Circular RNA ZBTB46 depletion alleviates the progression of Atherosclerosis by regulating the ubiquitination and degradation of hnRNPA2B1 via the AKT/mTOR pathway

Abstract

Background: CircZBTB46 has been identified as being associated with the risk of coronary artery disease (CAD) and has the potential to be a diagnostic biomarker for CAD. However, the specific function and detailed mechanism of circZBTB46 in CAD are still unknown.

Methods: The expression levels and properties of circRNAs were examined using qRT‒PCR, RNA FISH, and subcellular localization analysis. ApoE^-/- mice fed a high-fat diet were used to establish an atherosclerosis model. HE, Masson, and Oil Red O staining were used to analyze the morphological features of the plaque. CCK-8, Transwell, and wound healing assays, and flow cytometric analysis were used to evaluate cell proliferation, migration, and apoptosis. RNA pull-down, silver staining, mass spectrometry analysis, and RNA-binding protein immunoprecipitation (RIP) were performed to identify the interacting proteins of circZBTB46.

Results: CircZBTB46 is highly conserved and is significantly upregulated in atherosclerotic lesions. Functional studies revealed that knockdown of circZBTB46 significantly decreased the atherosclerotic plaque area, attenuating the progression of atherosclerosis. In addition, silencing circZBTB46 inhibited cell proliferation and migration and induced apoptosis. Mechanistically, circZBTB46 physically interacted with hnRNPA2B1 and suppressed its degradation, thereby regulating cell functions and the formation of aortic atherosclerotic plaques. Additionally, circZBTB46 was identified as a functional mediator of PTEN-dependent regulation of the AKT/mTOR signaling pathway and thus affected cell proliferation and migration and induced apoptosis.

Conclusion: Our study provides the first direct evidence that circZBTB46 functions as an important regulatory molecule for CAD progression by interacting with hnRNPA2B1 and regulating the PTEN/AKT/mTOR pathway.

Keywords: Atherosclerotic plaque; circular RNA; Coronary artery Disease; RNA-binding protein.

Fig. 1

CircZBTB46 is highly expressed in CAD, and circZBTB46 knockdown attenuates the progression of atherosclerosis. (A-B) Fluorescence in situ hybridization (FISH) was used to show the location and expression of circZBTB46 in human atherosclerotic plaques. Fluorescence intensity was measured using ImageJ software and the mean fluorescence intensity was represented as mean ± SEM. (C) The schedule of atherosclerosis model establishment and AAV intervention. (D) The relative expression of circZBTB46 in the heart in the two groups. (E-F) Oil red O staining of the intact aortas and cross-sections of aortic roots. (G) HE staining of the aortic sinus in the AAV-sh-NC and AAV-sh-circZBTB46. (H) Masson staining of the aortic sinus in the two groups of mice after AAV intervention. (I) α-SMA, (J) CD31, and (K) F4/80 staining in the aortic sinus in the AAV-sh-NC and AAV-sh-circZBTB46 groups. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Silencing circZBTB46 inhibits cell proliferation and migration and induces apoptosis. (A-B) The relative expression of circZBTB46 in HCAECs and HCASMCs transfected with si-NC, si-circZBTB46-1, and si-circZBTB46-2. (C-D) The CCK-8 assay revealed that circZBTB46 knockdown inhibited cell proliferation. (E) Western blot analysis showed that the protein levels of cleaved PARP and cleaved caspase 3 were significantly increased whereas, Cyclin D1 and Cyclin A were significantly decreased upon silencing of circZBTB46. (F-G) The migration of HCAECs and HCASMCs was inhibited by knockdown of circZBTB46. (H-I) Flow cytometric analysis was performed to determine showed the percentages of apoptotic cells after transfection with si-NC, si-circZBTB46-1, and si-circZBTB46-2. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

CircZBTB46 physically interacts with hnRNPA2B1. (A-C) The relative subcellular localization of circZBTB46 was assessed by qRT‒PCR and FISH. (D) Silver staining of the gel after SDS‒PAGE separation of the proteins that were immunoprecipitated with the 3′ biotin-labeled probe for circZBTB46 (probe) and the biotinylated antisense probe (NC) in RNA pull-down assays. The arrow shows the position of hnRNPA2B1. (E) Mass spectrometry analysis showed that hnRNPA2B1 was the top-ranked protein according to the peptide identification results. (F) Western blot analysis following the RNA pull-down assay and (G) qRT‒PCR analysis following the RIP assay confirmed the interaction between circZBTB46 and hnRNPA2B1. (H) FISH showed that circZBTB46 was colocalized with hnRNPA2B1 in cells and human coronary arteries. (I) 3‑Dimensional structure of the circZBTB46‑hnRNPA2B1 complex

Fig. 4

hnRNPA2B1 is upregulated in atherosclerotic plaques, and silencing hnRNPA2B1 inhibits cell proliferation and induces apoptosis. (A-B) The expression of hnRNPA2B1 in different stages of atherosclerosis was analyzed by immunohistochemical staining. (C) The relative expression levels of hnRNPA2B1 in PBMC samples from CAD patients and controls were determined by RT‒PCR. (D-E) The CCK-8 assay revealed that silencing hnRNPA2B1 inhibited cell proliferation. (F-G) Transwell and wound healing assays suggested that cell migration was dramatically suppressed by silencing hnRNPA2B1 expression. (H) Flow cytometric analysis indicated that the proportion of apoptotic cells was notably elevated in the si-hnRNPA2B1 groups. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

CircZBTB46 inhibits cell proliferation and migration through hnRNPA2B1 and the PTEN/AKT/mTOR pathway. (A-D) Overexpression of hnRNPA2B1 attenuated the inhibitory effects of circZBTB46 knockdown on the proliferation and migration of HCAECs and HCASMCs. (E) Overexpression of hnRNPA2B1 partially blocked circZBTB46-silencing induced cell apoptosis. (F-G) Silencing circZBTB46 or hnRNPA2B1 increased the expression of PTEN and inhibited AKT and mTOR phosphorylation. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

CircZBTB46 blocks the ubiquitination and degradation of hnRNPA2B1. (A) Western blot analysis of the expression level of hnRNPA2B1 after circZBTB46 knockdown or circZBTB46 overexpression. (B) RT‒PCR analysis of hnRNPA2B1 expression after transfection with si-circZBTB46 or the circZBTB46 overexpression plasmid. (C) Immunoblot analysis of the hnRNPA2B1 level in intact cells treated with CHX for 0, 3, 6 or 9 h. The relative fold changes were determined by comparison with the level at 0 h. (D) Cells transfected with si-circZBTB46 were treated with MG132 or CQ, and the hnRNPA2B1 level in the indicated cells was measured by Western blotting. (E) Lysates from circZBTB46-knockdown cells were immunoprecipitated (IP) with an anti-hnRNPA2B1 antibody and were then used for immunoblot analysis of ubiquitin and hnRNPA2B1. (F) Secondary structure prediction of circZBTB46 using the online web server RNAfold. The predicted binding sites of circZBTB46 with hnRNPA2B1 are shown in the blue circle. (G) The ubiquitination sites in hnRNPA2B1 were predicted via a Bayesian discriminant method and visualized using Discovery Studio 2021 (client version). Purple indicates the binding region, while yellow indicates the ubiquitination site