Study of the microRNA expression profile dysregulation by hydrogen peroxide on the retinal pigment epithelium cells: role of miR-205-5p

Age related macular degeneration (ADM) and diabetic retinopathy (DR) are common retina-related diseases leading to blindness. The retinal pigment epithelium (RPE) is essential for the vision, in fact is well documented that oxidative stress (OS) generated in RPE and choroid neovascularization are related to the origin of these disorders. The study of circulating miRNAs, small non-codding RNA, is opening new possibilities in terms of diagnosis and therapeutics. miRNAs can travel inside small Extracellular Vesicles (sEVs) playing an important role in intracellular communication. Concretely, miR205-5p is involved in VEGF-related angiogenesis and seems to regulate associated cell signaling pathways. One of the aims of the present work is to establish a miRNA expression profile of ARPE-19 cells and sEVs released from ARPE-19 cells under oxidative conditions (H2O2). Moreover, we want to evaluate the role of the identified miRNAs and sEVs in the progression of pathological processes related to retinal disorders. The miRNA expression profile was carried out using SeraMir Profiler that includes 384 miRNAs whose expression was analyzed in ARPE-19 cells and sEVs released by ARPE-19 cells, both in control conditions and treated with 600 M H2O2 for 24 hours. Additionally, the role of miRNAs and sEVs in angiogenic processes was studied using matrigel assays with HUVEC cells. We identify a different miRNA expression profile between oxidative stressed and control ARPE-19 cells (Cell miRNAs) and the sEVs (sEV miRNAs) released by them. As a result, 306 out of 384 Cell miRNAs were detected by the array and 7 were significantly overexpressed in H2O2-treated cells. Moreover, 218 miRNAs could be detected in control and H2O2-induced sEVs and only 2 of them were significantly under-expressed in H2O2-induced sEVs. Among the dysregulated Cell miRNAs we found the miR-205-5p. Our results indicate that miR-205-5p is modulated by OS and regulates VEGFAangiogenesis. We also found the role of 600 M H2O2-sEVs on oxidative stress induction, cell death increasing and angiogenesis stimulation in healthy ARPE-19 cells. The miRNA dysregulation observed suggest their implication in retinal disorders after oxidative challenge. Among the dysregulated miRNAs, miR-205-5p is proposed as a candidate against eye-related proliferative diseases. Therefore, results herein suggest the influence of sEV in neighboring cells and the possible utility of sEV miRNAs as biomarkers.La degeneración macular asociada con la edad (AMD) y la retinopatía diabética (RD) son alteraciones de la retina que llevan a la perdida de la visión. El epitelio pigmentario de la retina (RPE) es esencial para la visión, de hecho está bien documentado que el estrés oxidativo (OS) generado en el RPE y la neovascularización de la coroides están relacionados con el origen de estas alteraciones. El estudio de los microRNAs (miRNAs) circulantes, pequeños RNAs no codificantes, está abriendo nuevas posibilidades en el campo del diagnostico y de la terapia. Los miRNAs pueden viajar en el interior de pequeñas vesículas extracelulares (sEVs) jugando un papel importante en la comunicación intracelular. Concretamente, el miR-205-5p está involucrado en los procesos de angiogenesis relacionados con el VEGF y además parece regular las vías de señalización asociadas. Uno de los objetivos de este trabajo es determinar los perfiles de expresión de los miRNAs de las células ARPE-19 y de las sEVs liberadas por las ARPE-19 bajo condiciones oxidantes (H2O2). Además, queremos evaluar el papel de los miRNAs identificados y de las sEVs en la progresión de los procesos patológicos relacionados con las alteraciones de la retina. El perfil de expresión de los miRNAs se llevo a cabo utilizando SeraMir Profiler que incluye 384 miRNAs, cuya expresión se analizó en las células ARPE-19 y en las sEVs liberadas por estas células, tanto en condiciones control como sometidas a 600 M H2O2 durante 24 horas. Por otro lado, el papel de los miRNAs y las sEVs en los procesos angiogenicos se determino mediante el uso de ensayos de matrigel con células HUVEC.Identificamos un perfil de expresión de miRNAs diferente entre células ARPE-19 sometidas a estrés oxidativo y control (Cell miRNAs) y entre las sEVs liberadas por estas (sEV miRNAs). Como resultado, se detectaron 306 de los 384 Cell miRNAs analizados y 7 de ellos estaban significativamente sobrexpresados en las células tratadas con H2O2. Además, se detectaron 218 miRNAs en las sEVs liberadas por células controles y sometidas a H2O2 y solamente 2 de ellos estaban significativamente infraexpresados en las sEVs sometidas a H2O2. Entre los Cell miRNA desregulados encontramos el miR-205-5p. Nuestros resultados indican que el miR-205-5p está modulado por el OS y regula la angiogenesis mediada por el VEGFA. También determinamos el papel de las sEVs - H2O2 en la inducción del OS, el aumento de la muerte celular y la estimulación de la angiogenesis en células ARPE-19 sanas. Los miRNAs desregulados sugieren su implicación en los desordenes de la retina tras la exposición a OS. Entre los miRNAs desregulados, el miR-205-5p se propone como un candidato contra los procesos proliferativos relacionada con las alteraciones de la retina. Por otra parte, nuestros resultados demuestran la influencia de las sEVs en las células vecinas y la posible utilidad de los sEV miRNAs como biomarcadores.Generalitat Valenciana (PROMETEO/2016/094)Ayudas Internas Investigación de la Universidad Católica de Valencia San Vicente Mártir (2017-128-001 y 2018-128-001)Ayuda a Proyectos de Investigación San Alberto Magno de la Universidad Católica de Valencia San Vicente Mártir (2019-128-001)MedicinaCiencias de la Vida y del Medio Natura

