Study design

The purpose of this study was to find a target of therapy for atherosclerosis. Firstly, through the intersection of three GEO arrays (GSE28829, GSE43292 and GSE41571) differential genes, we found a common target, MYBL1. Secondly, MYBL1 was knocked down or overexpressed in Human umbilical vein endothelial cells (HUVECs) to clarify the role of MYBL1. Thirdly, transcriptome sequencing was used to find the downstream signaling pathway of MYBL1. Finally, Western blot, immunofluorescence, LC3 track, Flow cytometry, ChIP and CO-IP were used to study the pathogenesis of MYBL1 in atherosclerosis.

Tissue collection and cell culture

Human arterial tissues were obtained from patients undergoing dissection aneurysm surgery, and a total of 16 specimens were collected from December 10, 2018 to March 19, 2021. The sample size for the human were merely a convenience. HUVECs were obtained from ATCC (USA). 10% FBS (Gibco, Thermo Fischer Scientific, Bartlesville, OK, USA) and 60 µg/ml endothelial cell growth agent (BD Biosciences) were used to culture HUVECs.

Animals and treatments

Seven-week-old male ApoE−/− and ApoE+/+mice purchased from GemPharmatech Co., Ltd were used for the experiments. Two groups (ApoE−/− and ApoE+/+ mice) fed a high-fat diets (0.15% cholesterol and 21% fat, Shanghai Medical Laboratory Animal Center) for 12 weeks. The environment was maintained on a 12-h light–dark cycle, and the mice had access to water AD libitum.

Data

In order to study the expression changes of atherosclerotic plaque genes, the selected dataset must contain atherosclerotic plaque samples. The atherosclerosis expression profile datasets were obtained from the Gene Expression Omnibus (GEO) database, and the series of GSE28829 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE28829), GSE43292 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE43292), and GSE41571 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41571) data were extracted for subsequent research and analysis. These samples all contained atherosclerotic plaque samples with different pfirrmann grades, and GSE28829 contained 13 atherosclerotic plaque samples (EA) and 16 advanced atherosclerotic plaque samples (AA). GSE43292 consisted of 32 specimens of early and advanced carotid atherosclerotic plaques. GSE41571 consisted of 5 samples of ruptured atherosclerotic plaques and 6 samples of stable atherosclerotic plaques.

Small interfering RNA and Lentivirus transfection

Small interfering RNAs to MYBL1 (si-MYBL1, Gene Pharma, China) were mixed with Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) for 20 min in the serum-free medium and then added to HUVECs.

The LV3-pGLV-H1 + Puro plasmids with pcDNA-MYBL1, pcDNA-AMPK, pcDNA-GABARAP, pcDNA-PLEKHM1, shRNA-MYBL1 and shRNA-PLEKHM1 (Lenti-MYBL1, Lenti-PLEKHM1, Lenti-shMYBL1 and Lenti-shPLEKHM1) (Gene Pharma, China) were transfected into HUVECs following all manufacturer protocols.

Differentially expressed genes identification

The probe names of GSE28829, GSE43292, and GSE41571 were converted into gene names. DESeq2, edgeR, and limma were used for differential analysis. Determination conditions for | log2 (FC) |> 1.5, adjusted P-value ≤ 0.05.

Functional enrichment analysis

GO function (https://www.geneontology.org/) and KEGG (https://www.genome.jp/kegg/) pathway enrichment analysis was performed on differential genes, and important pathways were selected. Go enrichment analysis is a key tool to understand the molecular function of genes and the biological pathways involved. KEGG contains genome, chemistry, system function information and so on.

Immunohistochemistry

The tissues were fixed with 4% formaldehyde solution (Beyotime, Shanghai, China) and subsequently dehydrated with high concentrations of ethanol (Damao, Tianjin, China), and the ethanol in the cells was displaced by the addition of xylene (Damao, Tianjin, China) for 30 min at room temperature. The tissues were embedded in paraffin for 3 h, then the tissues were cut into 4–5 μm and placed in constant temperature water at 40° C after the addition of 5% ethanol, followed by baking in an oven at 60° C for 30 min before removal for staining. Endogenous peroxidase and nonspecifically bound sites were blocked with 3% hydrogen peroxide and 5%BSA respectively. The primary antibody containing MYBL1 (Affinity, AF9007, 1:100) and PLEKHM1 (Abcam, ab204437, 1:50) was added to the sections and incubated in 10% goat serum blocking solution. The following day, the secondary antibody was adhered to the primary antibody and counterstained with hematoxylin.

Transcriptome sequencing

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, California, USA), followed by mRNA isolation using Nuceolspin RNA columns (Macherey–Nagel, Duren, Germany). cDNA was then synthesized using the Omniscript Reverse Transcription kit (Qiagen, UK). To smoothly build library and subsequent sequencing, FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to detect the original read piece of raw reads the quality. After cDNA libraries were constructed, they were sequenced on Illumina HiSeq2500 V4 2 × 100 PE (Genewiz).

