CircRNA DOCK1 Regulates miR-409-3p/MCL1 Axis to Modulate Proliferation and Apoptosis of Human Brain Vascular Smooth Muscle Cells

Background Intracranial aneurysm is an abnormal expansion in the intracranial arteries, which is associated with growth and apoptosis of vascular smooth muscle cells. Circular RNAs (circRNAs) have implicated in the progression of intracranial aneurysms. The purpose of this paper is to study the function and mechanism of circRNA dedicator of cytokinesis 1 (circ_DOCK1) in regulating proliferation and apoptosis of human brain vascular smooth muscle cells (HBVSMCs). Methods HBVSMCs were exposed to hydrogen peroxide (H2O2). Cell proliferation and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and flow cytometry, respectively. Circ_DOCK1, microRNA (miR)-409-3p, and myeloid cell leukemia sequence 1 (MCL1) levels were examined by quantitative reverse transcription polymerase chain reaction or western blotting. The target association was assessed by dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation assays. Results Exposure to H2O2 decreased proliferation and increased apoptosis of HBVSMCs. Circ_DOCK1 expression was reduced in H2O2-treated HBVSMCs. Circ_DOCK1 overexpression rescued H2O2-caused reduction of proliferation and PCNA expression and attenuated H2O2-induced apoptosis and expression of Bcl-2, Bax, and cleaved PARP. MiR-409-3p was targeted by circ_DOCK1 and upregulated in H2O2-treated HBVSMCs. MiR-409-3p upregulation mitigated the role of circ_DOCK1 in proliferation and apoptosis of H2O2-treated HBVSMCs. MCL1 was targeted via miR-409-3p and downregulated via H2O2 treatment. Circ_DOCK1 overexpression enhanced MCL1 expression via modulating miR-409-3p. MiR-409-3p knockdown weakened H2O2-induced proliferation reduction and apoptosis promotion via regulating MCL1. Conclusion Circ_DOCK1 overexpression mitigated H2O2-caused proliferation inhibition and apoptosis promotion in HBVSMCs by modulating miR-409-3p/MCL1 axis.


INTRODUCTION
Intracranial aneurysm is an abnormal expansion in the intracranial arteries which could lead to aneurysm rupture (Brinjikji et al., 2016). The therapeutic strategies against intracranial aneurysm mainly include surgical and endovascular approaches (Lozano et al., 2019). However, the majority of cases with vascular remodeling undergo eventual rupture (Frosen et al., 2004). Smooth muscle cells are responsible in maintaining the vascular structure and are associated with cerebrovascular diseases, including intracranial aneurysm (Frosen and Joutel, 2018). The vascular smooth muscle cell apoptosis can lead to the degradation of vascular wall, thus inducing the development and rupture of intracranial aneurysm . Hence, exploring the mechanism of vascular smooth muscle cell proliferation and apoptosis may help in finding novel ways for intracranial aneurysm treatment.
Non-coding RNAs are important regulators for vascular smooth muscle cell processes in vascular diseases (Leeper and Maegdefessel, 2018). Circular RNAs (circRNAs) are a type of non-coding RNAs without 5 and 3 ends, which can function as microRNA (miRNA) sponges to take part in the regulation of vascular smooth muscle cell processes in intracranial aneurysm . For instance, hsa_circ_0021001 can act as a potential biomarker for intracranial aneurysm, and patients with low expression of hsa_circ_0021001 have the worse outcomes (Teng et al., 2017). The circRNA dedicator of cytokinesis 1 (circ_DOCK1, also called hsa_circ_0020397 according to the circRNA ID of circBase database) is downregulated in artery wall tissues and vascular smooth muscle cells of intracranial aneurysm patients, and it promotes vascular smooth muscle cell proliferation Yin and Liu, 2021). Although the reports also uncovered the miR-138/KDR and miR-502-5p/GREM1 networks underlying the regulation of circ_DOCK1, our understanding of its molecular basis is still limited.
MiRNAs are a group of short non-coding RNAs that modulate mRNA expression, which are involved in intracranial aneurysm progression (Liu et al., 2014) and are associated with the regulation of vascular smooth muscle cell proliferation and apoptosis (Wang and Atanasov, 2019). For instance, miR-448-3p and miR-205 are associated with the progression of intracranial aneurysm Zhong et al., 2019). Furthermore, miR-409-3p is a differentially expressed miRNA in intracranial aneurysm (Bekelis et al., 2016). Nevertheless, the function and mechanism of miR-409-3p in vascular smooth muscle cell dysfunction in intracranial aneurysm remains unknown.
Myeloid cell leukemia sequence 1 (MCL1) is a key member of B cell lymphoma-2 (Bcl-2) prosurvival family, which controls cell proliferation and apoptosis (Ertel et al., 2013). Furthermore, MCL1 contributes to vascular smooth muscle cell proliferation and inhibits apoptosis in vascular diseases, including intracranial aneurysm (Lee et al., 2015;Zhao W. et al., 2018). CircInteractome and starBase algorithms predict miR-409-3p might bind to circ_DOCK1 and MCL1. Thus, we hypothesized circ_DOCK1 might indirectly regulate MCL1 by miR-409-3p to participate in the regulation of vascular smooth muscle cell dysfunction in intracranial aneurysm.
Oxidative stress is well known as a contributor to the development and rupture of intracranial aneurysm (Starke et al., 2013). Hydrogen peroxide (H 2 O 2 ), an inducer of oxidative stress, is involved in apoptosis of vascular smooth muscle cells (Meng et al., 2018). Moreover, H 2 O 2 has been used to establish an in vitro of intracranial aneurysm via inducing the apoptosis of vascular smooth muscle cells (Zhao W. et al., 2018;Shi et al., 2019). In this study, we established the cellular model of intracranial aneurysm using H 2 O 2 -treated human brain vascular smooth muscle cells (HBVSMCs). Moreover, we analyzed the function of circ_DOCK1 on H 2 O 2 -caused HBVSMC dysfunction and explored the potential regulatory network of circ_DOCK1/miR-409-3p/MCL1. This study may propose novel insight into the vascular smooth muscle cell dysfunction in intracranial aneurysm.

