic showed reduced STIM1 expression. To test whether miR-146a-5p inhibition preserves STIM1 expression, INS1 cells were treated with miR-146a-5p inhibitor along with IL-1β and inhibition of miR-146a-5p led to partial preservation of STIM1 expression. Future studies will test the effect of miR-146a-5p mimics and inhibitors on SOCE. The results indicate that the stress induced by IL-1β leads to induction of miR-146a-5p, which may then target STIM1 mRNA. Such studies could enable broader implementation of miRNA in βcell dysfunction.

CRPS ist eine anhaltende Schmerzerkrankung, die nach Verletzungen auftritt und mit einer variierenden Kombination von Symptomen aus den Bereichen Sensorik, Vasomotorik, Sudomotorik/Ödemen und Motorik verbunden ist. Die Physiologie des Krankheitsbildes ist nicht abschließend geklärt, allerdings wird eine komplexe Interaktion mehrerer Pathomechanismen als Ursache angenommen. Deshalb stellt das CRPS sowohl eine diagnostische als auch eine therapeutische Herausforderung dar. miRNAs sind kurze, einsträngige und nicht-codierende RNAs, die durch Inhibierung der Translation und Degradierung von mRNAs an der posttranskriptionellen Regulation der Genexpression beteiligt sind. Sie werden auch von den Zellen durch Exosomen, Mikrovesikel, Lipoproteine und Apoptose freigesetzt, sodass sie in unterschiedlichen Körperflüssigkeiten nachgewiesen werden können. Bei vielen Erkrankungen kann eine Dysregulation der miRNA-Expression festgestellt werden, weshalb großes Interesse daran besteht, sie als Biomarker oder für therapeutische Ansätze nutzbar zu machen. Die miRNAs miR-183, -21, -29b, -144, - 223 wurden bereits im Zusammenhang mit entzündlichen und neuropathischen Prozessen und der Dysruption von Immunobarrieren beschrieben. In dieser Arbeit wurde untersucht, ob bei der Expression dieser miRNAs im Blut von CRPS-Patienten, Patienten mit einem komplikationslosen posttraumatischen Heilungsverlauf und von Kontrollen ohne ein vorangegangenes Trauma Unterschiede bestehen. Die Messungen erfolgten im Plasma, in Leukozyten und Exosomen, um dadurch auch die Regulation in den einzelnen Blutkomponenten vergleichen zu können. Tatsächlich fanden sich unterschiedliche miRNA-Expressionsprofile bei den verschiedenen Biomaterialien. Außerdem konnte bei einzelnen miRNAs ein Einfluss von Alter und Geschlecht auf die Expression nachgewiesen werden. Diese Beeinflussung war darüber hinaus auch abhängig vom untersuchten Biomaterial und vor allem bei den Exosomen besonders ausgeprägt. In den Exosomen ergab sich eine signifikante Hochregulation von miR-223-5p bei den FK im Vergleich mit den CRPS-Patienten. Bei der Zusammenfassung der Daten von CRPS und FK fand sich außerdem eine negative Korrelation zwischen der miR-223-5p-Expression und dem CSS, sodass ein erhöhtes Expressionsniveau mit einer milderen Krankheitsausbildung verbunden war. Hinsichtlich der traumanaiven Kontrollen war das Expressionsniveau der CRPS-Patienten hingegen unverändert. Diese Ergebnisse weisen darauf hin, dass beim CRPS im Vergleich mit einem regelrechten Heilungsverlauf eine insuffiziente beziehungsweise ausbleibende posttraumatische Anpassung des miRNA-Spektrums vorliegt. Diese posttraumatische Regulation stellt eventuell eine wichtige Voraussetzung für den Ablauf eines komplikationslosen Heilungsprozesses dar. Für miR-223-5p wurde bereits mehrfach eine antiinflammatorische Wirkung durch die Regulation von proinflammatorischen Rezeptoren und die Beeinflussung der Differenzierung von Makrophagen beschrieben. Eine verminderte Expression könnte somit zu einer Disposition für überschießende Entzündungsreaktionen führen und dadurch zur Entwicklung von CRPS beitragen. Diese Ergebnisse weisen auf die Beteiligung zirkulierender und vor allem exosomaler miRNAs bei der Pathophysiologie des CRPS hin. Zur weiterführenden Abklärung der pathophysiologischen