Patients and cells

Blood samples were collected from 21 atherosclerosis patients and 17 healthy volunteers from Shengjing Hospital of China Medical University to prepare serum samples, and the participants provided informed consent. All examined samples were stored at − 80 °C. This research was done under the Ethics Committee in Shengjing Hospital of China Medical University and was carried out according to the guidelines of the Declaration of Helsinki.

HUVECs were furnished by American Type Culture Collection (Manassas, VA, USA), and grown in Dulbecco’s modified Eagle’s medium (DMEM; cat.no. SG 12,100; Solarbio, Beijing, China) with 5% CO2 at 37 °C. 10% fetal bovine serum (FBS; cat.no.10082; Invitrogen, Paisley Scotland, UK) and 1% penicillin–streptomycin (cat. no. 15140–122; Invitrogen) were added to DMEM. To establish Atherosclerosis models, HUVECs were treated with 0 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL of ox-LDL (cat. no. H7950; Solarbio, Beijing, China) for 24 h.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA in cells and serum samples was extracted with the help of Trizol (cat.no.15596018; Thermo Fisher Scientific, Waltham, MA, USA), followed by incubation with RNase R (4 U/μg, cat. no. RNR07250; Epicentre, Illumina, San Diego, CA, USA) at 37 °C for 1 h. Then, cDNA was synthesized and amplified in PCRmax Alpha gradient PCR instrument (Staffordshire, UK) with SYBR Green PCR kit (cat. no. DRR820A; TaKaRa, Beijing, China). Relative RNA expression was normalized to GAPDH or U6, and calculated by the 2–ΔΔCt method. All primers were obtained from Beijing Genomics Institute (BGI, Shenzhen, China), as shown in Table 1.

Table 1 Primers sequences used for PCRCell proliferation

The impacts of ox-LDL and circ_0004104 on cell proliferation were evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT). Shortly, 0.5 g MTT (cat. no. M1020; Solarbio) was dissolved in phosphoric acid buffer (PBS; cat.no. 10010023; Thermo Fisher Scientific) of 100 mL and configured into 5 mg/mL MTT working solution. MTT was used to treat cells for 4 h, and then cells were added with dimethyl sulfoxide (DMSO; cat. no. D8371; Solarbio). OD value at 490 nm was detected by a microplate analyzer (Molecular Devices, Silicon Valley, California, USA).

Cell apoptosis assay

Flow cytometry and Annexin V-FITC/PI apoptosis detection kit (cat.no. CA1020; Solarbio) were used to estimate HUVECs apoptosis. Briefly, ox-LDL-treated HUVECS were incubated with 5 μL Annexin V-FITC and PI and mixed with 400 μL binding buffer. Then apoptosis cells and normal cells were distinguished by FACS CantoII flow cytometry (BD Biosciences, San Jose, CA, USA) with BD FACSDiva software.

Angiogenesis experiment

HUVECs were grown with serum-free medium for 16 h in advance, then the cells were inoculated into a 24-well plate containing Matrigel (cat. no. 354234; BD Biosciences) and cultured in serum-containing medium. The tube formation was observed under a microscope and photographed under the inverted microscope. The amounts of the formed tubes represented the tube forming capability of HUVECs.

Western blot assay

Proteins that were extracted from ox-LDL-treated cells were separated with Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (cat. no. P1200; SDS-PAGE, Solarbio) and polyvinylidene difluoride (PVDF) membrane (Amersham, Munich, Germany), and then PVDF membrane was blocked with TBST buffer containing 5% bovine serum albumin. The protein bands were incubated with primary antibodies: CyclinD1 (ab16663, 1:1000, Abcam, Cambridge, MA, USA), cleaved-caspase 3 (ab2302, 1:1000, Abcam), ROCK2 (ab125025, 1:1000, Abcam), GAPDH (ab9485, 1:1000, Abcam), and the secondary antibody (1:5000, ab205718, Abcam), respectively. Finally, the protein bands were incubated with ECL Western Blotting Substrate (cat. no. PE0010; Solarbio) and observed by a gel imaging analysis system. The original western blots were showed in Additional file 1.

