Identification of feline miRNAs and evaluation of their differential expression in Abyssinian amyloidosis

Introduction: Domestic felids represent one of the main species in which amyloidosis occurs. The disease is caused by the presence of protein complexes, known as amyloids, which form insoluble deposits in different organs. However, little is known about the pathogenic pathway and the genetic of the disease is still under exploration. Among cat breeds, amyloidosis is known to affect Abyssinian/Somali and Siamese/Oriental cats, where the main target organs for the deposit were found to be kidneys and liver, respectively. Objective: The aim of this study is to identify feline miRNAs and to evaluate their possible differential expression in amyloidosis-affected and healthy Abyssinian cats. The main objective is profiling miRNAs potentially involved in the disease onset or in the regulation of its pathogenesis. Materials & Methods: miRNAs were extracted from Formalin Fixed Paraffin Embedded (FFPE) samples collected from 4 healthy and 6 affected Abyssinians, using the miRNeasy Mini Kit (Qiagen). The sequencing of miRNAs was carried out using/by? XXX and its quality was assessed with FastQC. Cutadapter was used to remove the adapter sequences from the high-throughput sequencing reads. MiRDeep2 was then used to collapse the reads and map the fastq files against the reference sequence (mapper.pl function), to identify miRNAs (mirDeep2.pl function) and to determine their expression (quantifier.pl function). MiRNAs with less than 10 reads for each sample were filtered out. The TMM method (Trimmed mean of M values) was used for the normalization and data were expressed as log2 CPM (counts per million reads). The reference genome for Felis catus was retrieved from NCBI (genome assembly vs 9.0) and miRNAs related to cat were identified using the corresponding human miRNAs on miRbase (http://www.mirbase.org/Zp.shtml). Results: A total of 854 miRNAs were detected, and subsequently reduced to 341 after filtering. Considering the False Discovery Rate (FDR) at 5%, no miRNAs resulted differentially expressed between affected and healthy Abyssinians, whereas 22 miRNAs showed significance considering the nominal P-value. Among these 22 miRNAs, six are known to be involved in the development of Alzheimer Disease (AD), four of which with a P-value < 0.009. Suggestively, within the not significant miRNAs, miR-26a-5p (P-value 0.120) is one of the main miRNAs involved in the human light chain amyloidosis onset. Conclusions: recent studies in humans have been focusing on disclosing the potential role of miRNAs in the accumulation of amyloid fibrils, especially in the AD. It was shown that miRNAs significantly affect specific alterations rather than play a direct role in the onset and the development of the AD itself. In our study, some of the identified miRNAs were already found to be associated with AD in humans. These results are encouraging and could explain the role of these miRNAs in feline amyloidosis too. The genes directly regulated and involved in these pathways still have to be investigated. Further evidences could also be obtained using an integrative approach through a proteomic analysis