ANALYSIS OF MICRORNAS AND GENIC POLYMORPHISMS IN THE DIAGNOSIS OF NEOPLASTIC AND TRANSMISSIBLE DISEASES IN DOGS

During my PhD, I mainly worked on two different fields of research: 1. Analysis of microRNAs in canine hematopoietic malignancies for diagnostic purposes. MicroRNAs (miRNAs) are a new class of small non-coding RNAs involved in the negative regulation of gene expression. In humans, it has been shown that miRNAs play a key role in the modulation of the innate immune response and in the regulation of normal and neoplastic hematopoiesis. The expression profile of these molecules varies in the different stages of differentiation of the hematopoietic system cellular components and any alteration of these processes is directly associated with dysregulation of one or more miRNAs. The frequency and nature of some of these tumors are similar in dogs and humans. In dog, the studies of hemato-oncology performed to date have shown that the currently used methodologies are not always able to obtain comprehensive information about the staging, grading, immunophenotyping and prognosis of the diseases. This makes miRNAs very attractive as new potential diagnostic and prognostic markers allowing better subclassification of these tumors in dogs. The purpose of the study undertaken in this project was to examine, using stem-loop TaqMan real time RT-PCR, the expression profile of a panel of miRNAs in some hematological malignancies of the dog, such as chronic lymphocytic leukemia (CLL), lymph node lymphoma and splenic lymphoma, in relation to their non cancer tissues. Panels of miRNAs, already known in humans because of their importance in hematopoietic malignancies, have been selected for investigation, according to the information available in the literature and databases, and to their level of conservation in the canine genome. As a preliminary step, for each studied canine neoplasia, an accurate validation of suitable candidate endogenous control (EC) genes for normalization of miRNA expression levels in normal and neoplastic lymphoid tissues was made. This was performed by means of real time RT-PCR followed by statistical analysis using the algorithms NormFinder and geNorm. In all studied lymphoid malignancies, and for each miRNA investigated, a relative quantification (by calculating the delta-delta Ct to the endogenous control gene selected) and an absolute quantification (interpolating its Ct value on standard curves obtained from serial dilutions of an equimolar pool of synthetic miRNAs) was performed after selecting the best housekeeping genes. The results of this study confirm that in dogs, as already shown in humans, there is differential expression of some miRNAs in various hematologic malignancies, suggesting the potential utility of miRNA monitoring as a new diagnostic strategy in canine hematopoietic malignancies. In particular, in lymph node lymphoma samples miR-181a was upregulated in canine T-cell lymphoma and miR-17-5p was upregulated in canine B-cell lymphoma. Moreover, the molar ratio between miR-181a and miR-17-5p clearly distinguished between T-cell and B-cell lymphoma samples. In CLL samples, the molar ratio miR-150/miR-125b was correlated with the immunophenotype. Finally, molar ratio miR-17-5p/miR-155 was correlated with grading in canine splenic lymphoma. Furthermore, in dogs is often difficult to obtain a sufficient number of fresh clinical samples to perform large diagnostic studies. From here the need arises to carry out studies of retrospective nature based on appropriately validated methods of analysis that make possible to analyze archival samples. Therefore, one of the goals of this study was to develop a methodology for extraction and analysis of miRNAs from smears of aspirated lymph node (for the study of lymph node lymphoma) and peripheral blood (for the study of CLL) and from tissue sections fixed in formalin and included in paraffin (for the study of splenic lymphoma). The results obtained during this study confirm the possibility to analyze miRNAs for retrospective studies starting from such archival samples, as the data obtained from these samples are comparable to those obtained from fresh samples. 2. High Resolution Melting Analysis (HRMA) of genic polymorphisms for diagnostic purposes. High Resolution Melting (HRM) analysis is a new, post-PCR analysis method used for identifying genetic variation in nucleic acid sequences. Simple and fast, this method is based on DNA melting (dissociation) curve techniques and is enabled by the recent availability of improved double-stranded DNA (dsDNA)–binding dyes along with next-generation real-time PCR instrumentation and analysis software. HRM analysis can discriminate DNA sequences based on their composition, length, GC content, or strand complementarity. Accordingly, HRMA allows the detection of multiple types of variations in DNA sequences such as single base changes, insertions, deletions and duplications. In this work this new methodology was utilized as a successful rapid diagnostic technique for the evaluation of the resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples, and for the detection and discrimination of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood.