Circular non-coding LRP6 is a tissue-enriched circular RNA which regulates smooth muscle cell biology acting as a sponge for miR-145

Rationale: microRNAs (miRNAs) modulate gene expression by repressing translation of targeted genes. Previous work has established a role for miRNAs in regulating vascular smooth muscle cell (VSMC) activity. Whether circular RNAs (circRNA) are involved in the modulation of miRNA activity in VSMCs is unknown. Objective: We aimed to identify circRNAs interacting with miRNAs enriched in VSMCs and modulating the cells' activity. Methods and Results: RNA sequencing and bioinformatics identified several circRNAs enriched in VSMCs; however, only one, possessing multiple putative binding sites for miR-145, was highly conserved between mouse and man. This circRNA gemmed from alternative splicing of lipoprotein receptor 6 (Lrp6), a gene highly expressed in vessels and implicated in vascular pathologies, and was thus named circ_Lrp6. Its role as a miR-145 sponge was confirmed by determining reciprocal interaction through RNA immunoprecipitation, stimulated emission depletion microscopy, and competitive luciferase assays; functional inhibition of miR-145 was assessed by measuring expression of the target genes ITGβ8, FASCIN, KLF4, Yes1, and Lox. The interaction was preferentially localized to P-bodies, sites of mRNA degradation. Using loss- and gain-of-function approaches, we found that circ_Lrp6 hindered miR-145-mediated regulation of VSMC migration, proliferation, and differentiation. Differential expression of miR-145 and circ_Lrp6 in murine and human vascular diseases suggests that the ratio of circ_Lrp6 bound to miR145 vs. unbound could play a role in vascular pathogenesis. Viral delivery of circ_Lrp6 shRNA prevented intimal hyperplasia in mouse carotids. Conclusions: circ_Lrp6 is an intracellular modulator and a natural sponge for miR-145, counterbalancing the functions of the miRNA in VSMCs. mouse circular RNA DCC_circRNAs_Mouse.txt: circular RNAs detected with DCC package in Mouse Aorta