Regular paper

Circ_FOXO3 regulates KLF6 through sponge adsorption of
miR-122-5p to repress H2O2-induced HBVSMC proliferation,
thus promoting IA development in vitro model

Pei Dong Yue, Ya Nan Lu, Lei Zhang and Zheng Fei Ma✉

Department of Neurosurgery, Suzhou Hospital of Anhui Medical University (Suzhou Municipal Hospital of Anhui Province), SuZhou City, AnHui Province, 234000, China

Purpose: The phenotypic transformation of human brain vascular smooth muscle cells (HBVSMC) is widely involved in the appearance and progression of intracranial aneurysms (IA). Aneurysm (IA) Circular RNA circ_FOXO3 functions pivotally in vascular diseases and tumors, but its regulatory role as well as its molecular mechanism in IA is still uncertain. This research was to explore how circ_FOXO3 works and its mechanism in vitro model of HBVSMC IA induced by H2O2. Methods: Thirty-eight patients with IA and their normal tissues were clinically collected. Examination of endothelin-1, vascular hematoma factor, circ_FOXO3, microRNA (miR)-122-5p and KLF6 and the correlation of circ_FOXO3 with clinical case information were ensured. Establishment of an in vitro IA model was through HBVSMC induced by H2O2 and transfection with circ_FOXO3, miR-122-5p and KLF6 related plasmids was to figure out their roles in cell growth. The relationship among circ_FOXO3, miR-122-5p with KLF6 was detected. Results: Up-regulated circ_FOXO3 and KLF6 and reduced miR-122-5p were in IA tissues; Circ_FOXO3 was associated with smoking history, Hunt-Hess grading and endothelial injury degree. Repressive circ_FOXO3 or KLF6 and strengthening miR-122-5p facilitated H2O2-induced proliferation and repressed HBVSMC apoptosis, while elevation of circ_FOXO3 or depressive miR-122-5p was opposite. circ_FOXO3 bound to miR-122-5p, whose target was KLF6, which participated in controlling IA by mediating the circ_FOXO3/miR-122-5p axis. Conclusion: In summary, the findings suggest that circ_FOXO3 suppresses H2O2-induced proliferation of HBVSMC but promotes apoptosis via modulation of miR-122-5p/KLF6 axis. Targeted therapy of circ_FOXO3/miR-122-5p/KLF6 axis is supposed to be a promising treatment approach for IA patients.

Keywords: circular RNA circ_FOXO3; microRNA-122-5p; Promote; Intracranial aneurysm

Received: 12 November, 2021; revised: 23 December, 2021; accepted: 18 March, 2022; available on-line: 22 October, 2022

✉e-mail: mazfei817@hotmail.com

Abbreviations: circRNAs, circular RNAs; IA, intracranial aneurysm; miRNAs, microRNAs; ncRNAs, noncoding RNAs; VSMC, vascular smooth muscle cells

Introduction

Intracranial aneurysm (IA) is a serious cerebral vascular degeneration that often leads to fatal vessel rupture and subarachnoid hemorrhage (Maumus-Robert et al., 2020) . Despite decades of research, effective treatments for IA are still limited (Etminan et al., 2016). The molecular basis for the formation and rupture of IA is complex and the dysfunction of vascular smooth muscle cells (VSMC) is related to the pathogenesis of IA (Mandelbaum et al., 2013; Frösen et al., 2018). VSMC apoptosis can lead to vascular wall degradation, thus inducing the occurrence and rupture of IA (Liu et al., 2019). Therefore, exploring the mechanisms of proliferation and apoptosis of VSMC is helpful to find a new way to treat IA. Circular RNAs (circRNAs) are noncoding RNAs (ncRNAs) formed by covalently closed loops that are widely expressed in human cells (Salzman et al., 2012). In recent years, with the wide application of RNA sequencing technology, it has been found that many exon transcripts can accept nonlinear reverse splicing or gene rearrangement to form circRNAs (Chen et al., 2015). CircRNAs have been reported to be involved in the regulation of vascular smooth muscle cell processes in IA. For example, decreased expression of circ_0020397 in IA may reduce VSMC proliferation by increasing miR-138 expression and decreasing KDR expression (Wang et al., 2019). The cross-head box circRNA O3 (circ-Foxo3, hsa_circ_0006404), encoded by the human FOXO3 gene, is one of the most studied circRNAs and acts as a sponge for potential microRNAs (miRNAs). A study has clarified FOXO3 is involved in the regulation of many vascular diseases. For example, miR-30c-5p represses NLRP3 inflammasome-mediated endothelial cell apoptosis in atherosclerosis by down-regulating FOXO3 (Li et al., 2018). MiR-629 regulates hypoxic pulmonary vascular remodeling by targeting FOXO3 and PERP (Zhao et al., 2019). However, circ_FOXO3 in IA has not been fully studied. In recent years, research on IA has also focused on the regulation of miRNAs, which are endogenous 23 nt ncRNAs and negatively regulate gene expression. MiRNAs exert post-transcriptional functions mainly by directly binding to complementary messenger RNA and repressing the expression of target genes (Lewis et al., 2005; Lynam-Lennon et al., 2009; Bartel et al., 2009). Dysregulation of miRNA is associated with a variety of diseases, and more and more evidence indicates that miRNAs play a momentous role in vascular diseases. For example, miR-4735-3p regulates the phenotypic regulation of VSMC by targeting HIF-1-mediated IA autophagy (Gao et al., 2019). The down-regulation of MiR-29b induces the phenotypic regulation of VSMC, and its importance in the formation and progression of IA rupture is manifested (Sun et al., 2017), etc. Few studies have been conducted on miR-122-5p, and the action mechanism of miR-122-5p in IA needs to be further explored. The study confirmed that circ_FOXO3 repressed H2O2-induced proliferation and accelerated apoptosis of human cerebrovascular smooth muscle cells (HBVSMC). Further mechanistic studies manifested this role of circ_FOXO3 was realized by regulating the miR-122-5p/KLF6 axis, confirming the novel mechanism of circ_FOXO3 in IA process.

