The role of reciprocal regulation between TAL1 and miRNA expression in T-cell leukemia progression

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2015

STUDYING ABERRANT METHYLATION AND miRNA PROFILING FOR THE DETECTION OF LUNG CANCER BIOMARKERS

Lung cancer is a major chronic disease responsible for the highest mortality rate, among other types of cancer, and represents 29% of all deaths in Canada. The clinical diagnosis of lung carcinoma still requires a standard diagnostic approach, as there are no symptoms in its early stage. Therefore, it is usually diagnosed at a later stage, when the survival rate is low. With the recent advancement in molecular biology and biotechnology, a molecular biomarker approach for the diagnosis of early lung cancer seems to be a potential option. In this study, we aimed to investigate and standardize a promising Lung ,Cancer Biomarker by studying the aberrant methylation of two tumour suppressor genes, namely RASSFIA and RAR-B, and the miRNA profiling of four . commonly deregulated miRNA (miR-199a-3p, miR-182, miR-lOO and miR-221). Four lung cancer cell lines were used (two SCLC and two NSCLC), with comparisons being made with normal lung cell lines. Our results, we found that none of these genes were methylated. We then evaluated TP53, and found the promoter of this gene to be methylated in the cancer cell lines, as compared to the normal cell lines, indicating gene inactivation. We carried out miRNA profiling of the cancer cell lines and reported that 80 miRNAs are deregulated in lung cancer cell lines as compared to the normal cell lines. Our study was the first of its kind to indicate that hsa-mir-4301, hsa-mir-4707-5p and hsa-mir-4497 (newly discovered miRNAs) are deregulated in lung cancer cell lines. We also investigated miR-199a-3p, mir-lOO and miR-182, and found that miR-199a -3p and mir-l00 were down-regulated in cancer lines, whereas miR-182 was up-regulated in the cancer cell lines. In the final part of the study we observed that mir-221 could be a putative biomarker to distinguish between the two types of lung cancer because it was down-regulated in SCLC, and up-regulated in the NSCLC cell lines. In conclusion, we found four miRNA molecular biomarkers that possibly could be used in the early diagnosis of the lung cancer. More studies are still required with larger numbers of samples to effectively establish these as molecular biomarkers for the diagnosis of lung cancer

Potential Urinary miRNA Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients

MicroRNAs (miRNAs) are a class of short (similar to 22nt), single stranded RNA molecules that function as post-transcriptional regulators of gene expression. MiRNAs can regulate a variety of important biological pathways, including: cellular proliferation, differentiation and apoptosis. Profiling of miRNA expression patterns was shown to be more useful than the equivalent mRNA profiles for characterizing poorly differentiated tumours. As such, miRNA expression "signatures" are expected to offer serious potential for diagnosing and prognosing cancers of any provenance. The aim of this study was to investigate the potential of using deregulation of urinary miRNAs in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the miRNA signatures specific for PCa, miRNA expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using whole genome expression profiling. Differential expression of two individual miRNAs between healthy males and BPH patients was detected and found to possibly target genes related to PCa development and progression. The sensitivity and specificity of miR-1825 for detecting PCa among BPH individuals was found to be 60% and 69%, respectively. Whereas, the sensitivity and specificity of miR-484 were 80% and 19%, respectively. Additionally, the sensitivity and specificity for miR-1825/484 in tandem were 45% and 75%, respectively. The proposed PCa miRNA signatures may therefore be of great value for the accurate diagnosis of PCa and BPH. This exploratory study has identified several possible targets that merit further investigation towards the development and validation of diagnostically useful, non-invasive, urine-based tests that might not only help diagnose PCa but also possibly help differentiate it from BPH.

Estrategias metodológicas para mejorar la enseñanza de la historia mundial del módulo de ciencias sociales de preparatoria [por] Gloria Esparza Hernández y María Mirna Granat Ramos

Tesis (Maestría en Enseñanza Superior) U.A.N.L.

