Etwa 25 % der Nierenzellkarzinome (RCC) weisen bei Diagnosestellung bereits Metastasen auf. Aufgrund der schlechten Prognose des metastasierten RCC besteht ein dringender Bedarf an neuen Therapieformen sowie an prognostischen und diagnostischen Markern. microRNAs (miRNAs) bieten sich dabei als vielversprechende molekulare Biomarker an. Für den klarzelligen RCC-Subtypen (ccRCC) wurde bereits ein umfangreiches miRNA Expressionsprofil erstellt, mit dem ccRCC-relevante, vorwiegend herunterregulierte miRNAs identifiziert werden konnten. In der vorliegenden Arbeit wurde gezeigt, dass die Expression der miR-141 und miR-145 in RCC-Zelllinien durch epigenetische Mechanismen gehemmt ist und die Promotorbereiche dieser miRNAs stark methyliert vorliegen. In RCC-Zellen konnte eine tumorsuppressive Wirkung dieser miRNAs durch Hemmung der Migration (beide) und Invasion (miR-141) nachgewiesen werden. Durch die gleichzeitige Überexpression der beiden miRNAs kam es zu einer kooperativen Wirkung und so zu einer verstärkten Hemmung der Zellmigration. Weitere Untersuchungen konnten eine Reihe neuer onkogener Targets der miR 141 und miR 145 identifizieren. Dabei zeigte sich ein kooperativer Effekt durch Kombination beider miRNAs auf die Expression der Targets HS6ST2 und LOX. Die Targets LOX und MAP4K4 waren in ccRCC Gewebe auf mRNA-Ebene stark überexprimiert im Vergleich zum umliegenden Normalgewebe. Bei der anschließenden Tissue-Mikroarray-Analyse der Expression auf Proteinebene zeigte sich zudem ein prognostisches Potenzial der Targets LOX und MAP4K4 für das Gesamtüberleben von ccRCC Patienten. Diese Daten verdeutlichen den enormen Einfluss von epigenetisch dysregulierten miRNAs und deren spezifischen Targets auf tumorassoziierte Prozesse. Zudem bietet das Netzwerk aus Epigenetik, miRNAs und deren jeweiligen Targets nicht nur eine Reihe von diagnostischen und prognostischen Möglichkeiten, sondern liefert auch viele Ansatzpunkte für die Entwicklung von neuen therapeutischen Strategien. / Approximately 25 % of diagnosed renal cell carcinoma (RCC) have already metastasized. Due to poor prognosis of metastatic RCC, there is an urgent need for new therapies and prognostic and diagnostic markers to identify high-risk patients. Here microRNAs (miRNAs) might be promising new molecular biomarkers. For the clear cell RCC subtype (ccRCC) a comprehensive miRNA expression profile was already established. In this profiling several ccRCC-associated, predominantly down-regulated miRNAs were identified. In the present study, epigenetic mechanisms were identified to play a significant role in the down regulation of miR-141 and miR-145 in RCC cell lines. In addition, a strong methylation of the corresponding promoter regions was detected at molecular level. In RCC cells a tumor suppressive effect of these miRNAs was shown by decreasing migration (both) and invasion (miR-141) and furthermore, co overexpression of both miRNAs resulted in a cooperative effect with increased inhibition of cell migration. Several new oncogenic targets of miR-141 and miR-145 were identified by further investigations. Here the two miRNAs again showed a cooperative effect, as demonstrated by a significantly increased inhibition of HS6ST2 and LOX expression. In ccRCC tissue the expression of LOX and MAP4K4 was strongly enhanced on mRNA level compared to normal tissue. In the subsequent tissue microarray analysis of protein expression, LOX and MAP4K4 showed a prognostic impact for the overall survival of patients with ccRCC. These results illustrate a huge impact of epigenetically dysregulated miRNAs and of their specific targets on tumor-associated processes. Furthermore, the network of epigenetics, miRNAs and their respective targets will offer a number of diagnostic and prognostic capabilities, but will also provide many opportunities for the development of new therapeutic strategies.

Der Cholesterin-Stoffwechsel ist sehr streng auf zellulärer Ebene reguliert und ist essentiell für das Zellwachstum. MicroRNAs (miRNAs), eine Klasse nicht-kodierender RNAs, wurden als kritische Regulatoren der Genexpression identifiziert und entfalten ihre Wirkung vorwiegend auf posttranskriptioneller Ebene. Aktuelle Arbeiten aus der Gruppe um Fernández-Hernando haben gezeigt, dass hsa-miR-33a und hsa-miR-33b, miRNAs die in den Intronsequenzen der Gene für die Sterol-regulatorischen Element- Bindungsproteine (SREBP-2 und SREBP -1) lokalisiert sind, den Cholesterin-Stoffwechsel im Einklang mit ihren Wirtsgenen regulieren. Gleichermaßen inhibiert miR-33 Schlüsselenzyme in der Regulation der Fettsäureoxidation, einschließlich CROT, CPT1A, HADHB, SIRT6, AMPKα, genauso wie IRS2, eine wesentliche Komponente des Insulin-Signalwegs in der Leber. Diese Studie zeigt, dass hsa-miR-33 Familienmitglieder nicht nur Gene in Cholesterin- und Fettsäure-Stoffwechsel sowie Insulin-Signalwege regulieren, sondern zusätzlich die Expression von Genen des Zellzyklus und der Zellproliferation modulieren. miR-33 inhibiert die Expression der CDK6 und CCND1, wodurch sowohol die Zellproliferation als auch die Zellzyklusprogression verringert wird. Die Überexpression von miR-33 induziert einen signifikanten G1 Zellzyklusarrest. Durch eine Inhibierung der miR-33 Expression mittels 2''F/MOE-modifiziert Phosphorothioat-Backbone Antisense-Oligonukleotiden, wird die Leberregeneration nach partieller Hepatektomie (PH) in Mäusen verbessert, was auf eine wichtige Rolle für miR-33 in der Regulation der Hepatozytenproliferation während der Leberregeneration hinweist. Zusammengefasst zeigen diese Daten, dass Srebf/miR-33 Locus kooperieren, um Zellproliferation und Zellzyklusprogression zu regulieren, und könnte somit auch relevant für die menschliche Leberregeneration sein. / Cholesterol metabolism is tightly regulated at the cellular level and is essential for cellular growth. Cellular imbalances of cholesterol and fatty acid metabolism lead to pathological processes, including atherosclerosis and metabolic syndrome. MicroRNAs (miRNAs), a class of noncoding RNAs, have emerged as critical regulators of gene expression acting predominantly at posttranscriptional level. Recent work from Fernández-Hernando´s group and others has shown that hsa-miR-33a and hsa-miR-33b, miRNAs located within intronic sequences of the sterol regulatory element-binding protein (SREBP-2 and SREBP-1) genes, respectively, regulate cholesterol metabolism in concert with their host genes. Similarly, miR-33 targets key enzymes involved in the regulation of fatty acid oxidation including CROT, CPT1A, HADHB, SIRT6 and AMPKα, likewise, IRS2, an essential component of the insulin- signaling pathway in the liver. This study shows that hsa-miR-33 family members not only regulate genes involved in cholesterol and fatty acid metabolism and insulin signaling, but in addition modulate the expression of genes involved in cell cycle regulation and cell proliferation. Thus, miR-33 inhibited the expression of CDK6 and CCND1, thereby reducing cell proliferation and cell cycle progression. Over-expression of miR-33 induced a significant G1 cell cycle arrest and most importantly, inhibition of miR-33 expression using 2’F/MOE-modified phosphorothioate backbone antisense oligonucleotides improved liver regeneration after partial hepatectomy (PH) in mice, suggesting an important role for miR-33 in regulating hepatocyte proliferation during liver regeneration. Altogether, these data establish that Srebf/miR-33 locus may co-operate to regulate cell proliferation, cell cycle progression and may also be relevant to human liver regeneration.

Introdução: Os sarcomas uterinos constituem uma forma rara de neoplasia maligna que compreende cerca de 3% de todos os canceres uterinos. Os fatores de risco são pouco conhecidos e as formas de tratamento escassas, sendo o papel da quimioterapia e radioterapia limitado. Nesse contexto, torna-se importante a compreensão dos mecanismos moleculares envolvidos no processo de carcinogênese, tanto para diagnóstico e prognóstico mais precisos, quanto para ampliar os avanços terapêuticos. Um dos mecanismos moleculares envolvidos no desenvolvimento de canceres é a regulação da expressão gênica por microRNAs (miRNAs), que são pequenas moléculas de RNAs não-codificantes que desempenham papéis importantes em várias vias biológicas. Alterações na expressão de miRNAs podem acarretar em diversas doenças incluindo o câncer. Poucos trabalhos foram realizados, até o momento, no sentido de esclarecer a regulação ou expressão de miRNAs nos sarcomas uterinos. Objetivo: Avaliar a expressão de miRNAs relacionados ao desenvolvimento de tumores em amostras de carcinossarcomas e sarcomas do útero. Métodos: Foram selecionadas 100 amostras incluindo: 56 leiomiossarcomas (LMS), 24 carcinossarcomas (CS), 18 sarcomas do estroma endometrial (SEE) e 2 adenossarcomas (AS). O RNA total de amostras incluídas em parafina foi obtido e utilizado para realização da síntese de cDNA. As reações de PCR em Tempo Real foram realizadas utilizando o Kit miScript SYBR