Circ_Lrp6, a Circular RNA Enriched in Vascular Smooth Muscle Cells, Acts as a Sponge Regulating miRNA-145 Function
Circ_Lrp6, a Circular RNA Enriched in Vascular Smooth Muscle Cells, Acts as a Sponge Regulating miRNA-145 Function
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DOI：
10.1161/circresaha.118.314240
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发表时间：
2019-02-15
影响因子：
20.1
通讯作者：
Elia, Leonardo
中科院分区：
文献类型：
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作者：
Hall, Ignacio Fernando;Climent, Montserrat;Elia, Leonardo
基本原理：microRNAs（miRNAs）通过抑制靶基因的翻译来调节基因表达。先前的工作已经确定了miRNA在调节血管平滑肌细胞（VSMC）活性中的作用。目的：筛选与VSMCs中富集的miRNA相互作用并调控VSMCs细胞活性的环状RNA。方法和结果：通过RNA测序和生物信息学方法，鉴定了VSMCs中富集的几种环状RNA;然而，只有一个，具有多个推定的miR-145结合位点，在小鼠和人类之间高度保守。这种环状RNA由Lrp 6的选择性剪接产生（脂蛋白受体6），一种在血管中高度表达并与血管病理学有关的基因，因此被命名为circ_Lrp6。其作为miR-145海绵的作用通过RNA免疫沉淀、受激发射损耗显微镜和竞争性荧光素酶测定确定相互作用来证实;通过测量靶基因ITG β 8的表达来评估miR-145的功能抑制（整合素-β 8）、FASCIN（成束蛋白肌动蛋白束蛋白1）、KLF 4（Kruppel样因子4）、Yes 1（YES原癌基因1）和Lox（赖氨酰氧化酶）。这种相互作用优先定位于P体，mRNA降解的位点。使用功能丧失和获得的方法，我们发现circ_Lrp6阻碍了miR-145介导的VSMC迁移，增殖和分化的调节。miR-145和circ_Lrp6在小鼠和人血管疾病中的差异表达表明，与miR-145结合的circ_Lrp6与未结合的circ_Lrp6的比率可能在血管发病机制中起作用。结论：circ_Lrp6是miR-145的细胞内调节因子和天然海绵，可平衡VSMCs中miRNA的功能。
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 ITG beta 8 (integrin-beta 8), 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.