Human brain vascular smooth muscle cells (Cat. No. CP-H116) were purchased from Procell (Wuhan, China) and cultured in specific complete medium for vascular smooth muscle cell culture (Cat. No. CM-H116; Procell) at 37°C and 5% CO₂. To establish an in vitro of intracranial aneurysm as reported (Zhao W. et al., 2018; Shi et al., 2019), cells were incubated with 0, 30, 90, or 180 μM of H₂O₂ (Sigma, St. Louis, MO, United States) for 6 h.

Circular RNAs dedicator of cytokinesis 1 overexpression vector was constructed by Geneseed (Guangzhou, China), and the pCD5-ciR vector was regarded as a negative control (vector). MiR-409-3p mimic, mimic negative control (miR-NC), miR-409-3p inhibitor (anti-miR-409-3p), inhibitor negative control (anti-miR-NC), small interfering RNA (siRNA) for MCL1 (si-MCL1), and negative control of siRNA (si-NC) were generated by Genomeditech (Shanghai, China), and the oligonucleotide sequences are shown in Table 1. For cell transfection, HBVSMCs were incubated with 1 μg constructed vectors or 30 nM oligonucleotides and 5 μl Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, United States). After 24 h, transfected cells were harvested for expression analysis or subjected to H₂O₂ (180 μM) exposure.

Human brain vascular smooth muscle cells were lysed in Trizol (Thermo Fisher Scientific), and total RNA was isolated following the accompanying instructions. Then 1 μg RNA was reverse transcribed using miRNA Reverse Transcriptase kit or M-MLV Reverse Transcriptase kit (Thermo Fisher Scientific) according to the accompanying instructions. For quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, cDNA was mixed with SYBR (Vazyme, Nanjing, China) and designed primers. The primer pairs were synthesized by Sangon (Shanghai, China), and the sequences are shown in Table 2. The qRT-PCR was performed on CFX96^TM Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, United States). Relative expression level was detected by the 2^–ΔΔCt method with U6 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.

The circular structure of circ_DOCK1 was analyzed by RNase R digestion and actinomycin D analyses. For RNase R digestion analysis, RNA was treated with 2 U/μg RNase R (Geneseed) for 20 min, followed by reverse transcription and qRT-PCR for detection of circ_DOCK1 and linear DOCK1 expression.

For actinomycin D analysis, HBVSMCs were challenged by 2 μg/ml actinomycin D (Sigma) for 0, 8, 16, or 24 h, followed by collection for RNA isolation. The isolated RNA was used for qRT-PCR to measure circ_DOCK1 and linear DOCK1 expression.

Cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). After the treatment of H₂O₂ or not, 1 × 10⁴ HBVSMCs were placed in 96-well plates. After incubation for 0, 24, or 48 h, 10 μl 5 mg/ml MTT (Solarbio, Beijing, China) was added, and cells were continuously cultured for 4 h. The medium was then discarded, and 100 μl dimethyl sulfoxide (DMSO) (Beyotime, Shanghai, China) was added. Optical density (OD) value at 570 nm was determined via a microplate reader (Bio-Rad Laboratories).

Cell apoptosis was measured with an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime) following the instruction. After exposure to H₂O₂ or not, 2 × 10⁵ HBVSMCs were added in 12-well plates and cultured for 48 h. Next, cells were collected, interacted with Annexin V-binding buffer, and then dyed with 10 μl Annexin V-FITC and propidium iodide (PI). The apoptotic cells (with Annexin V-FITC positive and PI positive or negative) were measured with a flow cytometer (Agilent, Beijing, China).

Human brain vascular smooth muscle cells were lysed in RIPA buffer (Beyotime), and protein was obtained after a centrifugation at 10,000 × g for 5 min. The protein was quantified with a bicinchoninic acid kit (Thermo Fisher Scientific) according to the instructions. The samples (20 μg) were separated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred on nitrocellulose membrane (Bio-Rad Laboratories). The membranes were incubated in 3% bovine serum albumin (Solarbio) for 1 h, and then interacted with primary antibodies overnight and secondary antibody for 2 h. All antibodies were purchased from Abcam (Cambridge, United Kingdom), including proliferating cell nuclear antigen (PCNA) (ab152112, 1:2,000 dilution), Bcl-2 (ab194583, 1:500 dilution), Bcl-2-associated X (Bax) (ab53154, 1:500 dilution), cleaved poly-ADP ribose polymerase (PARP) (ab32064, 1:3,000 dilution), MCL1 (ab243136, 1:2,000 dilution), GAPDH (ab9485, 1:5,000 dilution), and horseradish peroxidase-labeled IgG (ab6721, 1:10,000 dilution). Next, the membranes were interacted with enhanced chemiluminescence (Solarbio), and the blots were analyzed via Quantity One software (Bio-Rad Laboratories) with GAPDH as a normalized reference.