Green PCR (Qiagen) para análise a partir de 84 sequências de miRNAs já descritos na literatura para outros tipos de cânceres. Foram analisados dados clínicos como idade, sintomas principais, uso de contraceptivo oral, estado menopausal, terapia de reposição hormonal, tabagismo, tratamento cirúrgico, tratamento adjuvante, metástase, grau histológico, recorrência, óbito e estadiamento. Os resultados foram tabulados nos softwares SPSS e GraphPad Prism 5.01. Resultados: Observamos miRNAs que apresentaram diferentes perfis de expressão nos tumores, em comparação com o tecido referência (miométrio). Dentre 42 miRNAs que apresentaram diferenças de expressão nos sarcomas e carcinossarcomas uterinos, 6 destacaram-se por apresentar diferenças significativas entre os tipos histológicos: miR-96-5p (p < 0,001; LMSxCS), miR-205-5p (p=0,016; CSxSEE), miR-181a-5p (p=0,010; CSxLMS), miR-135b-5p (p=0,018; CSxLMS), miR-183-5p (p=0,012; CSxLMS) e miR-193b-3p (p=0,008; CSxLMS). Diversas associações entre a hiper ou hipoexpressão dos miRNAs com as variáveis clínicas coletadas foram constatadas. A expressão de 12 miRNAs foram associadas com sobrevida global e 3 com sobrevida câncer específica. Maior expressão de miR-335-5p, miR-301a-3p e miR-210-3p foi encontrada em amostras de pacientes que realizaram tratamento adjuvante e desenvolveram metástase ou recidiva, para as quais observamos a expressão de miR-138-5p, miR-146b-5p e miR-218-5p associada com maior sobrevida livre de doença. Conclusão: Em conjunto, os resultados sugerem que a caracterização dos miRNAs possui papel promissor para fins diagnóstico, prognóstico e até terapêuticos nos sarcomas e carcinossarcomas uterinos / Introduction: The uterine sarcomas are a rare form of malignancy comprising about 3% of all uterine cancers. Risk factors are not well known and the treatment forms are scarce, being the role of chemotherapy and radiotherapy limited. In this context, it becomes important the comprehension of the molecular mechanisms involved in the carcinogenesis process, as for more accurate diagnosis and prognosis, as to broaden the therapeutic advances. One of the molecular mechanisms involved in cancer development is the regulation of gene expression by microRNAs (miRNAs), which are small molecules of non-coding RNAs that play important roles in some biological pathways. Alterations in miRNA expression could result in some diseases, including cancer. Few studies were performed until present time, to clarify the miRNAs regulation or expression in uterine sarcomas. Objective: To evaluate the expression of miRNAs related to development of tumors in carcinosarcoma and uterine sarcomas samples. Methods: 100 samples were evaluated including: 56 leiomyosarcomas (LMS), 24 carcinosarcomas (CS), 18 endometrial stromal sarcomas (ESS) and 2 adenosarcomas (AS). The total RNA from paraffin-embedded samples were obtained and used to cDNA synthesis. The real time PCR reactions, was performed using the miScript SYBR Green PCR Kit (Qiagen) for analysis of 84 sequences already described in literature in other types of cancer. Were analyzed clinical data such as age, main symptoms, oral contraceptive use, menopausal status, hormone replacement therapy, smoking, underlying pathology, surgical treatment, adjuvant treatment, metastasis, histological grade, recurrence, death and staging. The results were tabulated in the SPPS and Graphpad Prism 5.01. Results: We observed miRNAs that showed different expression profiles in tumors compared to the reference tissue (myometrium). Among 42 miRNAs that showed different expression in uterine sarcomas and carcinosarcomas, 6 stood out for showing significant differences among the histological types: miR-96-5p (p < 0,001; LMSxCS), miR-205-5p (p=0,016; CSxSEE), miR-181a-5p (p=0,010; CSxLMS), miR-135b-5p (p=0,018; CSxLMS), miR-183-5p (p=0,012; CSxLMS) e miR-193b-3p (p=0,008; CSxLMS). Many associations between overexpression or hypoexpression of miRNAs with the clinical variables collected were found. The expression of 12 miRNAs were associated with cancer-specific survival and 3 with disease-free survival. It were also found, increased expression of miR-335-5p, miR-301a-3p and miR-210-3p in samples of patients who underwent adjuvant treatment and developed metastasis or recurrence, and correlation analysis showed that the expression of miR-138-5p, miR-146b-5p and miR-218- 5p are associated with higher disease-free survival of treated patients. Conclusion: The results suggest that the characterization of miRNAs have a promising role for purposes of diagnosis, prognosis and therapeutic for the uterine sarcomas and carcinosarcomas