MicroRNA involvement in breast cancer susceptibility and progression

RESUMO: Os microRNAs (miRNAs) são pequenos RNAs não codificantes com função reguladora que regulam a expressão génica ao ligar-se a sequências específicas na região 3’ UTR dos mRNAs. Diversos estudos mostraram que os miRNAs regulam mecanismos fundamentais para o normal funcionamento celular, como crescimento celular, proliferação, diferenciação e apoptose. A expressão de alguns miRNAs é alterada em diversos tipos de cancro, nomeadamente em cancro da mama. Estudos de análise funcional em linhas celulares mostraram que os miRNAs podem funcionar como supressores de tumor ou ter actividade oncogénica. Assim, o valor clínico dos miRNAs como potenciais marcadores para cancro da mama está a ser amplamente estudado de momento. No entanto, apenas se conhecem efeitos de alguns miRNAs. A maior dificuldade, neste âmbito, depreende-se com a identificação de possíveis alvos com relevância biológica para cancro da mama. Visto que os programas bioinformáticos predizem um elevado número de falsos positivos e falsos negativos, é de extrema importância identificar experimentalmente alvos relevantes. Nesta tese procuramos explorar diferentes abordagens da influência de miRNAs em cancro da mama. Começamos por estudar os mecanismos que estão por trás da regulação dos próprios miRNAs. Colocámos a hipótese de serem mecanismos epigenéticos, tais como a metilação de citosinas no DNA, que estão a influenciar os níveis de expressão dos miRNAs. Para tal, tratámos linhas celulares de mama com um agente capaz de desmetilar o DNA e verificámos que os níveis de miRNAs são alterados. Contudo, não conseguimos encontrar uma associação entre a metilação de ilhas CpG nas regiões promotoras dos genes que codificam para os miRNAs. No entanto, não podemos excluir a possibilidade de os níveis de expressão de miRNAs estarem a ser regulados por metilação das suas zonas promotoras, dado que não estudámos todas as regiões promotoras existentes. De seguida, abordámos a influência de dois miRNAs, miR-200c e miR-203, na resistência para fármacos dirigidos a cancro da mama, nomeadamente, Paclitaxel e 5-fluoruacil. Para tal fizemos expressar ambos os miRNAs na linha celular MDA-MB-231 e inibir os mesmos na linha celular MCF-7. Infelizmente não fomos capazes de encontrar significado estatístico nos resultados obtidos. Contudo pudemos concluir que o miR-200c parece ter um efeito contrário nas linhas MCF-7 e MDA-MB-231 no que diz respeito ao tratamento com Paclitaxel e o miR-203 parece aumentar a resistência para o mesmo comporto na linha celular MDA-MB-231. O tratamento com 5-fluoruacil não mostrou qualquer diferença em ambas as linhas. Dado que os estudos in vitro, nesta área, devem ser transpostos para humanos e/ou tecidos humanos, seguidamente procurámos estudar os níveis de expressão do miR-200c e do miR-203 em tecido tumoral mamário, bem como a expressão de dois alvos hipotéticos encontrados utilizando ferramentas bioinformáticas, SIX1 e SOX2. Relativamente ao miR-200c, não encontrámos quaisquer diferenças entre tecido normal e tecido tumoral de mama, nem conseguimos relacionar este miRNA com características clinicopatológicas. Comparativamente detectaram-se diferenças para o miR-203 e conseguimos relacionar este com os estadios iniciais de desenvolvimento tumoral. Conseguimos também demonstrar que o miR-203 pode ser um potencial marcador para discriminar os tumores lobulares invasivos. No que diz respeito à expressão do SOX1 e SOX2, observámos que ambos possuem uma incidência baixa na nossa população e que não se associam com a expressão dos miRNAs em estudo. Por último, procurámos validar alguns alvos do miR-200c e do miR-203. Para tal, efectuámos um estudo comparativo de proteómica, onde fizemos expressar o miR-200c e o miR-203 na linha celular MDA-MB-231 e inibimos os mesmos miRNAs na linha celular MCF-10A. Este estudo exploratório, ainda por terminar, revelou aproximadamente 3000 proteínas diferentemente expressas nas linhas celulares. No entanto, até ao momento conseguimos reduzir esta vasta lista para uma menor com aproximadamente 10 proteínas para cada linha celular. De futuro, serão seguidas outras abordagens para validar estes alvos hipotéticos. Devido ao facto da população recolhida ser recente, o seguimento clínico nos próximos anos permitirá tirar algumas conclusões relativas à resistência à terapia