Human coronary artery tissues and cell culture

The human coronary artery tissues used in the present study were obtained from four donors at the Department of Human Anatomy at Nanjing Medical University. The bereaved families of the donors provided written informed consent, and the experiments were reviewed and approved by the ethics committees of Nanjing Medical University and the First Affiliated Hospital of Nanjing Medical University. Details of specimen processing were described in our previous studies [13]. Human coronary artery smooth muscle cells (HCASMCs; Sigma‒Aldrich, USA) and human coronary artery endothelial cells (HCAECs; ScienCell, USA) were cultured with their respective growth media in a humidified incubator with 5% CO2 at 37 °C. Cycloheximide (CHX), chloroquine (CQ) and MG132 were purchased from MedChemExpress (New Jersey, USA) and were used for protein stability assays.

Atherosclerosis mouse model and adeno-associated virus (AAV) intervention

ApoE−/− mice fed a high-fat diet (HFD) have been widely used for the establishment of atherosclerosis (AS) models in vivo [14, 15]. In the present study, seven- to eight-week-old male ApoE−/− mice (GemPharmatech Co., Ltd., Nanjing, China) were fed a HFD (Beijing Keao Xieli Feed Co.,Ltd., Beijing China) to establish an AS model. To investigate the impact of circZBTB46 silencing on the progression of atherosclerotic plaques, mice were injected with AAV9 vectors carrying sh-NC and sh-circZBTB46 (GeneChem Co., Ltd., Shanghai, China) via the tail vein at a titer of 8.0 × 1011 in a volume of 200 µl per mouse. After the injection, the mice were continually fed a HFD for another 6 weeks before being sacrificed for tissue collection. All mice were group-housed under SPF conditions at an indoor temperature of 25 ± 2 °C with free access to water and food. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. The design of the present study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Blood lipid analysis and atherosclerotic lesion analysis

Mice were euthanized by exsanguination at designated time points under anesthesia with isoflurane, and blood samples were collected for blood lipid analysis using triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) test kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The hearts and intact aortas from the aortic root to the bifurcation of the iliac artery were then promptly separated and fixed in 4% paraformaldehyde for subsequent pathological analysis. After tissue embedding and sectioning, the sections were stained with hematoxylin and eosin to analyze the morphological features of plaques, Masson staining was used to visualize the collagen fibers, and Oil Red O staining was performed to determine the lipid content. The staining intensity was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc. Rockville, MD, USA).

Plasmid and oligonucleotide transfection

The circZBTB46 small interfering RNA (siRNA) oligonucleotides and circZBTB46 overexpression plasmid, along with the corresponding negative controls, were acquired from GenePharma (Shanghai, China), and the hnRNPA2B1 overexpression plasmid was constructed by GeneChem Co., Ltd. (Shanghai, China). The sequences of the siRNAs used in the study are summarized in Supplementary Table 1. Plasmids and siRNAs were transfected into cells using Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, USA) in accordance with the manufacturer’s protocol.

Fluorescence in situ hybridization (FISH) and subcellular fractionation

FISH was carried out according to the manufacturer’s protocol. The human coronary artery specimen slices were baked at 62 °C for 2 h. The major steps of tissue FISH were as follows: slice dewaxing, rehydration, digestion, prehybridization, hybridization, post-hybridization washes, counterstaining with DAPI, and mounting. Cell culture-treated coverslips were prepared for the in vitro RNA FISH assay. After being rinsed in PBS, the cells were fixed, permeabilized, blocked, and incubated in hybridization buffer with FISH probes. The circZBTB46 and 18 S probes were designed and synthesized by Servicebio (Wuhan, China).

Cytoplasmic and nuclear RNAs molecules were purified using the Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek, Ontario, Canada) according to the manufacturer’s instructions. Briefly, cells were lysed by using Lysis Buffer J for 10 min on ice. Then, the lysate was centrifuged for 10 min at maximum speed in a benchtop centrifuge to separate the cell fractions. Subsequently, the cytoplasmic and nuclear RNA were purified and eluted using the respective spin column in accordance with the instructions. The expression level of circZBTB46 was measured by qRT‒PCR, with GAPDH as an internal reference for cytoplasmic RNAs and U6 for nuclear RNAs.

RNA isolation and quantitative real-time PCR (qRT‒PCR)

First, total RNA was extracted by using Trizol reagent (Vazyme, Nanjing, China), and the concentration and quantification of each sample were measured by a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, HiScript® III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Nanjing, China) was used to synthesize cDNA, which was subsequently amplified by using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a QuantStudio 7 Flex system (Thermo Fisher Scientific, Waltham, MA, USA) for 40 cycles. All primers used are listed in Supplementary Table 2.

Cell proliferation assay and cell migration assay

Cell proliferation was examined by a Cell Counting Kit 8 assay (CCK-8 kit; APExBIO, USA), while cell migration was assessed by Transwell and wound healing assays. A detailed description of the experimental procedures was presented in our previous articles [16].

Flow cytometric analysis of apoptosis

According to the instructions of the Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China), cells were detached with EDTA-free trypsin, and then, 1 × 105 cells were harvested after centrifugation at 1000 rpm, 4 °C for 5 min. Afterward, the cells were washed twice with precooled PBS at 1000 rpm and 4 °C for 5 min and then resuspended in 100 µl of 1× Binding Buffer. Subsequently, the cells were incubated with 5 µl of Annexin V-FITC and 5 µl of propidium iodide (PI) staining solution in the dark at room temperature (20–25 °C) for 10 min. Finally, 400 µl of 1× Binding Buffer was added to each tube, and the stained samples were detected by flow cytometry (FACScan, BD Biosciences, CA, USA) within 1 h.