No carcinoma papilífero de tiroide (CPT) ocorrem alterações na via MAPK (do inglês Mitogen-Activated Protein Kinase), sendo a mais frequente mutação a BRAFT1799A. A via MAPK é modulada pelo miRNA, como o let-7 que estão pouco expresso no CPT. A biogênese de let-7 é controlada por proteínas ligantes de RNA LIN28 que inibem o processamento das formas primária e precursora de let-7 (pri-let-7 e pre-let-7). Avaliamos a expressão gênica de LIN28, pri-let-7f, pre-let-7f e let-7f por rtPCR em tempo real, utilizando RNA total das linhagens TPC-1 e BCPAP de PTC e amostras tumorais de pacientes com CPT. A linhagem não tumoral Nthyori 3-1 foi utilizada para induzir LIN28. Observa-se redução de let-7f em 4 dos 5 pacientes analisados enquanto que LIN28A está aumentado em 2 pacientes com BRAFT1799A. Nota-se maior expressão de pri-let-7f em TPC-1 e menor expressão de pre-let-7f e let-7f. Observa-se maior expressão de LIN28A em TPC-1, enquanto que LIN28B está mais expresso em BCPAP com BRAFT1799A. A inibição de BRAFV600E em BCPAP diminui LIN28B e aumenta let-7f, enquanto que a indução de LIN28B reduz a expressão de let-7f. Concluímos que há relação inversa entre LIN28 e let-7f no CPT e LIN28 influencia a diminuição de let-7f podendo assim contribuir com tumorigênese tireoidiana. / In papillary thyroid cancer (PTC) changes in MAPK (Mitogen-Activated Protein Kinase) pathway are common being BRAFT1799A mutation the most frequent alteration. MAPK pathway is modulated by miRNA such as let-7, an under-expressed miRNA in PTC. The RNA binding protein LIN28 controls let-7 biogenesis, blocking primary and precursor let-7 (pri-let-7 e pre-let-7) processing. We evaluated LIN28, pri-let-7f, pre-let-7f and let-7f gene expression by real time RTPCR using total RNA of human PTC sample and PTC cell lines TPC-1 and BCPAP. Non-tumoral cell line Nthyori 3-1 was used to evaluate LIN28B influence in let-7f. We observed decreased let-7f expression in 4 out of 5 PTC patients, and increased LIN28A in 2 patients with BRAFT1799A mutation. TPC-1 cells express higher levels of pri-let-7f while pre-let-7f and let-7f are less expressed. We noted that LIN28A is more expressed in TPC-1, while LIN28B is more expressed in BCPAP cells with BRAFT1799A. The inhibition of BRAFV600E in BCPAP decreases LIN28B and increases let-7f, while the induction of LIN28B in Nthyori 3-1 reduces let-7f expression. We conclude there is an inverse association between LIN28 and let-7f in PTC, and that LIN28 influences let-7f reduction which could contribute to thyroid tumorigenesis.

Introdução: Os sarcomas uterinos constituem uma forma rara de neoplasia maligna que compreende cerca de 3% de todos os canceres uterinos. Os fatores de risco são pouco conhecidos e as formas de tratamento escassas, sendo o papel da quimioterapia e radioterapia limitado. Nesse contexto, torna-se importante a compreensão dos mecanismos moleculares envolvidos no processo de carcinogênese, tanto para diagnóstico e prognóstico mais precisos, quanto para ampliar os avanços terapêuticos. Um dos mecanismos moleculares envolvidos no desenvolvimento de canceres é a regulação da expressão gênica por microRNAs (miRNAs), que são pequenas moléculas de RNAs não-codificantes que desempenham papéis importantes em várias vias biológicas. Alterações na expressão de miRNAs podem acarretar em diversas doenças incluindo o câncer. Poucos trabalhos foram realizados, até o momento, no sentido de esclarecer a regulação ou expressão de miRNAs nos sarcomas uterinos. Objetivo: Avaliar a expressão de miRNAs relacionados ao desenvolvimento de tumores em amostras de carcinossarcomas e sarcomas do útero. Métodos: Foram selecionadas 100 amostras incluindo: 56 leiomiossarcomas (LMS), 24 carcinossarcomas (CS), 18 sarcomas do estroma endometrial (SEE) e 2 adenossarcomas (AS). O RNA total de amostras incluídas em parafina foi obtido e utilizado para realização da síntese de cDNA. As reações de PCR em Tempo Real foram realizadas utilizando o Kit miScript SYBR Green PCR (Qiagen) para análise a partir de 84 sequências de miRNAs já descritos na literatura para outros tipos de cânceres. Foram analisados dados clínicos como idade, sintomas principais, uso de contraceptivo oral, estado menopausal, terapia de reposição hormonal, tabagismo, tratamento cirúrgico, tratamento adjuvante, metástase, grau histológico, recorrência, óbito e estadiamento. Os resultados foram tabulados nos softwares SPSS e GraphPad Prism 5.01. Resultados: Observamos miRNAs que apresentaram diferentes perfis de expressão nos tumores, em comparação com o tecido referência (miométrio). Dentre 42 miRNAs que apresentaram diferenças de expressão nos sarcomas e carcinossarcomas uterinos, 6 destacaram-se por apresentar diferenças significativas entre os tipos histológicos: miR-96-5p (p < 0,001; LMSxCS), miR-205-5p (p=0,016; CSxSEE), miR-181a-5p (p=0,010; CSxLMS), miR-135b-5p (p=0,018; CSxLMS), miR-183-5p (p=0,012; CSxLMS) e miR-193b-3p (p=0,008; CSxLMS). Diversas associações entre a hiper ou hipoexpressão dos miRNAs com as variáveis clínicas coletadas foram constatadas. A expressão de 12 miRNAs foram associadas com sobrevida global e 3 com sobrevida câncer específica. Maior expressão de miR-335-5p, miR-301a-3p e miR-210-3p foi encontrada em amostras de pacientes que realizaram tratamento adjuvante e desenvolveram metástase ou recidiva, para as quais observamos a expressão de miR-138-5p, miR-146b-5p e miR-218-5p associada com maior sobrevida livre de doença. Conclusão: Em conjunto, os resultados sugerem que a caracterização dos miRNAs possui papel promissor para fins diagnóstico, prognóstico e até terapêuticos nos sarcomas e