The binding sites of miRNAs to circ_DOCK1 were predicted by the web-based program CircInteractome¹. The molecular targets of miR-409-3p were predicted using the online database starBase (which are based on miRNA target prediction programs, i.e., TargetScan, miRanda, microT, PITA, miRmap, and PicTar)². The wild-type (WT) sequence (…AACAUU…) of circ_DOCK1 or MCL1 was inserted in the pmir-GLO vector (Promega, Madison, WI, United States), generating the circ_DOCK1-WT and MCL1-WT luciferase reporter vectors. The mutant (MUT) luciferase reporter vectors (circ_DOCK1-MUT and MCL1-MUT) were constructed using the mutated sequence (…CCACGG…). These luciferase reporter vectors and miR-409-3p mimic or miR-NC were cotransfected into HBVSMCs. After 24 h, luciferase activity was measured with a dual-luciferase analysis kit (Promega).

A Pierce^TM Magnetic RNA-Protein Pull-Down kit (Thermo Fisher Scientific) was used for RNA pull-down assay. Briefly, the biotin-labeled circ_DOCK1-WT, circ_DOCK1-MUT, and negative control (bio-NC) were generated and interacted with the magnetic beads. HBVSMCs were lysed and incubated with the magnetic beads for 8 h. MiR-409-3p level enriched on the beads was detected by qRT-PCR.

A Magna RIP^TM RNA-Binding Protein Immunoprecipitation kit (Sigma) was used for RNA immunoprecipitation (RIP) analysis. In brief, HBVSMC lysates were incubated with anti-Ago2 or anti-IgG-coated magnetic beads for 6 h. MCL1 and miR-409-3p levels enriched on the beads were measured via qRT-PCR.

All experiments were repeated three times with four replicates. Results were expressed as mean ± standard deviation (SD). Statistical analysis was processed by GraphPad Prism 8 (GraphPad Inc., La Jolla, CA, United States) and SPSS version 19 software (SPSS Inc., Chicago, IL, United States). The difference was compared by Student’s t test or one-way analysis of variance followed by Tukey’s post hoc test, as appropriate. It was statistically significant at P < 0.05.

To analyze whether circ_DOCK1 was involved in intracranial aneurysms, a H₂O₂-caused cellular model was established using HBVSMCs. As shown in Figures 1A,B, stimulation of H₂O₂ led to obvious proliferation reduction and apoptosis promotion in a concentration-dependent pattern, suggesting the successful establishment of the in vitro model. Moreover, circ_DOCK1 expression was examined in this model. Results displayed that circ_DOCK1 abundance was evidently decreased in HBVSMCs after treatment of H₂O₂ in a dose-dependent pattern (Figure 1C). Additionally, the stability of circ_DOCK1 was analyzed via RNase R digestion and actinomycin D analyses. Circ_DOCK1, rather than DOCK1, was resistant to RNase R and actinomycin D, indicating circ_DOCK1 had a stable circular structure (Figures 1D,E). These results suggested that the downregulated circ_DOCK1 might be associated with H₂O₂-induced HBVSMC injury.

To study the function of circ_DOCK1 in H₂O₂-induced model, HBVSMCs were transfected with vector or circ_DOCK1 overexpression vector before the stimulation of H₂O₂. The transfection of circ_DOCK1 overexpression vector markedly elevated circ_DOCK1 abundance in HBVSMCs (Figure 2A). Furthermore, circ_DOCK1 overexpression mitigated H₂O₂-induced decrease of cell proliferation and proliferation-related PCNA expression (Figures 2B,C). Additionally, circ_DOCK1 upregulation weakened H₂O₂-caused apoptosis of HBVSMCs (Figure 2D). Moreover, the antiapoptotic Bcl-2 and proapoptotic Bax and cleaved PARP levels were detected in HBVSMCs. Results showed H₂O₂ significantly inhibited Bcl-2 abundance and increased Bax and cleaved PARP expression, and this effect was reversed by circ_DOCK1 overexpression (Figure 2E). These results indicated circ_DOCK1 mitigated H₂O₂-induced HBVSMC damage.