utilizada e a sua relação com a expressão dos miRNAs em estudo.ABSTRACT: MicroRNAs (miRNAs) are small non-coding regulatory RNAs that modulate gene expression by binding to their target mRNAs. By these means, miRNAs control normal rates of all major cellular pathways. A subset of miRNAs, which are differentially detected between normal and tumour tissue samples, has been identified in breast cancer, and functional analysis in cell line systems has revealed tumour suppressive and oncogenic functions of some of these miRNAs. Hence, the clinical value of these as novel biomarkers for cancer is being actively investigated. However, the function of only a few of these miRNAs in breast cancer has been investigated. One major difficulty is the identification of target mRNAs and proteins with biological significance in breast cancer and consequently the identifications of the pathways they influence. Given the relatively high rates of both false-positives and false-negatives from current miRNAs targets prediction programs, it is critically important to experimentally identify relevant miRNAs targets and the pathways involved in carcinogenesis. In this thesis our main goal was to study the role of miRNAs in breast cancer. Thus, we started by addressing the mechanisms behind regulation of miRNAs expression levels. We hypothesized if that epigenetic mechanisms, such as DNA methylation, could influence miRNAs expression levels. Therefore, we treated breast cell lines with demethylating agents and observed that miRNAs expression levels were altered. However, we failed to prove a direct correlation between the methylation of CpG islands in promoter regions of the miRNAs studied and their expression. Nevertheless, we cannot exclude the possible regulation of miRNAs levels by methylation since we did not study all possible promoter regions of miRNAs genes as their promoter regions have not been fully identified. Next, we addressed the possible effect of miRNAs in breast cancer therapy resistance. For that, we treated breast tumour cell lines with Paclitaxel and 5-fluoruacil, two known chemotherapeutics used in breast cancer, with the ectopic expression or inhibition of miR-200c and miR-203. Unfortunately, we did not find any statistical difference between untreated and treated cells. However, miR-200c seems to have contrary effects in MCF-7 and MDA-MB-231 cells regarding treatment with Paclitaxel and miR-203 seem to augment resistance to Paclitaxel in MDA-MB-231 cells. Both miRNAs did not show any effect in cells treated with 5-Fluoruacil. Since in vitro studies, always lack studies using human tissues. We subsequently studied the expression levels of miR-200c and miR-203 in breast tumour tissues and two putative targets found by bioinformatics tools, SIX1 and SOX2. Concerning miR-200c, we did not detect significant differences between normal breast and tumour tissues in our population. Additionally, we failed to correlate miR-200c with clinicopathological features. Regarding miR-203, we detect statistically differences between normal and tumour tissue and it seems that miR-203 is involved in breast cancer development, mainly in early stages of development. We also show that miR-203 might be a potential marker to discriminate stages in invasive lobular carcinoma. About the expression levels of SIX1 and SOX2, we observe relatively low levels of both proteins through immunohistochemistry and do not found any statistically difference between their expression and their regulators, miR-200c and miR-203. Finally, we address the validation of putative targets of the miR-200c and miR-203. Thus, we conducted a comparative proteomic study to find differently expressed proteins when miR-200c and miR-203 were ectopically expressed or inhibited in breast cell lines. This exploratory study, until now, revealed a small list out of approximately 3000 proteins that are putative targets of both miRNAs and are differently expressed. Further studies will be conducted in order to validate these putative targets. With this thesis, we believe we provide new insight into the involvement of miRNAs in breast cancer and also important knowledge of how miRNAs levels are being regulated and also in the discovery of new targets. We also gathered a considerable study population during this thesis, which allow future correlations on therapy outcomes and survival with the biomarkers studied