carcinossarcomas uterinos / Introduction: The uterine sarcomas are a rare form of malignancy comprising about 3% of all uterine cancers. Risk factors are not well known and the treatment forms are scarce, being the role of chemotherapy and radiotherapy limited. In this context, it becomes important the comprehension of the molecular mechanisms involved in the carcinogenesis process, as for more accurate diagnosis and prognosis, as to broaden the therapeutic advances. One of the molecular mechanisms involved in cancer development is the regulation of gene expression by microRNAs (miRNAs), which are small molecules of non-coding RNAs that play important roles in some biological pathways. Alterations in miRNA expression could result in some diseases, including cancer. Few studies were performed until present time, to clarify the miRNAs regulation or expression in uterine sarcomas. Objective: To evaluate the expression of miRNAs related to development of tumors in carcinosarcoma and uterine sarcomas samples. Methods: 100 samples were evaluated including: 56 leiomyosarcomas (LMS), 24 carcinosarcomas (CS), 18 endometrial stromal sarcomas (ESS) and 2 adenosarcomas (AS). The total RNA from paraffin-embedded samples were obtained and used to cDNA synthesis. The real time PCR reactions, was performed using the miScript SYBR Green PCR Kit (Qiagen) for analysis of 84 sequences already described in literature in other types of cancer. Were analyzed clinical data such as age, main symptoms, oral contraceptive use, menopausal status, hormone replacement therapy, smoking, underlying pathology, surgical treatment, adjuvant treatment, metastasis, histological grade, recurrence, death and staging. The results were tabulated in the SPPS and Graphpad Prism 5.01. Results: We observed miRNAs that showed different expression profiles in tumors compared to the reference tissue (myometrium). Among 42 miRNAs that showed different expression in uterine sarcomas and carcinosarcomas, 6 stood out for showing significant differences among the histological types: miR-96-5p (p < 0,001; LMSxCS), miR-205-5p (p=0,016; CSxSEE), miR-181a-5p (p=0,010; CSxLMS), miR-135b-5p (p=0,018; CSxLMS), miR-183-5p (p=0,012; CSxLMS) e miR-193b-3p (p=0,008; CSxLMS). Many associations between overexpression or hypoexpression of miRNAs with the clinical variables collected were found. The expression of 12 miRNAs were associated with cancer-specific survival and 3 with disease-free survival. It were also found, increased expression of miR-335-5p, miR-301a-3p and miR-210-3p in samples of patients who underwent adjuvant treatment and developed metastasis or recurrence, and correlation analysis showed that the expression of miR-138-5p, miR-146b-5p and miR-218- 5p are associated with higher disease-free survival of treated patients. Conclusion: The results suggest that the characterization of miRNAs have a promising role for purposes of diagnosis, prognosis and therapeutic for the uterine sarcomas and carcinosarcomas

No câncer, alterações em microRNAs (miRNAs), pequenos RNAs que regulam a tradução protéica, exerce efeito oncogênico (oncomiR). Os oncomiRs regulam genes chave para a proliferação celular e apoptose, sendo importantes para a biologia do câncer. O carcinoma papilífero de tiróide apresenta alterações genéticas alinhadas na via MAPK (RET>RAS>BRAF>ERK). Observamos que a indução do oncogene RET/PTC diminui a expressão de let-7 em células foliculares tiroidianas. Na linhagem TPC-1 (com RET/PTC-1), a introdução de let-7 diminui a proliferação celular e a fosforilaçãode ERK, indicando papel de gene supressor tumoral. No carcinoma anaplásico, avaliamos o papel da introdução do cluster miR-17-92 na linhagem ARO. Observamos que in vitro miR-17-92 atua de forma oncogênica aumentando proliferação e viabilidade celular de ARO. No entanto, estas células apresentam diminuição no crescimento em soft-agar. No xenotransplante, os tumores de ARO-miR-17-92 apresentam menor volume e expressam MMP-9 de forma reduzida, indicando também um papel de gene supressor tumoral para o cluster. / In cancer, alteration in microRNA, small RNAs (~22nt) that regulate post-transcriptionally protein levels, exerts oncogenic role (oncomiR). OncomiRs control genes involved in cell proliferation and apoptosis, influencing cancer biology. Papillary thyroid cancer displays activating genetic alterations in MAPK signaling pathway (RET>RAS>BRAF>ERK). Using conditional induction of oncogenes in thyroid cells, we observed that RET/PTC decreases let-7 miRNA expression. In papillary thyroid cancer cell TPC-1 (with RET/PTC-1) we observed that let-7 introduction inhibits cell proliferation and ERK phosphorylation, indicating tumor suppressor role for let-7. In anaplastic thyroid cancer, we evaluate the role of introduction of miR-17-92 cluster in ARO cell line. We observed in vitro that miR-17-92 increases ARO cell proliferation and viability, acting as oncogene. However, these cells show impaired soft agar growth. In xenotransplant, ARO-miR-17-92 tumors are smaller in volume and express reduced levels of MMP-9, indicating a tumor suppressor role for the cluster.

Submitted by admin tede (tede@pucgoias.edu.br) on 2018-06-19T18:25:16Z No. of bitstreams: 1 DANIEL RODRIGUES DE BASTOS.pdf: 1922893 bytes, checksum: 51e3aa82ceb630f944c3c65627dcffaa (MD5) / Made available in DSpace on 2018-06-19T18:25:16Z (GMT). No. of bitstreams: 1 DANIEL RODRIGUES DE BASTOS.pdf: 1922893 bytes, checksum: 51e3aa82ceb630f944c3c65627dcffaa (MD5) Previous issue date: 2017-05-04 / Introduction: Breast cancer is the most frequent neoplasm among women worldwide and represents the leading cause of death in this population. Important biomarkers have been studied in order to better define the prognosis of patients affected by this cancer. MicroRNAs are small molecules of non-coding RNAs composed of 21 to 25 nucleotides that play an important role in the post-transcriptional regulation of several genes. Objective: The objective of this study was to evaluate the expression of microRNAs (miRNAs: hsa-miR-7 and hsa-miR-10b) and BRCA1 protein in breast cancer samples, as well as the possible associations between expression of these markers with clinicopathological and prognostic aspects. Method: The study included 92 cases of breast carcinoma from Hospital Araújo Jorge, Associação de Combate ao Câncer em Goiás. Formalin fixed paraphin embedded samples were used for the analisis. MicroRNA was extracted from the samples and used for cDNA synthesis. The cDNA samples were adjusted to the same concentration and submitted to quantitative real-time PCR (qRT-PCR). Samples were further evaluated by immunohistochemistry for BRCA1 expression. Results: From a group of 234 immunohistochemical records, 56 cases of non-triple-negative and 36 triple-negative breast carcinomas were selected. Five-year overall survival was significantly associated to triple negative phenotype (p = 0.044), advanced stages (p = 0.005), lymph node involvement (p = 0.038), presence of distant metastasis (p = 0, 0008) and absence of BRCA1 expression (p = 0.039). Significant associations were demonstrated between the absence of BRCA1 and the triple-negative phenotype (p = 0.0346), as well as the absence of estrogen receptor expression (p = 0.006) and absence of progesterone receptor expression (p = 0.0163). The analysis by qRTPCR demonstrated different levels of miR-10b and miR-7 expression in the tumors, with significant associations with triple-negative phenotype (p = 0.021, p = 0.042) and the absence of BRCA1 (p = 0.039, p = 0.006). The comparison between absence and presence of human epidermal growth factor receptor expression showed a significant difference for miR-7 (p = 0.031), and the expression of miR-10b in these cases was not statistically different (p = 0.926). Conclusion: Significant associations were demonstrated between the absence of BRCA1 and the triple-negative phenotype. Five-year overall survival was reduced for the triple-negative phenotype patients, clinical stages III and IV, the presence of lymph node metastasis, the presence of distant metastasis and the absence of BRCA1 expression. This study also demonstrated that hsa-miR-7 and hsa-miR-10b are significantly associated with the absence of BRCA1 expression and triple-negative phenotype, with poorer survival in these patient profiles. Studies with more cases and with cell lines should be performed in order to confirm the role of hsa-miR-7 and hsa-miR-10b in the modulation of BRCA1 expression. / Introdução: O câncer de mama é a neoplasia mais frequente entre as mulheres de todo o mundo e representa a principal causa de morte nesta população. Importantes biomarcadores têm sido estudados, a fim de definir melhor o prognóstico de pacientes acometidas por esta doença. Os microRNAs são pequenas moléculas de RNAs não codificantes, compostos por 21 a 25 nucleotídeos e desempenham importante papel na regulação pós-transcricional de diversos genes. Objetivo: O objetivo do estudo foi avaliar a expressão de microRNAs (miRNAs: hsa-miR-7 e hsamiR- 10b) e da proteína BRCA1 em amostras de câncer de mama, bem como as possíveis associações entre a expressão desses marcadores e os aspectos clinicopatológicos e prognósticos. Método: O estudo foi composto por 92 casos de carcinoma de mama, provenientes do Hospital Araújo Jorge, da Associação de Combate ao Câncer em Goiás. Foram utilizados fragmentos de tumores incluídos em parafina. A extração de microRNA das amostras foi realizada e o produto gerado foi utilizado para a síntese de cDNA. As amostras de cDNA foram ajustadas para igual concentração e submetidas à PCR quantitativa em tempo real (qRT-PCR). As amostras foram avaliadas ainda por meio de imuno-histoquímica para expressão de BRCA1. Resultados: Um total de 234 registros de imuno-histoquímica foi avaliado, resultando em 56 casos de carcinomas de mama apresentando fenótipo não triplonegativo e 36 com fenótipo triplo-negativo. A sobrevida das pacientes em função das características clinicopatológicas demonstrou associações com os casos triplonegativos (p=0,044), estádios mais avançados (p=0,005), acometimento linfonodal (p=0,038), presença de metástase à distância (p=0,0008) e ausência da expressão de BRCA1 (p=0,039). Associações significativas foram demonstradas entre a ausência de BRCA1 e o fenótipo triplo-negativo (p=0,0346), entre a ausência da expressão do receptor de estrógeno e ausência da expressão de BRCA1 (p=0,006), e entre o receptor de progesterona e a ausência da expressão de BRCA1 (p=0,0163). A análise por qRT-PCR demonstrou diferentes níveis de expressão de miR-10b e de miR-7, com associações significativas ao fenótipo triplo-negativo (p=0,021; p=0,042) e à ausência de BRCA1 (p=0,039; p=0,006). A comparação entre ausência e presença da expressão do receptor do fator de crescimento epidérmico humano demonstrou diferença significativa para miR-7 (p=0,031), sendo que a expressão de miR-10b nestes casos não foi estatisticamente diferente (p=0,926). Conclusão: Associações significativas foram demonstradas entre a ausência de BRCA1 e o fenótipo triplo-negativo. A sobrevida em cinco anos foi inversamente associada ao fenótipo triplo-negativo, aos estádios clínicos III e IV, à presença de metástase linfonodal, à presença de metástase à distância e à ausência da expressão de BRCA1. Este estudo demonstrou ainda que hsa-miR-7 e hsa-miR- 10b estão significativamente associados à ausência da expressão de BRCA1 e ao fenótipo triplo-negativo, sendo observada pior sobrevida nestes perfis de pacientes. Estudos com maior número de casos e com linhagens celulares devem ser realizados para constatar o papel de hsa-miR-7 e hsa-miR-10b na modulação da expressão de BRCA1.

Chez les patients souffrant d'insuffisance rénale chronique, les calcifications vasculaires représentent la première cause de mortalité. Elles résultent de la trans-différenciation des cellules musculaires lisses (CMLs) en cellules de type ostéoblastique et/ou chondrocytaire, en réponse à des cytokines inflammatoires ou à une hyperphosphatémie. Les CMLs forment alors des cristaux par l'activité de la phosphatase alcaline non-spécifique du tissu (TNAP). A la lumière de résultats récents, nous avons émis l'hypothèse que la TNAP module la trans différenciation des CMLs. Nos objectifs étaient donc de déterminer l'effet de la TNAP dans la trans-différenciation des CMLs, et d'étudier les mécanismes impliqués dans son induction, avec un intérêt particulier pour les microRNAs. Nous avons observé que l'ajout de phosphatase alcaline purifiée ou la surexpression de TNAP stimule l'expression de marqueurs chondrocytaires en culture de CMLs et de cellules souches mésenchymateuses. De plus, l'inhibition de la TNAP bloque la maturation de chondrocytes primaires. Nous excluons un rôle des cristaux formés par la TNAP, puisque l'ajout de cristaux seuls ou associés à une matrice collagénique n'a pas reproduit les effets de la TNAP. Nous suspectons que la TNAP agit en hydrolysant le pyrophosphate inorganique (PPi). En effet, c'est la TNAP qui hydrolyse le PPi en culture de CMLs et de chondrocytes, et le PPi mime les effets de l'inhibition de TNAP en culture de chondrocytes. Enfin, nous rapportons le profil de microRNA des artères cultivées en conditions hyperphosphatémiques. Ces résultats pourraient être particulièrement importants dans le développement de nouvelles approches thérapeutiques / In patients with chronic kidney disease (CKD), vascular calcification represents the main cause of mortality. Vascular calcification results from the trans-differentiation of vascular smooth muscle cells (VSMCs) into cells similar to osteoblasts and/or chondrocytes, in response to inflammatory cytokines or hyperphosphatemia. Calcifying VSMCs form calcium phosphate crystals through the activity of tissue nonspecific alkaline phosphatase (TNAP). In light of recent findings, we hypothesized that TNAP also modulates VSMC trans-differentiation. Our objectives were therefore to determine the effect of TNAP activity on VSMC trans-differentiation, and secondly to investigate the molecular mechanisms involved in TNAP expression in aortas, with a particular interest in microRNAs. We