Circular RNA circEsyt2 regulates vascular smooth muscle cell remodeling via splicing regulation
Xue Gong, … , Gengze Wu, Chunyu Zeng
Xue Gong, … , Gengze Wu, Chunyu Zeng
Published December 15, 2021
Citation Information: J Clin Invest. 2021;131(24):e147031. https://doi.org/10.1172/JCI147031.
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Research Article Cardiology Vascular biology

Circular RNA circEsyt2 regulates vascular smooth muscle cell remodeling via splicing regulation

Abstract

Circular RNAs (circRNAs) have been recently recognized as playing a role in the pathogenesis of vascular remodeling–related diseases by modulating the functions of miRNAs. However, the interplay between circRNAs and proteins during vascular remodeling remains poorly understood. Here, we investigated a previously identified circRNA, circEsyt2, whose expression is known to be upregulated during vascular remodeling. Loss- and gain-of‑function mutation analyses in vascular smooth muscle cells (VSMCs) revealed that circEsyt2 enhanced cell proliferation and migration and inhibited apoptosis and differentiation. Furthermore, the silencing of circEsyt2 in vivo reduced neointima formation, while circEsyt2 overexpression enhanced neointimal hyperplasia in the injured carotid artery, confirming its role in vascular remodeling. Using unbiased protein–RNA screening and molecular validation, circEsyt2 was found to directly interact with polyC-binding protein 1 (PCBP1), an RNA splicing factor, and regulate PCBP1 intracellular localization. Additionally, circEsyt2 silencing substantially enhanced p53β splicing via the PCBP1–U2AF65 interaction, leading to the altered expression of p53 target genes (cyclin D1, p21, PUMA, and NOXA) and the decreased proliferation of VSMCs. Thus, we identified a potentially novel circRNA that regulated vascular remodeling, via altered RNA splicing, in atherosclerotic mouse models.

Authors

Xue Gong, Miao Tian, Nian Cao, Peili Yang, Zaicheng Xu, Shuo Zheng, Qiao Liao, Caiyu Chen, Cindy Zeng, Pedro A. Jose, Da-Zhi Wang, Zhao Jian, Yingbin Xiao, Ding-Sheng Jiang, Xiang Wei, Bing Zhang, Yibin Wang, Ken Chen, Gengze Wu, Chunyu Zeng

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Figure 1

Identification of circEsyt2 based on circRNA profiling of mouse atherosclerotic aortae.

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Identification of circEsyt2 based on circRNA profiling of mouse atherosc...
(A) Heat map of differentially expressed circRNAs in atherosclerotic (HFD + ApoE–/–) and control groups (CD + ApoE–/– or CD + C57BL/6J). CD: chow diet. HFD: high fat diet. ApoE–/–: ApoE knockout mice. n = 2. (B) PCR amplification using divergent or convergent primers against complementary DNA (cDNA) or genomic DNA (gDNA) from aortae. (C) qRT-PCR to check for the expression of circRNAs in aortae. *P < 0.05, **P < 0.01 vs. control. n = 4. (D) Confirmation of the backsplicing junction site within the circEsyt2 by Sanger sequencing. (E) mRNA expressions after RNase-R digestion in mouse VSMCs followed by qRT-PCR. **P < 0.01, ***P < 0.001 vs. RNase-R. n = 3. (F) Actinomycin D (Act D) treatment of VSMCs followed by qRT-PCR. Linear mRNA controls: GAPDH and Esyt2. **P < 0.01 vs. DMSO. n = 3. (G) qRT-PCR to check for the expression of circEsyt2 in mouse tissues. n = 3. (H) Subcellular localization of circEsyt2 in VSMCs. Left: qRT-PCR to check for the expression of circEsyt2, Esyt2, linear mRNA in the cytoplasmic and nuclear fractions; GAPDH, cytoplasmic control; snoR41, nuclear control. Right: images of FISH for circEsyt2 (red) and cofluorescence with α-SMA (green) and DAPI (blue). 18S, cytoplasmic control; U6, nuclear control; antisense, negative control. Scale bars: 20 μm. Data are mean ± SEM. Two-sided unpaired t test or Kolmogorov-Smirnov test for C, E, and F.