Cell Culture and H 2 O 2 Treatment
Human brain vascular smooth muscle cells  were purchased from Procell (Wuhan, China) and cultured in specific complete medium for vascular smooth muscle cell culture Procell) at 37 • C and 5% CO 2 . 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 H 2 O 2 (Sigma, St. Louis, MO, United States) for 6 h.

RNase R Digestion and Actinomycin D Analyses
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.

Flow Cytometry
Cell apoptosis was measured with an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime) following the instruction. After exposure to H 2 O 2 or not, 2 × 10 5 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).

Dual-Luciferase Reporter Assay
The binding sites of miRNAs to circ_DOCK1 were predicted by the web-based program CircInteractome 1 . 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

RNA Pull-Down and RNA Immunoprecipitation Assays
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.

Statistical Analysis
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.

Circ_DOCK1 Expression Is Reduced in H 2 O 2 -Treated HBVSMCs
To analyze whether circ_DOCK1 was involved in intracranial aneurysms, a H 2 O 2 -caused cellular model was established using HBVSMCs. As shown in Figures 1A,B, stimulation of H 2 O 2 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 H 2 O 2 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 H 2 O 2induced HBVSMC injury.

Circ_DOCK1 Overexpression Attenuates H 2 O 2 -Induced HBVSMC Injury
To study the function of circ_DOCK1 in H 2 O 2 -induced model, HBVSMCs were transfected with vector or circ_DOCK1 overexpression vector before the stimulation of H 2 O 2 . The transfection of circ_DOCK1 overexpression vector markedly elevated circ_DOCK1 abundance in HBVSMCs (Figure 2A). Furthermore, circ_DOCK1 overexpression mitigated H 2 O 2induced decrease of cell proliferation and proliferation-related PCNA expression (Figures 2B,C). Additionally, circ_DOCK1 upregulation weakened H 2 O 2 -caused apoptosis of HBVSMCs ( Figure 2D). Moreover, the antiapoptotic Bcl-2 and proapoptotic Bax and cleaved PARP levels were detected in HBVSMCs. Results showed H 2 O 2 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 H 2 O 2 -induced HBVSMC damage.

MiR-409-3p Is Targeted by circ_DOCK1 and Upregulated in H 2 O 2 -Treated HBVSMCs
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 H 2 O 2 ( Figure 3E). These results suggested that miR-409-3p was targeted via circ_DOCK1.