Western blot analysis

RIPA lysis buffer containing 1 mM PMSF reagent (Beyotime, Shanghai, China) was used to extract total protein from cell samples. Protein concentrations in the lysates were then quantified by a bicinchoninic acid assay (Beyotime, Shanghai, China) after sonication and centrifugation. Then, loading buffer was added to the lysates, and proteins were denatured at 95° C for 10 min. Subsequently, proteins were separated on SDS‒PAGE gels and transferred onto a polyvinylidene difluoride (PVDF) membrane (0.22 μm, Millipore, USA), which was then blocked with 5% skim milk and subjected to immunoblot analysis. Finally, an ECL Chemiluminescence Kit (Vazyme, Nanjing, China) was used to visualize the protein bands through signal detection. The anti-hnRNPA2B1, anti-CyclinD1, anti-β-actin, anti-α-tubulin, anti-Ubiquitin, anti-AKT, anti-mTOR, and anti-p-mTOR antibodies were purchased from Proteintech (Wuhan, China); the anti-cleaved caspase 3 antibody was obtained from Cell Signaling Technology (Danvers, MA, USA); the anti-cleaved PARP and anti-GAPDH antibodies were obtained from HUABIO (Hangzhou, China); and the anti-p-AKT, anti-Cyclin A and anti-PTEN antibodies were obtained from Wanlei Biotechnology Company (Shenyang, China).

RNA pull-down, silver staining, and mass spectrometry analysis

The biotin-labeled circZBTB46 probe and antisense were synthesized by GenePharma (Shanghai, China), and RNA pull-down assay was performed using a Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA). In brief, RNA probe (100 pmol) was mixed with 50 ul prewashed streptavidin magnetic beads and incubated for 30 min at room temperature with agitation. Then, the magnetic beads were washed with an equal volume of 20 mM Tris (pH 7.5), and 100 µL of 1X Protein-RNA Binding Buffer was added to the beads and mixed well. Subsequently, RNA-Protein Binding Reaction Master Mix was prepared and added to the RNA-bound beads, and the mixture was then incubated for 60 min at 4 °C with agitation. Finally, RNA-binding protein complexes were eluted with 50 µL of Elution Buffer and separated on SDS‒PAGE gels, and the gels were then subjected to silver staining (Beyotime, Shanghai, China). The selected bands were identified by mass spectrometry (BiotechPack, Beijing, China) and Western blot analysis.

RNA-binding protein immunoprecipitation (RIP) assays

Under the guidance of the manufacturer’s instructions, we performed RIP assays by using an EZ-Magna RIP Kit (Millipore, Billerica, USA). Briefly, 5 µg of the anti-hnRNPA2B1 antibody and negative control IgG (Millipore, Billerica, USA) were incubated with magnetic beads for 30 min at room temperature with agitation. Then, the supernatant of the cell lysate was incubated overnight at 4 ℃ with antibody-conjugated magnetic beads for each RIP reaction. Finally, proteins in each sample were digested by proteinase K, and RNA was purified for subsequent downstream analysis.

Modeling of the circZBTB46‑hnRNPA2B1 interaction

The secondary structure of circZBTB46 was predicted via the online web server RNAfold [17] (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). Then, the 3dRNA/DNA Web Server [18] (http://biophy.hust.edu.cn/new/3dRNA) and I-TASSER [19] (https://zhanggroup.org/I-TASSER/) were used to predict the three-dimensional structures of circZBTB46 and hnRNPA2B1, respectively. Moreover, the ubiquitination sites in the hnRNPA2B1 protein were predicted via a Bayesian discriminant method (http://bdmpub.biocuckoo.org/prediction.php). In addition, the interaction sites in circZBTB46 and hnRNPA2B1 were identified with catRAPID [20] (http://service.tartaglialab.com/page/catrapid_group) and the HDOCK server [21] (http://hdock.phys.hust.edu.cn/) and was then visualized using Discovery Studio 2021 (client version).

Immunohistochemical staining (IHC)

We conducted immunohistochemical staining in accordance with a standard protocol, which included deparaffinization, rehydration, antigen retrieval, and blocking. Then, the sections were incubated with a primary antibody at 4 °C overnight and subsequently incubated with a secondary antibody for 30 min at room temperature prior to color development with DAB. After counterstaining with hematoxylin, the slides were dehydrated, transparentized using xylene, and mounted with neutral resin.

Coimmunoprecipitation (Co-IP) assay

We performed a co-IP assay using an Immunoprecipitation Kit with Protein A + G Magnetic Beads (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Protein A/G beads were first incubated with the anti-hnRNPA2B1 antibody at room temperature for 2 h with agitation. Then, cell lysates were added to the protein A/G bead–antibody complexes and incubated overnight at 4 °C with rotation. Finally, SDS‒PAGE Sample Loading Buffer (1X) was used to elute precipitated proteins at 95 °C for 5 min, and the supernatants were collected for WB analysis with anti-hnRNPA2B1 or anti-ubiquitin (Proteintech, Wuhan, China) antibodies.

Statistical analysis

Statistical analysis of the data in the present study was performed by GraphPad Prism 8, and the data are presented as the means ± SEMs. Comparisons between two groups were conducted by two-tailed Student’s t test for normally distributed data or the Kruskal-Wallis test for nonnormally distributed data, while comparisons among more than two groups were conducted by one-way ANOVA test. A p value < 0.05 was considered to indicate statistical significance.