Western blot

Extracted protein were obtained from tissues in atherosclerotic plaque tissue and HUVECs. Its concentration was tested with a BCA kit (Beyotime, Shanghai, China). The details refered to a previous study (Ding et al. 2020). The primary antibodies were listed as follow: MYBL1 (Affinity, AF9007, 1:1000), PLEKHM1 (Proteintech, 16202–1-AP, 1:1000), β-actin (Proteintech, 81115–1-RR, 1:5000), AMPK (Proteintech, 10929–2-AP, 1: 1000), GABARAP (Proteintech, 18723–1-AP, 1:500), Cleaved-Caspase3 (CST, 9664, 1:1000), Bcl-2 (Proteintech, 26593–1-AP, 1:1000), Bax (Proteintech, 50599–2-Ig, 1:1000), hVps41 (Proteintech, 13869–1-AP, 1:500), hVps11 (Proteintech, 19140–1-AP, 1:500), Rab7 (Proteintech, 55469–1-AP, 1:500), LC3 (Novus, NB100-2220, 1:1000) and p62 (CST, 88588, 1:1000).

RT-PCR

RT-PCR were performed according to previous study (Ding et al. 2020).

Transwell assay

Serum-free medium containing cells were added into the upper chamber (8 μm filter, Costar, Cambridge, MA, United States), followed by the addition of diluted Matrigel matrix gel (Corning, USA) in an incubator overnight. The medium containing 20% FBS was spread in the lower chamber. After the cell suspension was prepared, the cells were spread in the upper chamber. Non-migrating cells in the upper chamber were erased, and the migrated cells were fixed with 4% paraformaldehyde and stained with crystal violet (Procell, Wuhan, China).

TUNEL assay

After washing the cells once with PBS (Procell, Wuhan, China), the cells were fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 30 min and then washed once more with PBS. PBS with 0.1% Triton X-100 (Beyotime, Shanghai, China) was added and incubated for 2 min on ice. 50 μl of the ready-made TUNEL assay solution (Beyotime, Shanghai, China) was added and incubated at 37° C in the dark for 60 min before washing three times with PBS. TUNEL-positive cells were observed under fluorescence microscope after being treated with anti-fluorescence quenching solution.

CCK-8

The protocol of CCK-8 was according to previous study (Ding et al. 2020).

Immunofluorescence

Cells that had been seeded onto glass coverslips were fixed in 4% paraformaldehyde (Beyotime, Shanghai, China) for 10 min and then washed with PBS. The details referred to our previous study (Ding et al. 2020).

LC3 track

GFP-RFP-LC3 lentivirus was purchased from Hanhang Biotech (Shanghai, China) and then transfected into cells and incubated at 37° C for 72 h. LSM-510 (Zeiss) confocal fluorescence microscope was used to observe the change of GFP-RFP-LC3 fusion protein in the autophagy stream. GFP (green light) was quenched in acidic environment, indicating that autophagy activity in cells was strong.

Flow cytometry

Annexin V-FITC Apoptosis Detection Kit was purchased from Beyotime, Shanghai, China. The details referred to our previous study (Ding et al. 2020).

ChIP assays

Chromatin Immunoprecipitation (ChIP) Assay Kit (Beyotime, Shanghai, China) was used to performe ChIP. The cultured cells were fixed with 37% formaldehyde (final concentration 1%) for 10 min, then the cross-linking reaction was terminated by adding 2 mol/L glycine solution (final concentration 0.125 mol/L), and the cells were incubated for 10 min. The solution was washed twice by adding PBS, centrifuged at 1000 × g for 20 min and the precipitate was subsequently resuspended in M2 buffer. This step was performed twice. DNA was interrupted with an ultrasonic crusher and diluted threefold with ChIP dilution buffer. 100 μL of Protein A/G Dynabeads was washed with 1 mL ChIP dilution buffer, and magnetic beads resuspended in 100 μL ChIP dilution buffer were added to prehybridize chromatin for 1 h. 20 μL of 5 mol/L NaCl was added, and the cross-linking was deactivated at 65 °C for 6 h. After decross-linking, the cells were placed in a -20 °C refrigerator for temporary storage. 20 mg/mL glycogen sedimentation aid was added, then 3 mol/L NaAc (pH5.2) was added at a 1:10 volume ratio, and finally 1 mL absolute ethanol was added, and precipitated at -20 °C for 3 h. The DNA was purified by washing twice with 70% ethanol. The degree of DNA enrichment was analyzed by quantitative PCR.

CO-IP

After washing the cells twice with PBS, precooled RIPA (Beyotime, Shanghai, China) was added. Cells and suspensions were separated. Centrifugation was performed at 14000 rpm for 15 min at 4° C, and the supernatant was transferred to a new centrifuge tube. A 50% Protein A/G agarose bead working solution was added to the sample and the Protein A/G agarose beads were removed after centrifugation for 15 min. Antibodies were added and the mixture was incubated overnight. 100 μl Protein A agarose beads were added to capture the antibody-antibody complex, the antibody-antibody mixture was slowly shaken at 4° C for 1 h, and the precipitate was collected by transient centrifugation at 14000 rpm for 5 s, and the precipitate was washed three times with precooled wash buffer. The agarose beads-antigen–antibody complex was suspended in 60 μl of 2 × loading buffer and gently mixed. Free antigens, antibodies and beads were centrifuged, the supernatant was electrophorized, and the remaining agarose beads were collected. Later electrophoresis was carried out. Before electrophoresis, it should be boiled for 5 min again.

Cholesterol analysis

Cholesterol was detected according to manufacturer’s protocol (Beyotime, Shanghai, China).

Statistical analysis

SPSS 20.0 statistical software was used for statistical analysis, and all experiments were repeated at least three times. Data were expressed as mean ± standard deviation (mean ± SD). Analysis of variance and Tukey post hoc were used to analyze differences among multiple groups. When the p-value < 0.05. ***p < 0.001, **p < 0.01, *p < 0.05, the difference was considered statistically significant.