环状 RNA Lrp6 在血管平滑肌细胞中富集，作为一种海绵调节 miRNA-145 的功能。
Circ_Lrp6, a Circular RNA Enriched in Vascular Smooth Muscle Cells, Acts as a Sponge Regulating miRNA-145 Function.
机构信息
From the Humanitas Research Hospital, Rozzano, Milan, Italy (I.F.H., M.C., M.Q., F.M.F., T.S., S.Z., P.C., P.K., E.C., G.C., L.E.).
Humanitas University, Rozzano, Milan, Italy (I.F.H., S.Z., P.C., E.C., G.C.).
出版信息
Circ Res. 2019 Feb 15;124(4):498-510. doi: 10.1161/CIRCRESAHA.118.314240.
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.
背景
microRNAs（miRNAs）通过抑制靶向基因的翻译来调节基因表达。先前的研究已经确立了 miRNAs 在调节血管平滑肌细胞（VSMC）活性中的作用。环状 RNA 是否参与调节 VSMCs 中的 miRNA 活性尚不清楚。
目的
我们旨在鉴定与富含 VSMC 的 miRNAs 相互作用并调节细胞活性的环状 RNA。
方法和结果
RNA 测序和生物信息学方法鉴定了几种在 VSMCs 中富集的环状 RNA，但只有一种环状 RNA，它具有多个可能与 miR-145 结合的位点，在小鼠和人类之间高度保守。这种环状 RNA 来源于 Lrp6（脂蛋白受体 6）的可变剪接，Lrp6 在血管中高度表达，与血管病变有关，因此被命名为 circ_Lrp6。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来确定其作为 miR-145 海绵的相互作用；通过测量靶基因 ITGβ8（整合素-β8）、FASCIN（成束蛋白 1）、KLF4（Kruppel 样因子 4）、Yes1（YES 原癌基因 1）和 Lox（赖氨酰氧化酶）的表达来评估 miR-145 的功能抑制。这种相互作用主要定位于 P 体，即 mRNA 降解的部位。通过使用失活和功能获得的方法，我们发现 circ_Lrp6 阻碍了 miR-145 对 VSMC 迁移、增殖和分化的调节。miR-145 和 circ_Lrp6 在小鼠和人类血管疾病中的差异表达表明，circ_Lrp6 与 miR-145 结合的比率与未结合的比率可能在血管发病机制中发挥作用。环状 RNA circ_Lrp6 的病毒传递可防止小鼠颈动脉内膜增生。
结论
circ_Lrp6 是 miR-145 的一种细胞内调节剂和天然海绵，平衡了 miRNA 在 VSMCs 中的功能。
Zotero 插件