miR-9/9* in Myeloid Development and Acute Myeloid Leukemia

miR-9/9* have been shown to be deregulated in different types of human cancer including lymphoid and myeloid malignancies. Nevertheless, we still lack the more comprehensive knowledge about the impact of miR-9/9* expression on normal hematopoietic cell function and their possible impact in the biology of AML. In Chapter 2, we aim to elucidate the function of miR-9/9* in normal myeloid differentiation. We use murine myeloid 32D cell line model and murine primary HSPCs to investigate the impact of miR-9/9* overexpression on granulocytic differentiation induced by G-CSF. We also examine the effect of miR-9/9* expression in human primary AML samples. Further, we set out to identify miR-9/9* targets that are common for murine and human cells and that may be relevant for the observed phenotype. In Chapter 3, we broaden our quest for direct and indirect targets that are regulated by miR-9/9* by analyzing proteome changes in 32D cells. We examine the deregulated proteins and the involved pathways that could potentially influence different aspects of normal hematopoietic cell function. Our observations are further extended by in vivo and in vitro experiments presented in Chapter 4, we investigate the influence of miR-9/9* expression on homing of normal HSPCs in the BM. Until now, few miRNAs have been shown to exhibit a prognostic value in AML. In Chapter 5, based on our results about the expression of miR-9/9* in AML patients (shown in Chapter 1), we set out to investigate whether the expression of miR-9/9* may predict patient outcome in AML. Finally, the results presented in this thesis are summarized and discussed in Chapter 6. Here we put the accumulated insights about the functions of miR-9/9* into the broader perspective of human cancer

The versatile nature of miR-9/9* in human cancer

miR-9 and miR-9* (miR-9/9*) were first shown to be expressed in the nervous system and to function as versatile regulators of neurogenesis. The variable expression levels of miR-9/9* in human cancer prompted researchers to investigate whether these small RNAs may also have an important role in the deregulation of physiological and biochemical networks in human disease. In this review, we present a comprehensive overview of the involvement of miR-9/9* in various human malignancies focusing on their opposing roles in supporting or suppressing tumor development and metastasis. Importantly, it is shown that the capacity of miR-9/9* to impact tumor formation is independent from their influence on the metastatic potential of tumor cells. Moreover, data suggest that miR-9/9* may increase malignancy of one cancer cell population at the expense of another. The functional versatility of miR-9/9* emphasizes the complexity of studying miRNA function and the importance to perform functional studies of both miRNA strands in a relevant cellular context. The possible application of miR-9/9* as targets for miRNA-based therapies is discussed, emphasizing the need to obtain a better understanding of the functional properties of these miRNAs and to develop safe delivery methods to target specific cell populations

The importance of frequent follow-up in the first year after pacemaker implantation upon detection of atrial tachyarrhythmia

Introduction: Newer pacemakers have data storage capabilities that permit detection of multiple episodes of atrial tachyarrhythmias (AT). Aim of the research : To document the frequency, time to first AT detection after implantation, and risk factors for symptomatic episodes and to determine the prevalence and predictors of pacemaker-detected AT. Material and methods: Patients (n = 62), from a single centre, with an implanted pacemaker that automatically recorded the cumulative daily AT burden were included in the analysis. The clinical history regarding AT was collected before implantation. Follow-ups were done at 3, 6, and 12 months after implantation with interrogation and symptoms reported from patients. Echocardiography was performed at baseline and at 12 months. Results: Atrial tachyarrhythmia was detected by the pacemaker in 16 (25.81%) of 62 patients. AT/atrial fibrillation (AF) burden was detected after 3 months in 13 out of 16 (81.25%) patients, and in 38.46% of them there was no clinical history of AF. Asymptomatic episodes occurred in 7 patients (43.75% – 3 men and 4 women), and 2 (28.57%) of them did not have a clinical history of AF. History of AF prior to implantation was significant for the appearance of AT/AF burden (p = 0.006), and use of β-blockers significantly limited AT/AF burden (p = 0.040). Other analysed data was statistically non-significant. Conclusions: Pacemaker-detection of AT is often the first paroxysmal AT diagnosis in asymptomatic patients. This suggests that AT data, collected from capable pacemakers and frequently reviewed, can lead to new diagnosis and early treatment in that group of patients, which could influence mortality and morbidity

Expression of a passenger miR-9* predicts favorable outcome in adults with acute myeloid leukemia less than 60 years of age

In double-stranded miRNA/miRNA* duplexes, one of the strands represents an active miRNA, whereas another, known as a passenger strand (miRNA*), is typically degraded. MiR-9* is not detectable in normal myeloid cells. Here we show that miR-9* is expressed in 59% of acute myeloid leukemia (AML) cases and we investigate its clinical impact in 567 adults with de novo AML (age⩽60 years). AML cases with detectable miR-9* included a lower percentage of cases with favorable risk (P<0.001) as compared with those with no detectable miR-9*. High levels of miR-9* expression independently predicted for higher complete remission (odds ratio=1.28, P=0.013) and better event-free survival (EFS) (hazard ratio (HR)=0.86, P=0.001), relapse-free survival (RFS) (HR=0.84, P=0.008) and overall survival (OS) (HR=0.86, P=0.002). Among the subgroup of adverse risk patients, high miR-9* expressers had strikingly longer median survival than low miR-9* expressers (EFS: 16 vs 5 months, P=0.020; RFS: 12 vs 4, P=0.060; OS: 23 vs 8, P=0.021). Comparative transcriptome analysis suggests that miR-9* regulates genes involved in leukemogenesis, for example, MN1 and MLLT3. This is the first report showing that an miRNA* has prognostic value in AML

Increased expression of miR-23a mediates a loss of expression in the RAF kinase inhibitor protein RKIP.

RAF kinase inhibitor protein (RKIP) is a seminal regulator of intracellular signaling and exhibits both anti-metastatic and anti-tumorigenic properties. Decreased expression of RKIP has been described in several human malignancies, including acute myeloid leukemia (AML). As the mechanisms leading to RKIP loss in AML are still unclear, we aimed to analyze the potential involvement of micro-RNAs (miRNAs) within this study. miRNA microarray and qPCR data of more than 400 AML patient specimens revealed correlation between decreased expression of RKIP and increased expression of miR-23a, a member of the miR-23a/27a/24-2 cluster. In functional experiments, overexpression of miR-23a decreased RKIP mRNA and protein expression, whereas miR-23a inhibition caused the opposite effect. By employing an RKIP 3&#39;UTR luciferase reporter construct with and without mutation or deletion of the putative miR-23a binding site, we could show that RKIP modulation by miR-23a is mediated via direct binding to this region. Importantly, miR-23a overexpression induced a significant increase of proliferation in hematopoietic cells. Simultaneous transfection of an RKIP expression construct lacking the miR-23a binding sites reversed this phenotype, indicating that this effect is truly mediated via downregulation of RKIP. Finally, by analyzing more than 4300 primary patient specimens via database retrieval from The Cancer Genome Atlas (TCGA), we could highlight the importance of the miR-23a/RKIP axis in a broad range of human cancer entities. In conclusion, we have identified miR-23a as a negative regulator of RKIP expression in AML and have provided data that suggest the importance of our observation beyond this tumor entity