Cardiovascular diseases remain a leading cause of morbidity and mortality worldwide, with incidence rates steadily rising. Among them, coronary artery disease (CAD) is the primary contributor to cardiovascular-related deaths. Despite significant progress in CAD diagnosis and treatment both surgically and medically, over the past decades, there remains an urgent need for new diagnostic biomarkers and therapeutic targets. The transformation of smooth muscle cells in the vascular middle layer from a contractile to a secretory phenotype, along with their migration into the intimal region, is a physiological response. However, the abnormal proliferation and migration of these smooth muscle cells in coronary atherosclerotic lesions can accelerate disease progression.
In recent years, many proteins or peptides encoded by noncanonical human open reading frames (NHORFs) have been recognized for their pathological roles in various diseases. However, until now, no NHORF-encoded proteins or peptides had been identified to play a biological role in the atherosclerotic process.
On November 13, Professor Jia Enzhi's research team from the First Affiliated Hospital of Nanjing Medical University (NMU) published a research paper titled CircBTBD7-420aa encoded by hsa_circ_0000563 regulates the progression of atherosclerosis and construction of circBTBD7-420aa engineered exosomes in JACC: Basic to Translational Science. The study elucidated the specific mechanism by which circBTBTD-420aa, encoded by the NHORF (circular RNA hsa_circ_0000563), regulates the proliferation and migration of smooth muscle cells in atherosclerosis. This finding provides new insights and potential therapeutic targets for the prevention and treatment of atherosclerosis. Furthermore, the team developed engineered exosomes designed to target smooth muscle cells with circBTBD7-420aa, laying the foundation for clinical applications. Additionally, the molecular function of circBTBTD-420aa has been granted a national patent by the China National Intellectual Property Administration (Patent No. ZL 2022 1 1314975.2, Announcement Date: December 5, 2023, Announcement No. CN 115896101 B).
To confirm the circular structure and characteristics of circBTBD7, the authors employed Sanger sequencing, qPCR, nuclear-cytoplasmic separation experiments, and fluorescence in situ hybridization. Database analysis suggested that circBTBD7 contains a noncanonical open reading frame, indicating its potential to encode an undetected protein, which the authors named circBTBD7-420aa. To verify its existence and activity, they constructed blank control plasmids, circBTBD7-420aa-flag plasmids, and circBTBD7-420aa-mut-flag plasmids. Using a BTBD7 antibody that recognizes an immunogenic sequence shared by both BTBD7 and circBTBD7-420aa, the authors successfully validated the endogenous presence of circBTBD7-420aa through Western blotting. Mass spectrometry further identified the specific amino acid sequence, LLDPGLK, providing definitive proof of the protein’s exisitence.
To demonstrate the role of circBTBD7-420aa in atherosclerosis, the authors induced abnormal proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) using oxidized low-density lipoprotein (ox-LDL). They found that overexpression of circBTBD7 or circBTBD7-420aa, unlike their respective mutated forms, significantly inhibited the abnormal proliferation and migration of HCASMCs. To further explore the molecular mechanism by which circBTBD7-420aa inhibits HCASMC proliferation and migration, the authors used IP-MS to identify potential targets of circBTBD7-420aa. They confirmed the existence of circBTBD7-420aa in the mass spectrometry results and identified SLC3A2 as a potential target. Further co-IP experiments validated the interaction between circBTBD7-420aa and SLC3A2. Functional assays including EdU, CCK8, Transwell, and scratch assays demonstrated that circBTBD7-420aa primarily exerts its functions through SLC3A2. The authors further confirmed that circBTBD7-420aa promotes the ubiquitination and degradation of SLC3A2, thereby reducing its stability.
The authors used ApoE-/- mice fed a high-fat diet to construct an atherosclerosis model. H&E and Masson staining experiments suggested that injection of circBTBD7-420aa lentivirus significantly alleviated aortic occlusion and collagen deposition in the mice, providing compelling evidence of its theraputic potential.
Finally, the authors successfully engineered exosomes targeting smooth muscle cells with circBTBD7-420aa, providing a promising new strategy for the targeted treatment of CAD.
This study reveals that circBTBD7-420aa, encoded by NHORF (circular RNA hsa_circ_0000563), affects the protein expression of SLC3A2 at the post-translational modification level, thereby inhibiting the abnormal proliferation and migration of human coronary artery smooth muscle cells. In vivo experiments further confirm the role of circBTBD7-420aa in slowing the progression of atherosclerosis. Moreover, the successful construction of engineered exosomes targeting smooth muscle cells with circBTBD7-420aa provides a new perspective for the diagnosis and treatment of CAD, positioning circBTBD7-420aa as a potential novel biomarker and therapeutic target.
Professor Jia Enzhi from the First Affiliated Hospital of Nanjing Medical University is the corresponding author of the paper. The first author is Gan Xiongkang (currently a PhD candidate at the School of Basic Medical Sciences, NMU), with PhD candidate Chi Boyu as the co-first author. This study was funded by the National Natural Science Foundation of China (Grant Nos. 81170180, 30400173, 30971257, and 81970302).
Original paper link: https://doi.org/10.1016/j.jacbts.2024.09.003
[Translation revised by Zhang Bei]
