Hsa_circ_0030042 regulates abnormal autophagy and protects atherosclerotic plaque stability by targeting eIF4A3

Rationale: Abnormal autophagic death of endothelial cells is detrimental to plaque structure as endothelial loss promotes lesional thrombosis. As emerging functional biomarkers, circular RNAs (circRNAs) are involved in various diseases, including cardiovascular. This study is aimed to determine the role of hsa_circ_0030042 in abnormal endothelial cell autophagy and plaque stability. Methods: circRNA sequencing and quantitative polymerase chain reaction were performed to detect hsa_circ_0030042 expression in coronary heart disease (CHD) and human umbilical vein endothelial cells (HUVECs). Transfection of stubRFP-sensGFP-LC3 adenovirus, flow cytometry, and electron microscopy were used to identify the role of hsa_circ_0030042 in ox-LDL‒induced abnormal autophagy in vitro. Bioinformatic analysis, RNA immunoprecipitation, immunofluorescence assay and other in vitro experiments were performed to elucidate the mechanism underlying hsa_circ_0030042-mediated regulation of autophagy. To evaluate the role of hsa_circ_0030042 in atherosclerotic plaques and endothelial function, we measured the carotid artery tension and performed histopathology and immunohistochemistry analysis. Results: hsa_circ_0030042 was significantly downregulated in CHD, while upon overexpression, it acted as an endogenous eukaryotic initiation factor 4A-III (eIF4A3) sponge to inhibit ox-LDL-induced abnormal autophagy of HUVECs and maintain plaque stability in vivo. Furthermore, hsa_circ_0030042 influenced autophagy by sponging eIF4A3 and blocking its recruitment to beclin1 and forkhead box O1 (FOXO1) mRNA, while hsa_circ_0030042-induced inhibition of beclin1 and FOXO1 was counteracted by eIF4A3 overexpression or decreased hsa_circ_0030042 binding. In high-fat-diet fed ApoE-/- mice, hsa_circ_0030042 also ameliorated plaque stability and counteracted eIF4A3-induced plaque instability. Conclusions: These results demonstrate a novel pathway involving hsa_circ_0030042, eIF4A3, FOXO1, and beclin1; hence, modulating their levels may be a potential therapeutic strategy against CHD.

OD260/OD280 ratios between 1.8 and 2.1 were deemed acceptable. Five micrograms of RNA per sample was used as input material for RNA sample preparation. First, we used an Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA) to obtain rRNA-depleted ribosomal RNAs. rRNAdepleted RNAs were further treated with RNase R (Epicentre, USA). Subsequently, sequencing libraries were generated from the rRNA-depleted and RNase R digested RNAs using an NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations. Briefly, the RNA was fragmented using divalent cations at an elevated temperature in NEBNext First Strand Synthesis Reaction Buffer. First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNaseH−).
Second-strand cDNA synthesis was then completed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activity. After adenylation of the 3' ends of DNA fragments, NEBNext Adaptors with hairpin loop structures were ligated to prepare for hybridization.
To select cDNA fragments 150-200 bp in length, the library fragments were purified using an AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl of USER Enzyme (NEB, USA) was allowed to react with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by 5 min at 95°C before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primers. Finally, the library was purified (AMPure XP system) and then qualified using an Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System with a HiSeq PE Cluster Kit v4 cBot (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq 2500 platform, and 125-bp paired-end reads were generated. We defined a circRNA expressed or not by junction reads with software CIRI2 (version 2.0.5), CIRI AS (version 1.2) and find_circ (version 1.2). We defined the statistical criteria with software DESeq2 (version1.10.1) for significant differently expressed circRNA as having a p-value＜0.05 and a fold change ≥2.0 or ≤0.5

Transfection of stubRFP-sensGFP-LC3 adenovirus
To observe the autophagy of HUVECs, stubRFP-sensGFP-LC3 adenovirus (Genechem, Shanghai, China) were transfected into cells according to the manufacturer's instructions. Following transfection for 72 h, HUVECs were stimulated with 100ng/ml ox-LDL for 0 h, 3 h and 24 h. After fixed with 4% formaldehyde, the autophagosomes were photographed using confocal laser scanning microscopy.