DISCUSSION
Intracranial aneurysm is local dilatation in cerebral arteries, and about 2-5% cases can develop to rupture .
Smooth muscle cells are one key cell type-forming media in intracranial arteries and have significant roles in intracranial aneurysm formation and rupture (Starke et al., 2014). The apoptosis and impaired proliferation of brain vascular smooth muscle cells are related to intracranial aneurysm progression (Miyata et al., 2020;Wei et al., 2020). In our study, we established an in vitro model of intracranial aneurysm using H 2 O 2 -challenged HBVSMCs as previously reported (Zhao W. et al., 2018;Shi et al., 2019). We found that circ_DOCK1 could attenuate H 2 O 2 -induced apoptosis promotion and proliferation inhibition in HBVSMCs. Moreover, we provided a novel molecular explanation, the miR-409-3p/MCL1 axis, for the function of circ_DOCK1 (Figure 7). Such analysis was hampered at present by the lack of in vivo assays using the animal models of intracranial aneurysm.
Circular RNAs are relevant to vascular smooth muscle cell dysfunction and intracranial aneurysm development Maguire and Xiao, 2020). Multiple evidences have reported that circ_DOCK1 could facilitate cell proliferation and constrain apoptosis in various cancers, like thyroid cancer, oral squamous cell carcinoma, bladder cancer, and colorectal cancer (Zhang et al., 2017;Wang et al., 2018;Liu P. et al., 2019;Cui and Xue, 2020). Moreover, circ_DOCK1 could increase proliferation of human umbilical artery smooth muscle cells . These all suggested the pro-proliferation and antiapoptotic functions of circ_DOCK1 in various cell lines. PCNA is a proliferation-related factor that regulates cell cycle  process and DNA replication (Strzalka and Ziemienowicz, 2011). The antiapoptotic Bcl-2 and proapoptotic Bax are important players in intrinsic apoptosis (Cui and Placzek, 2018;Carpenter and Brady, 2021). PARP is a multifunction protein associated with DNA damage and cell apoptosis (Kumar et al., 2020). By combining the detection of these biomarkers, we found that circ_DOCK1 mitigated H 2 O 2 -driven proliferation inhibition and apoptosis promotion in HBVSMCs.
Next, we wanted to explore a regulatory network mediated by circ_DOCK1. Bekelis et al. (2016) identified 20 upregulated miRNAs in aneurysm tissues. After analyzing the interaction between them and circ_DOCK1 using CircInteractome, we found that only miR-409-3p had potential to bind to circ_DOCK1. Hence, we analyzed and confirmed that miR-409-3p was targeted by circ_DOCK1. Previous studies reported miR-409-3p could repress cell proliferation in papillary thyroid carcinoma, breast cancer, tongue squamous cell carcinoma, and osteosarcoma (Zhang et al., 2016;Chen and Dai, 2018;Zhao Z. et al., 2018;Wu et al., 2019). These reports all suggested the antiproliferation function of miR-409-3p in various cells. Similarly, our study validated the antiproliferation and proapoptotic roles of miR-409-3p in H 2 O 2 -treated HBVSMCs. Moreover, we further confirmed that circ_DOCK1 exhibited the protective function on H 2 O 2 -induced HBVSMC damage by inhibiting miR-409-3p.
We further explored the downstream targets of miR-409-3p, and found the anti-apoptotic MCL1 was targeted by miR-409-3p. Previous reports suggested that MCL1 could promote cell proliferation and inhibit apoptosis in pulmonary artery smooth muscle cells and rat thoracic aortic smooth muscle cells (Lee et al., 2015;. Moreover, Zhao W. et al. (2018) showed MCL1 attenuated HBVSMC apoptosis by regulating the mitochondrial apoptotic pathway. Our results first identified MCL1 as a functional target of miR-409-3p. Furthermore, we first demonstrated that circ_DOCK1 could modulate MCL1 expression through miR-409-3p.
Previous work showed that H 2 O 2 enhanced apoptosis of vascular smooth muscle cells depending on the regulation of miR-26a/PTEN/AKT/mTOR pathway (Peng et al., 2018). Moreover, Zhao W. et al. (2018) reported that H 2 O 2 induced miR-29a expression in HBVSMCs and miR-29a knockdown abolished H 2 O 2 -dirven HBVSMC apoptosis, suggesting that H 2 O 2 promoted HBVSMC apoptosis by upregulating miR-29a. Our data suggested that H 2 O 2 drove HBVSMC apoptosis partially by regulating miR-409-3p/MCL1 axis via downregulating circ_DOCK1. With these findings, we envision that circ_DOCK1 may be a starting point for the development of circRNA-based therapies against intracranial aneurysm.

CONCLUSION
In conclusion, circ_DOCK1 promoted cell proliferation and inhibited apoptosis in H 2 O 2 -treated HBVSMCs at least in part by regulating miR-409-3p/MCL1 axis. This study proposed the importance of circ_DOCK1/miR-409-3p/MCL1 axis in regulating HBVSMC dysfunction and provided a potential therapeutic target for intracranial aneurysm treatment.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
XD designed and performed the experiments and obtained the data. XW and LH performed the statistical analysis. ZZ and SJ wrote the sections of the manuscript. YT wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.