Metabolism and Epigenetic Interplay in Cancer: Regulation and Putative Therapeutic Targets

Alterations in the epigenome and metabolism affect molecular rewiring of cancer cells facilitating cancer development and progression. Modulation of histone and DNA modification enzymes occurs owing to metabolic reprogramming driven by oncogenes and expression of metabolism-associated genes is, in turn, epigenetically regulated, promoting the well-known metabolic reprogramming of cancer cells and, consequently, altering the metabolome. Thus, several malignant traits are supported by the interplay between metabolomics and epigenetics, promoting neoplastic transformation. In this review we emphasize the importance of tumour metabolites in the activity of most chromatin-modifying enzymes and implication in neoplastic transformation. Furthermore, candidate targets deriving from metabolism of cancer cells and altered epigenetic factors is emphasized, focusing on compounds that counteract the epigenomic-metabolic interplay in cancer

Genetic deletion and inhibition of selenoprotein K reduces melanoma growth and metastasis by altering intracellular calcium

Ph.D.Ph.D. Thesis. University of Hawaiʻi at Mānoa 201

Advances in the Diagnosis and Treatment of Thyroid Carcinoma

This reprint is related to the latest research in the field of thyroid surgery, including molecular and imaging diagnosis, surgical treatment, and the treatment of recurrent disease and advanced thyroid carcinoma

