Background 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 and t-test analyses were used to determine the predicted target of miR-4520a, Rheb, after cloning its 3'UTR region containing miR-4520a recognition element into PGL3-prom vector. The expression levels of pyrin, miR-4520a and its putative target Rheb were validated in monocytes from FMF patients (n=10) 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 quantitative PCR. Expression levels of mRNAs and miRNA were determined with the 2-ΔΔCt method after normalizing to 18S RNA and RNU6B housekeeping 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. Stratification of patients group by genotype revealed an intriguing difference in miR-4520a relative expression. Specifically, miR-4520a expression was lower in carriers of M694V variant (combined group of homo- and heterozygotes), whereas the rest of FMF genotypes had higher levels (p<0.05). Subsequent comparison between the M694V-group and healthy controls showed a significant reduction in miR-4520a expression levels (p<0.01). Interestingly, one of the homozygote M694V patients with the highest fold change in miR-4520a expression experienced an FMF-attack while on study. Quantitative real time PCR analysis of the miRNA-enriched fraction of total RNA isolated from FMF patient's monocytes during the attack revealed a surprisingly increased miR-4520a expression (FC=0.85) compared to the attack-free period (FC=5.2). Bioinformatic analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of Rheb/mTOR signaling and of Suppressor of IKBKE (SIKE1). Expression levels of the first target we chose to investigate, Rheb, was confirmed by luciferase reporter gene assay as a direct target of miR-4520a (p<0.01). Validation of pyrin and Rheb mRNA and protein expression levels in monocytes from FMF patients is in progress.
Conclusions These findings provide initial evidence that Rheb is a valid target of miR-4520a and suggest that a dysfunctional pyrin due to M694V variant may be associated with a reduction in miR-4520a expression levels thus contributing to the low basal levels of autophagy shown in FMF patients.
M. F. Mashreghi, H. Latsoudis, J. Gruen et al. (2014). Ann Rheum Dis 73(Suppl2):344-345.
Acknowledgements Supported by the Hellenic Ministry of Education and Gen. Secret. for Research-Technology (ESPA project/No 898).
Disclosure of Interest None declared
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.