Circ_Lrp6, a Circular RNA Enriched in Vascular Smooth Muscle Cells, Acts as a Sponge Regulating miRNA-145 Function

Abstract

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 are involved in the modulation of miRNA activity in VSMCs is unknown. Objective: We aimed to identify circular RNAs interacting with miRNAs enriched in VSMCs and modulating the cells’ activity. Methods and Results: RNA sequencing and bioinformatics identified several circular RNAs enriched in VSMCs; however, only one, possessing multiple putative binding sites for miR-145, was highly conserved between mouse and man. This circular RNA gemmed from alternative splicing of Lrp6 (lipoprotein receptor 6), 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 (integrin-β8), FASCIN (fascin actin-bundling protein 1), KLF4 (Kruppel-like factor 4), Yes1 (YES proto-oncogene 1), and Lox (lysyl oxidase). 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 miR-145 versus 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.

Circular RNAs: A Novel Class of Functional RNA Molecules with a Therapeutic Perspective

“…Just 1 year later, simultaneously published by two groups, 3,12 circRNA CDR1as was shown to act as a competing RNA molecule by sponging miRNAs. Shortly afterward, several other circRNAs were reported and shown to act as miRNA sponges also, [61][62][63][64][65][66][67] strengthening the general miRNA sponge function of circRNAs. However, most circRNAs do not contain multiple miRNA-binding sites, 13 thus miRNA sponging is just one of multiple potential functions.…”

“…68 Subsequent to the cdr1as publications in 2013, multiple other circR-NAs were shown to act as miRNA sponges. [61][62][63][64][65][66][67] Using bioinformatics tools, miRNA-binding sites can be predicted within the circRNA sequence (e.g., by using CircInteractome 55 ). Therefore, circRNAs that hold conspicuous many miRNA-binding sites for the same miRNA can be relatively easily uncovered as miRNA sponges.…”

“…A small hairpin RNA (shRNA)-mediated knockdown of circ_Lrp6 was also shown to be beneficial in a mouse carotid artery stenosis model. 63 Moreover, Boeckel et al 106 investigated hypoxia-regulated circRNAs, and they identified the hypoxia-induced circRNA cZNF292. siRNAmediated knockdown of cZNF292 reduced tube formation and spheroid sprouting in vitro, suggesting a pro-angiogenic role of cZNF292.…”

Upregulation of miR‑423 improves autologous vein graft restenosis via targeting ADAMTS‑7

“…Increasing evidence has indicated that the abnormal expression of miRNAs serves an important role in transplanted vascular diseases and they have received much interest in the research of various diseases (21,(45)(46)(47). Proliferation, migration and phenotypic transformation of SMcs or endothelial cells are associated with various miRNAs (48) including miR-145 (49,50), miR-222 (51), miR-195 (52) and miR-21 (53). For example, Liu et al (54) confirmed that the upregulation of miR-378a targeted cyclin-dependent kinase 1 to promote the proliferation and migration of vascular SMcs and increase the incidence of in-stent restenosis following stenting.…”

Hsa_circ_0000345 regulates the cellular development of ASMCs in response to oxygenized low‐density lipoprotein

“…In addition, recent studies have shown that circ_Lrp6 regulated the proliferation and migration of VSMCs by sponging miR-145. 9 More importantly, circRNAs also participated in modulating cell fates in VMSCs under hypoxic conditions. For instance, silencing of circ_000595 decreased hypoxia-induced VSMCs apoptosis by augmenting miR-19a, which has been shown to arrest the cell cycle by impeding cyclinD1 expression.…”