Rationale: microRNAs (miRNAs) modulate gene expression by repressing translation of targeted genes. Several reports have established the role of short non-coding RNAs such as miRNAs in regulating vascular smooth muscle cell (VSMC) biology. Nonetheless, whether circular RNAs, previously described as potential endogenous modulators of miRNAs, 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 their 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 human. This circular RNA gemmed, in both species, from an 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 miR145 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, Klf4 (Kruppel-like factor 4), Yes1 (YES proto-oncogene 1), and Lox (lysyl oxidase). The interaction was preferentially localized into P-bodies, sites of mRNA storage/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 different 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.
Circ_Lrp6: novel circRNA playing a central role in VSMC biology orchestration by acting as a miR-145 sponge / HALL BALCAZAR, IGNACIO FERNANDO. - (2020 Mar 09).
Circ_Lrp6: novel circRNA playing a central role in VSMC biology orchestration by acting as a miR-145 sponge
HALL BALCAZAR, IGNACIO FERNANDO
2020-03-09
Abstract
Rationale: microRNAs (miRNAs) modulate gene expression by repressing translation of targeted genes. Several reports have established the role of short non-coding RNAs such as miRNAs in regulating vascular smooth muscle cell (VSMC) biology. Nonetheless, whether circular RNAs, previously described as potential endogenous modulators of miRNAs, 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 their 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 human. This circular RNA gemmed, in both species, from an 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 miR145 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, Klf4 (Kruppel-like factor 4), Yes1 (YES proto-oncogene 1), and Lox (lysyl oxidase). The interaction was preferentially localized into P-bodies, sites of mRNA storage/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 different 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
