Objectives: The development and maintenance of interstitial fibrosis, which is considered the basis for structural remodeling in atrial fibrillation (AF), involves expressional changes in cardiac microRNAs (miRs). Our previous investigations showed that various pro- and anti-fibrotic miRNAs are differentially expressed in the RA of AF patients compared to sinus rhythm (SR) patients. Among these we observed a strong downregulation of miR-146b-5p.

Methods: miR-146b-5p and target gene expression were analyzed in RA tissue and RA fibroblasts from AF and SR patients. Fibroblasts were isolated from RA tissue and employed at passage 1 for further analyses. Luciferase assays and miR-146b-5p overexpression were utilized for target gene analyses. Proliferation and migration assays of human cardiac fibroblast were used to determine the functions of miR-146b-5p. Signaling pathway analyses were performed by Western blotting.

Results: Mature miR-146b was identified as substantially downregulated in RA tissue and RA fibroblasts of AF patients. A miR-146b-5p mimic inhibited RA fibroblast migration and proliferation in vitro, while the according inhibitor promoted both. Target gene analyses predicted SIAH2, PRKAA2, EGFR, IRAK1, TRAF6 and SMAD4 as top candidates but only SIAH2, PRKAA2, EGFR were differentially expressed in human RA fibroblasts. Overexpression of miR-146b-5p resulted in a strong downregulation of SIAH2 and EGFR while the according inhibitor had an opposite effect. Luciferase reporter gene assays confirmed binding of miR-146b-5p to the 3’UTR of SIAH2 and EGFR. Stable knockout of SIAH2 and EGFR via CRISPR/Cas9 in vitro resulted in significantly altered proliferation, migration and activation of the ERK signaling pathway, indicative of a role in AF-associated fibrosis. The in vivo observed downregulation of miR-146b-5p in RA fibroblasts from AF patients could be caused by an upregulation of the lncRNA MALAT-1, which is known to act as a sponge for various miRs including miR-146. Indeed RA fibroblasts from AF patients demonstrated a strongly increased MALAT-1 expression. TGF-beta 1, a major mediator of atrial fibrotic remodeling, increases MALAT-1 expression in human RA fibroblasts in vitro, resulting in reduced miR-146b-5p but increased SIAH2 and EGFR mRNA and protein expression. This effect on miR-146b-5p expression is largely prevented by MALAT-1 siRNA, suggesting its causal role.

Conclusion: miR-146b-5p is a novel regulator of atrial fibrosis. Further in vivo analyses should investigate whether interventions targeting miR-146b-5p may offer therapeutic options for anti-fibrotic atrial re-remodeling.