Introduction

MicroRNA signature of THP1 cells revealed a 5.9-fold decreased expression of miR-4520a following siRNA-mediated knockdown of MEFV gene that encodes pyrin [1].

Objectives

We herein sought to validate the expression levels of miR-4520a in monocytes isolated from peripheral blood mononuclear cells (PBMCs) of FMF patients.

Methods

Dual luciferase assay was used to validate a predicted miR-4520a recognition element in the 3'UTR region of the Rheb gene. The expression levels of pyrin, miR-4520a and its putative target Rheb were validated in monocytes from FMF patients (n=9) and compared with healthy controls (n=8). Patients were off colchicine for two days (attack-free period) and monocytes were isolated from PBMCs. Total RNA together with the respective miRNA-enriched fractions were isolated from monocytes and used for mRNA and miR-4520a quantitation by real-time PCR using the 2-ΔΔCt method after normalizing to 18S RNA and RNU6B genes, respectively. Protein levels of pyrin and Rheb were detected by western blotting.

Results

The relative expression levels of miR-4520a were variable among FMF patients and not significantly different between patients and controls. However, when patients that did not harbor any mutations in MEFV were excluded from the analyses, the expression of miR-4520a was statistically different between FMF patients and controls (p<0.05), indicating an association between miR-4520a expression and mutations in the MEFV gene. Moreover, stratification of patients group by genotype revealed an intriguing difference in miR-4520a relative expression, with carriers of M694V variant (combined group of homozygotes, heterozygotes and compound heterozygotes) showing the highest increase (p<0.05). Subsequent comparison between the M694V group and healthy controls showed a significant increase in miR-4520a expression levels that remained significant even after bonferroni correction (p<0.01). Interestingly, one of the homozygote M694V patients with the highest fold change in miR-4520a expression (FC=7.8) experienced an FMF-attack while on study, with a concomitant decrease in miR-4520a relative expression (FC=0.45). Bio-informatic analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of Rheb/mTOR signaling. Expression levels of Rheb were confirmed by luciferase reporter gene assays providing further evidence that Rheb is a direct target of miR-4520a (p<0.01). Validation of pyrin and Rheb protein expression levels in monocytes from FMF patients is in progress.

Conclusion

Our findings provide initial evidence that Rheb is a valid target of miR-4520a and suggest that a dysfunctional pyrin due to gain of function mutations with a dosage effect [2], especially of M694V variant, may be associated with an increase in miR-4520a expression levels, thus contributing to deregulated mTOR signaling and subsequently IL-1β release [3].