EIciRNA CEROD upregulation exacerbates coronary artery calcification through promoting DHX36 ubiquitination
R Wang, L K Ma, L Chen, C GaoAbstract
Background
Coronary artery calcification (CAC), a hallmark of advanced atherosclerosis, serves as a robust independent predictor of major adverse cardiovascular events. Circular RNAs (circRNAs) have emerged as key regulators of pathological processes, yet their roles and underlying mechanisms in CAC remain largely uncharacterized, especially regarding the less-studied exon-intron circRNAs (EIciRNAs) subtype. This study was designed to investigate the mechanisms of EIciRNA CEROD (Calcification-associated EIciRNA Regulator Of DHX36), and the relevant roles and potential application of CEROD in CAC.
Methods
Genome-wide RNA sequencing of RNase R-resistant RNA from blood of 3 CAC patients and 3 controls identified dysregulated circRNAs. The conserved CEROD was validated via qRT-PCR, FISH, and RNase R digestion. Gain/loss-of-function assays in vascular smooth muscle cells (VSMCs) assessed VSMC calcification. RNA pulldown, RIP, ubiquitination assays assessed the mechanisms. In vivo, atherosclerotic mice received AAV9-mCEROD shRNA; aortic changes were evaluated via ultrasonography and immunohistochemistry staining.
Results
We discovered that CEROD is a mammalian-conserved EIciRNA, significantly upregulated in CAC patients’ blood, calcified arteries, and in vitro calcified VSMCs. It was an independent CAC risk factor and correlated with coronary stenosis severity. CEROD overexpression enhanced calcified nodule formation, calcium accumulation, ALP activity, and expression of pro-calcific markers, while knockdown exerted the opposite effects. Mechanistically, CEROD bound the 243–292 nt region of DHX36 and recruited the E3 ubiquitin ligase STUB1, promoting DHX36 ubiquitin-dependent degradation. Reduced DHX36 relieved its repression of PAPPA2 mRNA, stabilizing PAPPA2 and amplifying pro-calcific signaling. Notably, CEROD did not affect transcription of its parental gene (MCU). AAV9-mediated mCEROD knockdown in HFD-fed mice reduced aortic plaque burden and calcification, improved vascular remodeling, upregulated DHX36, and downregulated PAPPA2, RUNX2 and BMP2.
Conclusions
This study demonstrates conserved CEROD regulation of pro-calcific signaling via STUB1-mediated DHX36 ubiquitination and PAPPA2 mRNA stabilization. Targeting CEROD exhibits protective effects in atherosclerotic mice. These findings highlight CEROD as a potential diagnostic biomarker and therapeutic target for atherosclerotic calcification, expanding the functional landscape of EIciRNAs in cardiovascular pathology.clinical features of CERODThe working model of CEROD