THU0467 Silencing of the MEFV Gene in Familial Mediterranean Fever and Transcriptomic Analysis in Human Monocytes: Evidence for the Involvement of Pyrin in Innate Immune Responses and Autophagy

Background Mutations in MEFV gene encoding pyrin [1] account for Familial Mediterranean fever FMF, a recessively inherited disease but the high number of heterozygote patients is puzzling and requires additional investigation [2,3]. Objectives Elucidation of the pathophysiology of the disease, through the identification of novel genes and miRNAs that interact with or regulate the MEFV gene. Methods Silencing of MEFV gene by AccellsiRNAs was performed in THP-1 cell line that expresses endogenous pyrin. Global changes in mRNA and microRNA expression were assessed by GeneChip HuGene-1.0-ST-v1 and GeneChipmiRNA 3.0 Array, respectively. Putative target genes of the differentially regulated miRNAs were identified by using appropriate algorithms (TargetScan, PicTar). Functional classifications and biological processes of differentially expressed mRNAs and miRNA target genes were assigned according to Gene Ontology (GO) and the updated Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. mRNA and miRNA target genetic or protein interactions were constructed using Cytoscape 3.0.2 (Plugins GeneMania and BioGrid). Results We achieved 62% knockdown of the MEFV mRNA as measured by Taqman PCR, following siRNA-mediated knockdown of the respective gene.Global transcriptome analysis revealed 89 differentially expressed genes and 25 deregulated miRNAs involved in inflammatory-mediated responses, cell cycle, cytoskeleton/microtubule dynamics, intracellular trafficking and survival. Specifically, miR-4520a and miR-19b demonstrate altered expression levels during TLR-mediated signaling, which corroborate the role of pyrin in the regulation of inflammatory responses. Interestingly, the aforementioned deregulated miRNAs are also shown to play a significant role in intracellular membrane trafficking and subsequently autophagy through regulation of small RabGTPases. Bioinformatic analyses predicted high cooperativity within the significantly deregulated miRNAs, with the clustering of 8 (out of 11) members of the miR-378/422a superfamily being the most pertinent example. Conclusions These findings reiterate the involvement of pyrin in innate immune responses and suggest a potential role of this protein in autophagy, probably through regulation of membrane trafficking RabGTPases. Validation of this data in monocytes from FMF patients is in progress. References The International FMF Consortium (1997). Cell 90:797–807. Booty M.G., Chae J.J., et al (2009). Ann. Rheum. Dis. 60:1851