Activité anti-tumorale de la voie XBP1 dans les leucémies aiguës myéloïdes

Les Leucémies Aigües Myéloïdes (LAM) sont des hémopathies malignes, caractérisées par un l'expansion de blastes leucémiques, des progéniteurs myéloïdes bloqués à différents stades de leur différenciation, qui colonisent la moelle osseuse, puis le sang, pour enfin s'infiltrer dans différents tissus et former des métastases. Le traitement de référence de ces hémopathies repose sur une chimiothérapie classique associant de l'Aracytine à une anthracycline. Cependant, de fréquentes rechutes sont observées et sont marquées par une chimiorésistance des patients. De façon intéressante, une étude clinique menée par Schardt et collaborateur a révélé un lien entre l'activation de XBP1, un facteur de transcription exprimé dans le cadre du stress du Réticulum Endoplasmique, et un meilleur pronostic chez les patients atteint de LAM. Dans le but de préciser le rôle de XBP1 dans ces pathologies, nous avons développé un modèle permettant l'expression conditionnelle de XBP1, sous contrôle d'un promoteur sensible à la doxycycline, dans des lignées cellulaires de LAM. Nos résultats démontrent qu'une activation soutenue de XBP1 conduit à l'apoptose des cellules in vitro et in vivo. De plus, une activation plus modérée de XBP1 conduit à une chimiosensibilisation à l'Aracytine in vitro et in vivo. Enfin, une activation chronique de XBP1 déverrouille la différenciation myéloïde in vitro et in vivo. Par le biais d'analyses à grande échelle, nous avons identifiées deux cibles transcriptionnelles non-codantes de XBP1, miR-22-3p et miR-148a-3p, qui récapitulent à elles seules les phénotypes liés à l'expression de XBP1. L'expression de miR-22 induit l'apoptose et sensibilise à l'Aracytine via la diminution de l'expression de la protéine SIRT1. De plus, l'expression chronique de miR-22 et miR-148 inhibent respectivement l'expression de c-MYB et HEX, deux facteurs de transcription impliqués dans la répression de la différenciation myéloïde. L'ensemble de nos résultats démontrent non seulement une activité anti-leucémique du facteur de transcription XBP1, mais mettent également en lumière l'importance de ses cibles non codantes dans les mécanismes moléculaires sous-jacents.Acute Myeloid Leukaemia (AML) is a rare and highly fatal haematological disorder, characterized by progressive invasion of bone marrow, circulating blood and peripheral organs by abnormal myeloid progenitors named blasts. Blasts are defined by differentiation blockade, and keep typical features of myeloid progenitors, like high proliferation rate and survival increased. AML treatment is based on chemotherapy combining Aracytin with anthracyclin, which mainly target proliferative cells. However, relapses are frequently observed, associated with chemoresistance. Interestingly, a clinical study links ER-stress-related transcription factor XBP1 expression to favourable outcome in AML. In order to precise the role of XBP1 on AML, we developed a tetracycline-inducible XBP1 model in leukemic cell lines. Our studies clearly demonstrate that the sustained activation of XBP1 expression induce apoptosis both in vitro and in vivo, whereas a moderate XBP1 expression sensitizes cells to chemotherapeutic treatments. Finally, a chronic activation of XBP1 in AML cells expression unlock myeloid differentiation both in vitro and in vivo. Using ChIP-sequencing, RNA-sequencing and micro-RNA sequencing data, we identified two specific-XBP1s-non-coding targets, miR-22 and miR148a-3p, which recapitulate XBP1-related phenotypes. XBP1 transcriptional activation of miR-22 significantly suppresses viability and sensitizes leukemic cell to chemotherapy, through SIRT1 protein knockdown. Moreover, chronic expression of miR-22 and miR148a-3p inhibit respectively C-MYB and HEX expression, two transcription factors related to repression of myeloid differentiation, leading to myeloid differentiation progression. Taking together, these results show XBP1 anti-leukemic effects, through its non-coding targets implication in molecular mechanisms

Urological Cancer 2020

This Urological Cancer 2020 collection contains a set of multidisciplinary contributions to the extraordinary heterogeneity of tumor mechanisms, diagnostic approaches, and therapies of the renal, urinary tract, and prostate cancers, with the intention of offering to interested readers a representative snapshot of the status of urological research