Northern blot
Northern blot was performed as others described. In brief, the samples were run on a 1% formaldehyde-polyacrylamide-urea gel, transferred to positively charged Hybond N+ membranes (Amersham) followed by cross-linking through UV irradiation. The membranes were subjected to hybridization with 100 pmol 3'-digoxigenin (DIG)-labeled probe for hsa_circ_0030042 overnight at 50°C. hsa_circ_0030042 probe was synthesized with PCR DIG Probe Synthesis Kit (Roche). The detection was performed using a DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) according to the protocol. The forward primer for hsa_circ_0030042 probe sequence was 5'agtgacttggatggcatgtt-3'; the reverse primer was 5'-tctggattgagcatccaccaaga-3'. DIG-labeled GAPDH probe was used as control, and its forward primer was 5′-aatcccatcaccatcttcc-3'; the reverse primer was 5'-catcacgccacagtttcc-3'.

Western blot
HUVECs and aortic tissue samples were lysed using RIPA buffer (Solarbio, Beijing, China) for 20 min and collected by centrifugation at 12000 rpm for 10 min at 4℃. Nuclear and cytoplasmic proteins were extracted (Extraction Reagents, Thermo, USA) as needed according to the attached protocol. Equal amounts of proteins and pre-stained protein ladder (Thermo Fisher Scientific) were separated through 12% SDS-PAGE gels (TGX FastCast Acrylamide Kit, Bio-Rad, USA) Then, proteins were transferred to methanol-activated polyvinylidene fluoride membranes with a 0.2 μm pore size (Millipore, Billerica, MA, USA), and incubated with primary antibodies overnight at 4 °C.
The membranes were incubated with secondary antibodies (ProteinTech, Rosemont, Penn., USA) the next day for 1 h at room temperature. Bands with antigen-antibody complexes were visualized

Electron Microscopy
Stable hsa_circ_0030042 overexpression HUVECs (c0030042) and empty vector transfected HUVECs (circ-N.C) were transiently transfected with eIF4A3 siRNA for 48 h and 100ug/ml ox-LDL treated 24h. Then the cells were collected, 1,000 rpm centrifuge 5 min, the supernatant was discarded, 1 mL PBS resuspended, centrifuged 10 min, the supernatant was discarded, fixed with 2.5% glutaraldehyde, and then the the cells were embedded in spur resin after dehydration. Thin sections were cut on a Reichert Ultracut E microtome. Sectioned grids were stained with saturated solution of uranyl acetate and lead citrate. Sections were examined at 80 kV with a Hitachi transmission electron microscope.

Indirect Immunofluorescence Assay and Confocal Microscopy
circ-N.C and c0030042 group cells were grown on cover slips in 24-well plates. Upon reaching 70-80% confluence, cells were fixed in 4% paraformaldehyde for 30 min at room temperature and washed with PBS. Subsequently, permeabilized with PBS containing 0.5% Triton-X-100 for 15 min and blocked with PBS containing 5% bovine serum albumin for 1 h at room temperature. Then, samples were incubated with anti-eIF4A3 antibody (1:500, ab180573) for 4°C overnight, followed by incubation with anti-rabbit IgG Alexa Fluor 594-conjugated antibody for 1 h, and cells nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). All fluorescence images were acquired on an Olympus confocal microscope.

Actinomycin D treatment
To block transcription, cell culture medium was added with 10 ug/ml Actinomycin D (Sigma-Aldrich, St. Louis, MO, USA) in 0h, 3h, 6h, 9h and 12h. After treatment with Actinomycin D for different time points, the remaining of mRNA was assessed using qRT-PCR.

Enzyme-Linked Immunosorbent Assay (ELISA)
The level of IL-1β in mice plasma was determined using a mouse IL-1β ELISA Kit (ab197742, abcam, UK). The level of IL-6 in mice plasma was assessed using a mouse IL-6 ELISA Kit (ab222503). The level of MCP-1 in mice plasma was tested using a mouse MCP-1 ELISA Kit (R&D system, Minneapolis, MN, USA). All kits were used according to the manufacturer's instructions.
The OD value was recorded at 450 nm (with reference of 570 nm) in an ELISA plate reader.  Table S1 Top 50 significantly upregulated and 50 down regulated in CHD PBMCs were shown in details. The red color represented the up-regulated circRNAs. The green color represented the down-regulated circRNAs in CHD. The blue color showed the selected exonic CHD related circRNA, hsa_circ_0030042. Differential expression analysis between the two groups was performed using DESeq2. The adjusted p-value (padj) is the p-value adjusted for multiple testing using Benjamini-Hochberg to estimate the false discovery rate (FDR). circRNAs with a padj＜0.05 and a fold change ≥2.0 or ≤0.5 were considered differentially expressed.