Relevanz von miR-223-5p sind jedoch zusätzliche Untersuchungen erforderlich. Dabei bleibt es zu prüfen, ob eine verminderte miR-223-5p-Expression mit verstärkten Entzündungsmarkern und einer verstärkten proinflammatorischen Differenzierung von Makrophagen verbunden ist. Eine Abklärung der Herkunft der Exosomen könnte dabei helfen, zwischen einer lokalen und einer systemischen Reaktion zu unterscheiden. Die Beantwortung dieser Fragen könnte zu einem besseren Verständnis beitragen, warum manche Patienten nach einem Extremitätentrauma ein CRPS entwickeln und keinen normalen Heilungsverlauf erfahren. / CRPS is a lasting pain condition, which appears after an injury and manifests as a varying combination of sensoric, vasomotoric, sudomotoric/edema and motoric symptoms. The physiology of CRPS is not fully understood since there is a complex interaction of several possible underlying pathomechanisms. Therefore, CRPS presents a diagnostic as well as a therapeutic challenge. miRNAs are short, single-stranded, non-coding RNAs, which are involved in the posttranscriptional regulation of gene expression by inhibiting the trans- lation and causing the degradation of mRNA. They can be exported by the cells using exosomes, microvesicels, lipoproteins und apoptosis. Extracellular miRNAs can be found in several in different body fluids. Dysregulation of miRNA-expression has been found in several diseases, causing in an interest to utilize them as biomarkers or for therapeutic applications. The miRNAs miR-183, -21, -29b, -144 and -223 have been described in inflammatory und neuropathic processes as well as disruption of immunobarriers. This work investigated, if there is a difference in the expression of these miRNAs in the blood of CRPS patients, patients with a normal posttraumatic recovery und controls without trauma. miRNAs were measured in plasma, leukocytes and exosomes to additionally compare these blood components ...

Prostate cancer is the second most common cancer in the United States that affects men today. To better treat this disease accurate biomarkers and successful therapeutic treatments are needed. A novel approach to understand the mechanisms behind prostate cancer tumor formation lies in identifying dysregulated micro-RNAs (miRNAs), which are a class of small (18-24 nucleotides) non-coding RNAs that regulate gene expression posttranscriptionally by either inhibiting protein synthesis or signaling messenger-RNA for degradation. Multiple miRNAs were discovered in our highly tumorigenic and metastatic prostate cancer progression model M12 cell line compared to its weakly tumorigenic P69 parental cell line. Various analyses such as human panel analyses, single-miR analyses and patient tumor biopsy samples were analyzed to determine dysregulated miRNAs that contributed to the progression and metastasis of prostate cancer. Together with performing experiments to identify miRNAs, a de novo next generation sequencing approach was applied to identify miRNAs naturally present in biological fluids of normal and healthy subjects. Since, these miRNAs are highly dysregulated in many diseases, including cancer, they can act as potential biomarkers or therapeutic targets to improve treatments for prostate cancer. Essential miRNAs studied for this research were miR-17-3p that is known to target the ErbB2 mRNA; miR-299-5p that directly targets osteopontin (OPN) mRNA, and miR-147b that directly targets many mRNAs, such as COL4A2, ALDH5A1, NDUFA4, SDHD, and IER5. A wide range of miRNAs were identified in six biological fluids: venous blood, menstrual blood, vaginal fluid, semen, saliva, and feces. There were some miRNAs that were common to all 6 body fluids, some unique to each body fluid, and some miRNAs that literature suggested could potentially be biomarkers or normalizers for body fluid characterization.