Rationale: Neuregulin-1 (NRG-1) includes an extracellular epidermal growth factor–like domain and an intracellular domain (NRG-1-ICD). In response to transforming growth factor-β1, its cleavage by proteolytic enzymes releases a bioactive fragment, which suppresses the vascular smooth muscle cell (VSMC) proliferation by activating ErbB (erythroblastic leukemia viral oncogene homolog) receptor. However, NRG-1-ICD function in VSMCs remains unknown.

Objective: Here, we characterize the function of NRG-1-ICD and underlying mechanisms in VSMCs.

Methods and Results: Immunofluorescence staining, Western blotting, and quantitative real-time polymerase chain reaction showed that NRG-1 was expressed in rat, mouse, and human VSMCs and was upregulated and cleaved in response to transforming growth factor-β1. In the cytoplasm of HASMCs (human aortic smooth muscle cells), the NRG-1-ICD participated in filamentous actin formation by interacting with α-SMA (smooth muscle α-actin). In the nucleus, the Nrg-1-ICD induced circular ACTA2 (alpha-actin-2; circACTA2) formation by recruitment of the zinc-finger transcription factor IKZF1 (IKAROS family zinc finger 1) to the first intron of α-SMA gene. We further confirmed that circACTA2, acting as a sponge binding microRNA (miR)-548f-5p, interacted with miR-548f-5p targeting 3′ untranslated region of α-SMA mRNA, which in turn relieves miR-548f-5p repression of the α-SMA expression and thus upregulates α-SMA expression, thereby facilitating stress fiber formation and cell contraction in HASMCs. Accordingly, in vivo studies demonstrated that the localization of the interaction of circACTA2 with miR-548f-5p is significantly decreased in human intimal hyperplastic arteries compared with normal arteries, implicating that dysregulation of circACTA2 and miR-548f-5p expression is involved in intimal hyperplasia.

Conclusions: These results suggest that circACTA2 mediates NRG-1-ICD regulation of α-SMA expression in HASMCs via the NRG-1-ICD/circACTA2/miR-548f-5p axis. Our data provide a molecular basis for fine-tuning α-SMA expression and VSMC contraction by transcription factor, circular RNA, and microRNA.

基本原理：神经调节蛋白1（NRG-1）包括细胞外表皮生长因子样结构域和细胞内结构域（NRG-1-ICD）。响应转化生长因子-β1，其被蛋白水解酶的切割释放出生物活性片段，该片段通过激活ErbB（促红细胞白血病病毒癌基因同源物）受体来抑制血管平滑肌细胞（VSMC）增殖。但是，VSMC中的NRG-1-ICD功能仍然未知。

目的：在这里，我们描述了NRG-1-ICD的功能及其在VSMC中的潜在机制。

方法和结果：免疫荧光染色，Western印迹和定量实时聚合酶链反应显示，NRG-1在大鼠，小鼠和人VSMC中表达，并响应转化生长因子-β1而被上调和切割。在HASMC（人类主动脉平滑肌细胞）的细胞质中，NRG-1-ICD通过与α-SMA（平滑肌α-肌动蛋白）相互作用来参与丝状肌动蛋白的形成。在细胞核中，Nrg-1-ICD通过将锌指转录因子IKZF1（IKAROS家族锌指1）募集到α-SMA基因的第一个内含子来诱导环状ACTA2（α-actin-2； circACTA2）的形成。我们进一步证实，充当海绵结合microRNA（miR）-548f-5p的circACTA2与靶向α-SMAmRNA 3'非翻译区的miR-548f-5p相互作用，反过来缓解了miR-548f-5p对α-SMA表达的抑制，从而上调了α-SMA表达，从而促进了HASMCs中应激纤维的形成和细胞收缩。因此，体内研究表明，与正常动脉相比，人内膜增生性动脉中circACTA2与miR-548f-5p相互作用的定位显着降低，这暗示circACTA2和miR-548f-5p表达失调与内膜增生有关。 。

结论：这些结果表明，circACTA2通过NRG-1-ICD / circACTA2 / miR-548f-5p轴介导NSMC-1-ICD对HASMC中α-SMA表达的调节。我们的数据为通过转录因子，环状RNA和microRNA微调α-SMA表达和VSMC收缩提供了分子基础。