To explore the regulatory mechanism addressed by circ_DOCK1, the downstream miRNAs were predicted by CircInteractome. MiR-409-3p was a potential target, and the target sites are shown in Figure 3A. To validate their target relationship, the circ_DOCK1-WT and circ_DOCK1-MUT vectors were constructed. Moreover, miR-409-3p mimic effectively reduced the luciferase activity of circ_DOCK1-WT, but it induced little effect on the activity of circ_DOCK1-MUT when the binding sites (AACAUU) were mutated to CCACGG (Figure 3B). In addition, miR-409-3p could enrich with bio-circ_DOCK1-WT, but little enrichment was induced in bio-circ_DOCK1-MUT (Figure 3C). Additionally, miR-409-3p abundance in HBVSMCs was markedly decreased via circ_DOCK1 overexpression (Figure 3D). Furthermore, miR-409-3p abundance was evidently enhanced in HBVSMCs after exposure to H₂O₂ (Figure 3E). These results suggested that miR-409-3p was targeted via circ_DOCK1.

To analyze whether miR-409-3p was required for circ_DOCK1 to regulate HBVSMC injury, HBVSMCs were transfected with vector, circ_DOCK1 overexpression vector, circ_DOCK1 overexpression vector + miR-NC, or miR-409-3p mimic prior to exposure to H₂O₂. After the transfection, miR-409-3p expression was markedly reduced by circ_DOCK1 overexpression, which was rescued via addition of miR-409-3p mimic (Figure 4A). Moreover, miR-409-3p upregulation abolished the influence of circ_DOCK1 on cell proliferation and PCNA expression in HBVSMCs under H₂O₂ (Figures 4B,C). Additionally, miR-409-3p overexpression reversed the influence of circ_DOCK1 on apoptosis and abundances of related proteins (Bcl-2, Bax, and cleaved PARP) in H₂O₂-treated HBVSMCs (Figures 4D,E). These results indicated that circ_DOCK1 modulated H₂O₂-induced HBVSMC damage by targeting miR-409-3p.

To further explore the regulatory network, the molecular targets of miR-409-3p were analyzed via starBase. MCL1 was a potential target, and the target sites of miR-409-3p on MCL1 are exhibited in Figure 5A. To confirm this interaction, the MCL1-WT and MCL1-MUT vectors were constructed. MiR-409-3p mimic caused significant loss of luciferase activity of MCL1-WT, but it did not change the activity of MCL1-MUT (Figure 5B), and lots of MCL1 and miR-409-3p could be enriched in Ago2-based complex (Figure 5C). Furthermore, the effect of miR-409-3p on MCL1 expression was investigated in HBVSMCs transfected with miR-NC, miR-409-3p mimic, anti-miR-NC, or anti-miR-409-3p. The overexpression or knockdown efficacy of miR-409-3p mimic or anti-miR-409-3p is validated in Figure 5D. In addition, MCL1 expression was markedly decreased via miR-409-3p overexpression and increased by miR-409-3p knockdown (Figures 5E,F). Moreover, MCL1 abundance in HBVSMCs was evidently decreased by treatment of H₂O₂ (Figures 5G,H). Additionally, the influence of circ_DOCK1 on MCL1 expression was analyzed in HBVSMCs transfected with vector, circ_DOCK1 overexpression vector + miR-NC, or miR-409-3p mimic. Results showed circ_DOCK1 overexpression significantly upregulated MCL1 expression, which was decreased by miR-409-3p overexpression (Figures 5I,J). These results indicated that circ_DOCK1/miR-409-3p axis could target MCL1.

To study the function of miR-409-3p/MCL1 axis in HBVSMC injury, HBVSMCs were transfected with anti-miR-NC, anti-miR-409-3p, anti-miR-409-3p + si-NC, or si-MCL1 prior to exposure to H₂O₂. MCL1 abundance was obviously enhanced by miR-409-3p knockdown in HBVSMCs, which was reduced via addition of si-MCL1 (Figures 6A,B). In addition, miR-409-3p knockdown attenuated H₂O₂-mediated proliferation inhibition by rescuing cell proliferation and PCNA level, and this function was abrogated via interference of MCL1 using si-MCL1 (Figures 6C,D). Moreover, miR-409-3p downregulation weakened H₂O₂-induced apoptosis by decreasing apoptotic rate and expression of Bax and cleaved PARP and increasing Bcl-2 abundance, and these events were reversed by interference of MCL1 (Figures 6E,F). These findings suggested that miR-409-3p regulated H₂O₂-induced HBVSMC damage by targeting MCL1.