Cambios verticales de los maxilares y la oclusión en pacientes clase II en crecimiento tratados con headgear cervical comparado con el headgear de Cervera por Mirna Elizabeth Molina Salinas

Tesis (Maestría en Ciencias Odontológicas con Especialidad en Ortodoncia) U.A.N.L.

MEF2C-MYOCD and leiomodin1 suppression by miRNA-214 promotes smooth muscle cell phenotype switching in pulmonary arterial hypertension

Background Vascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis. Methods and Results In SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis. Conclusions Our findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH.

miRNA expression in human lung cancer and fetal lung: a comparative study

Trabalho apresentado na São Paulo Advanced School of Comparative Oncology, Águas de São Pedro, SP, 2012.

Differential MiRNA expression in childhood acute lymphoblastic leukemia and association with clinical and biological features

The present study aimed to analyze the expression profile of the microRNAs previously described as associated with childhood ALL, miR-92a, miR-100, miR-125a-5p, miR-128a, miR-181b, miR-196b and let-7e, and their association with biological/prognostic features in 128 consecutive samples of childhood acute lymphoblastic leukemia (ALL) by quantitative real-time PCR. A significant association was observed between higher expression levels of miR-196b and T-ALL, miR-100 and patients with low white blood cell count at diagnosis and t(12;21) positive ALL. These findings suggest a potential activity of these microRNAs in pediatric ALL biology. (C) 2011 Elsevier Ltd. All rights reserved.

A possible new mechanism for the control of miRNA expression in neurons

The control of gene expression by miRNAs has been widely investigated in different species and cell types. Following a probabilistic rather than a deterministic regimen, the action of these short nucleotide sequences on specific genes depends on intracellular concentration,which in turn reflects the balance between biosynthesis and degradation. Recent studies have described the involvement of XRN2, an exoribonuclease, in miRNA degradation and PAPD4, an atypical poly(A) polymerase, in miRNA stability. Herein, we examined the expression of XRN2 and PAPD4 in developing and adult rat hippocampi. Combining bioinformatics and real-time PCR,we demonstrated that XRN2 and PAPD4 expression is regulated by the uncorrelated action of transcription factors, resulting in distinct gene expression profiles during development. Analyses of nuclei position and nestin labeling revealed that both proteins progressively accumulated during neuronal differentiation, and that they are weakly expressed in immature neurons and absent in glial and endothelial cells. Despite the differences in subcellular localization, both genes were concurrently identified within identical neuronal subpopulations, including specific inhibitory interneurons. Thus, we cope with a singular circumstance in biology: an almost complete intersected expression of functional-opposed genes, reinforcing that their antagonistically driven actions on miRNAs “make sense” if simultaneously present at the same cells. Considering that the transcriptome in the nervous system is finely tuned to physiological processes, it was remarkable that miRNA stability-related genes were oncurrently identified in neurons that play essential roles in cognitive functions such as memory and learning. In summary, this study reveals a possible new mechanism for the control of miRNA expression.

MiRNA-208a and miRNA-208b are triggered in thyroid hormone-induced cardiac hypertrophy - role of type 1 Angiotensin II receptor (AT1R) on miRNA-208a/α-MHC modulation

Hyperthyroidism promotes cardiac hypertrophy and the Angiotensin type 1 receptor (AT1R) has been demonstrated to mediate part of this response. Recent studies have uncovered a potentially important role for the microRNAs (miRNAs) in the control of diverse aspects of cardiac function. Then, the objective of the present study was to investigate the action promoted by hyperthyroidism on β-MHC/miR-208b expression and on α-MHC/miR-208a expression, as well as the possible contribution of the AT1R in this event. The findings of this study confirmed that AT1R is a key mediator of the cardiac hypertrophy induced by hyperthyroidism. Additionally, we demonstrated that like β-MHC, miR-208b was down-regulated in the hyperthyroid group. Similarly, like the expression of its host gene, α-MHC, miR-208a expression was up-regulated in response to hyperthyroidism. Finally, our data suggest for the first time that AT1R mediates the hyperthyroidism-induced increase on cardiac miRNA-208a/α-MHC levels, while does not influence on the reduction of miRNA-208b/β-MHC levels.

Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.

MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.