Enzyme‑linked immunosorbent assay (ELISA)

Cell supernatant of Atherosclerosis models was collected, and TNF-α (human) ELISA Kit (cat. no. 589201; Cayman Chemical, Ann Arbor, Michigan, USA) and IL-1β (human) ELISA Kit (cat. no. BMS224HS; eBioscience, San Diego, California, USA) were used to detect TNF-α and IL-1β contents in the supernatant, respectively.

Cell transfection

Geneseed (Guangzhou, China) provided the stable circ_0004104 overexpression vector (pCD5-circ_0004104, circ_0004104) and the negative control (pCD5-ciR). Circ_0004104 specific small interfering RNA (si-circ_0004104), si-NC, miR-942-5p mimics (miR-942-5p), miR-NC, anti-miR-942-5p, anti-miR-NC, pcDNA-ROCK2 (ROCK2) and pcDNA were constructed by Sangon Biotech (Shanghai, China), followed by transfection into HUVECs using Lipo3000 reagent (cat. no. L3000015; Invitrogen).

Dual-luciferase reporter assay

The forecasted circ_0004104 and ROCK2 sequences that bound miR-942-5p and site-directed mutation sequences were cloned into psiCHECK2 plasmid (Promega, Madison, WI, USA) and expressed as WT-circ_0004104, WT-ROCK2 3’UTR, MUT-circ_0004104 and MUT-ROCK2 3’UTR. Then plasmids were co-transfected into HUVECs with miR-942-5p or miR-NC, respectively. After 24 h, the luciferase activities of plasmids were detected according to Dual-Luciferase Reporter Assay System (cat. no. E1910; Promega).

RNA immunoprecipitation (RIP) assay

The RIP experiment was implemented by taking advantage of the RNA Immunoprecipitation Kit (cat. no. 17–700; Sigma-Aldrich, St. Louis, MO, USA) and qRT-PCR. In short, HUVECs were lysed in complete RIP lysis buffer, followed by a mixture with RIP buffer containing magnetic beads conjugated with anti-Argonaute2 (Ago2, ab186733, 1:100, Abcam) antibody or normal mouse IgG (ab172730, 1:100, Abcam). Finally, beads were washed twice with PBS, and total RNA was isolated and subjected to qRT-PCR analysis of circ_0004104, miR-942-5p, and ROCK2.

RNA pull-down assay

For testing the interaction between miR-942-5p and circ_0004104 or ROCK2 in HUVECs, RNA pull-down assay was conducted. In short, HUVECs transfected with biotinylated- miR-942-5p (bio-miR-942-5p) and bio-miR-NC (GenePharma, Shanghai, China) were harvested and lysed. After that, magnetic beads (cat. no. 112-05D; Invitrogen) was utilized to mix with the harvested cell lysates, followed by RT-qPCR analysis.

Isolation and identification of exosomes

Ox-LDL treated and untreated HUVECs were centrifuged at 300 ×g for 10 min, the sediments were discarded and liquid supernatant was collected with a 0.22 µm filter mini-column (Sigma-Aldrich). The supernatant was centrifuged at 110,000 ×g for 70 min, the bottom precipitate was re-suspended with PBS, centrifuged at 110,000 ×g for 70 min, the precipitate was dissolved with 200 μL PBS, and stored at − 80 °C. Total exosome RNA and protein isolation kit (cat. no. 4478545; Invitrogen) was used to isolate RNA and proteins from exosomes and the protein levels of exosome surface markers CD9 (1:2000, ab92726, Abcam) and CD63 (1:2000, ab216130, Abcam) was measured by western blot.

Statistical analysis

All data were analyzed with SPSS 19.0 (IBM, Chicago, IL, USA) and illustrations were made with GraphPad Prism 8.0 (GraphPad Inc., LaJolla, California, USA). Pearson correlation analysis was used to analyze the expression association. Student’s t-test and one or two-way analysis of variance (ANOVA) were used to analyze differences between three or more groups. The results were shown as “mean ± standard deviation”. Statistical significance was identified as P value less than 0.05.