first observed that addition of purified alkaline phosphatase or TNAP over-expression stimulates the expression of chondrocyte markers in culture of the mouse and rat VSMC lines, and of mesenchymal stem cells. Moreover, TNAP inhibition blocks the maturation of mouse primary chondrocytes and reduces mineralization. We exclude a role for crystals in TNAP effects, since addition of crystals alone or associated to a collagenous matrix fails to mimic TNAP effects. We rather suspect that TNAP acts through the hydrolysis of inorganic pyrophosphate (PPi). Indeed, PPi is hydrolyzed by TNAP in VSMCs and chondrocytes and addition of PPi mimics the effects of TNAP inhibition on chondrocyte maturation. Finally, we report microRNA signature of aortic explants treated under hyperphosphatemic conditions that induce vascular calcification. These results could be of particular importance in patients with CKD

No description available.

Les microARN (miARN) sont de petits ARN non-codants qui répriment la traduction de leurs gènes cibles par hybridation à leur ARN messager (ARNm). L'identification de cibles biologiquement actives de miARN est cruciale afin de mieux comprendre leurs rôles. Ce problème est cependant difficile parce que leurs sites ne sont définis que par sept nucléotides. Dans cette thèse je montre qu'il est possible de modéliser certains aspects des miARN afin d'identifier leurs cibles biologiquement actives à travers deux modélisations d'un aspect des miARN. La première modélisation s'intéresse aux aspects de la régulation des miARN par l'identification de boucles de régulation entre des miARN et des facteurs de transcription (FT). Cette modélisation a permis, notamment, d'identifier plus de 700 boucles de régulation miARN/FT, conservées entre l'humain et la souris. Les résultats de cette modélisation ont permis, en particulier, d'identifier deux boucles d'auto-régulation entre LMO2 et les miARN miR-223 et miR-363. Des expériences de transplantation de cellules souches hématopoïétiques et de progéniteurs hématopoïétiques ont ensuite permis d'assigner à ces deux miARN un rôle dans la détermination du destin cellulaire hématopoïétique. La deuxième modélisation s'intéresse directement aux interactions des miARN avec les ARNm afin de déterminer les cibles des miARN. Ces travaux ont permis la mise au point d'une méthode simple de prédiction de cibles de miARN dont les performances sont meilleures que les outils courant. Cette modélisation a aussi permis de mettre en lumière certaines conséquences insoupçonnées de l'effet des miARN, telle que la spécificité des cibles de miARN au contexte cellulaire et l'effet de saturation de certains ARNm par les miARN. Cette méthode peut également être utilisée pour identifier des ARNm dont la surexpression fait augmenter un autre ARNm par l'entremise de miARN partagés et dont les effets sur les ARNm non ciblés seraient minimaux. / microRNAs (miRNAs) are small non coding RNAs that repress the translation of their target genes by pairing to their messenger RNA (mRNA). The identification of miRNAs' biologically active targets is a difficult problem because their binding sites are defined by only seven nucleotides. In this thesis, I show that it is possible to model specific aspects of miRNAs to identify their biologically active targets through two modeling of each one aspect of miRNAs. The first modeling considers the miRNAs regulations through the identification of regulatory loops between miRNAs and transcription factors (TFs). Through this modeling, we identified over 700 miRNA/TF regulatory loops conserved between human and mouse. With the results of this modeling, we were able to identify, in particular, two regulatory loops between LMO2 and the miRNAs miR-223 and miR-363. Using hematopoietic stem cells and progenitor cells transplantation experiment we showed that miR-223 and miR-363 are involved in hematopoietic cell fate determination. The second modeling focuses directly on the interaction between miARN and messenger RNA (mRNA) to determine the miRNA targets. With this work, we developed a simple method for predicting miRNA targets that outperforms the current state of the art tool. This modeling also highlighted some unsuspected consequences of miRNA effects such as the cell context specificity and the saturation of mRNA targets by miRNA. This method can also be used to identify mRNAs whose overexpression increases the expression level of another mRNA through their shared miRNA and whose global effects on other genes are minimal.