Materials and methods

Research objects

Thirty-eight IA patients (the IA group) with imaging diagnosis and neurosurgical clippings from the Suzhou Hospital of Anhui Medical University, Department of Neurosurgery, Suzhou Hospital of Anhui Medical University were selected as experimental subjects. Meanwhile, temporal polar temporal cortical artery tissue was removed from 38 patients (the control group) with temporal lobe epilepsy caused by amygdala and hippocampal sclerosis and was normal arterial tissue with postoperative histopathological examination. No obvious difference was presented in gender with age in the IA and the control. Venous blood (2 tubes) was taken from all subjects on an empty stomach simultaneously in the morning prior to surgery. The approval of the research was obtained through the Institutional Review Committee of the Suzhou Hospital of Anhui Medical University, following the principles of the Declaration of Helsinki. Written informed consent was obtained from the participants in the investigation.

Enzyme-linked immunosorbent assay (ELISA)

The examination of serum-related indicators was performed using an ELISA kit (NanJing JianCheng Institute of Bioengineering, Nanjing, China). After centrifugation of the blood samples, the examination of endothelin-1 (ET-1) and vascular hematoma factor (vWF) was based on the kit instructions.

Cell culture

HBVSMC (Cat. No. CP-H116) were purchased from Procell (Wuhan, China) and grown in a humidified incubator (Roewe Instrument Co., Ltd. Shanghai, China) at 37°C with 5% CO2. Dulbecco’s modified eagle medium containing 10% fetal bovine serum (FBS), 100 mg/L gentamicin and 2 mmol/L glutamine were applied. To establish IA in vitro (Shi et al., 2019; Zhao et al.,2018), cells were incubated with 0, 30, 90, or 180 μM H2O2 (Sigma, St. Louis, MO, USA) for 6 h.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

Culture medium consisting of 10% serum was adopted for the preparation of a single cell suspension; The 5×103 cells per well were seeded in a 96-well plate. After separate transfection of cells with circ_FOXO3 and NC for 0, 24, and 48 h, incubation was carried out with 10 μL per well 5 mg/ml MTT. After removing the supernatant, cells were added with 100 μL dimethyl sulfoxide (DMSO, Beyotime, Shanghai, China) was present. Finally, the detection of the absorbance value was carried out using a microplate reader (Thermo Labsystems) with wavelength of 570 nm.

Cell colony assay

The application of cells in logarithmic growth phase was for the preparation of a dispersed single cell suspension and the culture in a six-well plate medium was with 10% FBS. After removing the culture medium and transfection of the cells with circ_FOXO3 and NC, the culture medium was replaced with a new one and continued culture was conducted. Finally, after discarding the supernatant, the suspension was fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet to count colonies.

Real-time polymerase chain reaction (Rt-PCR)

The application of Trizol RNAiso (Takara) was for the extraction of total cellular RNA, and Thermo Nano Drop 2000 was used to detect the concentration and purity of total RNA, and Agilent-2100 sulfate polyacrylamide gel electrophoresis was applied to detect the integrity of total RNA. RNA was retrotranscribed to cDNA using the M-MLV reverse transcriptase kit (Thermo Fisher Scientific), and the final adoption of the quantitative real-time PCR kit was for fluorescence quantification (Takara, Japan). Primers were constructed and synthesized via Beijing Kinco. The adoption of GAPDH and U6 was performed as internal controls. Primer sequences were manifested in the following:

Circ_FOXO3:

MiR-122-5p:

KLF6:

GAPDH:

U6:

Luciferase report assay

The subclone of the circ_FOXO3 sequence or the 3’untranslated region (UTR) fragment of KLF6 containing the forecast binding site of miR-122-5p was included in the pmirglol-Luciferase target expression vector. After construction of the wild-type carriers circ_FOXO3 (pmirglol – circ_FOXO3-WT) and wild-type carriers KLF63 3’UTR (pmirglo-KLF63 3’UTR-WT) carriers, and plasmids pmirglo- KLF63 3’UTR-MUT or pmirGLO-circ_FOXO3- MUT, HBVSMC transfection was with KLF6 or circ_FOXO3 WT, MUT vectors, and miR-122-5p mimic or its NC for detection of luciferase activity via the dual luciferase Assay Kit (Promega, Madison, WI, USA).

RNA-pull down

For verification of the binding of miR-122-5p with circ_FOXO3, the assay was conducted. After the design of 3 biotin-labeled miRNA sequences (Bio-miR-122-5p-WT, -MUT, Bio-miR-NC, GenePharma, Shanghai, China), transfection was with biotinylated oligonucleotides and then specific lysate products (Ambion, Austin, Texas, USA). After incubation of the dissolved products with m-280 plates, RNase-free streptomycin beads and yeast tRNA (from σ), the wash was with a cold solution, low- and high-salt buffer. The antagonistic miR-122-5p probe was as NC. After Trizol extracted total RNA, detection of circ_FOXO3 enrichment was by Rt-PCR.

RNA immunoprecipitation (RIP) assay

A Magna RIPTM RNA-binding protein immunoprecipitation kit (Sigma) was used for RIP analysis. Briefly, HBVSMC lysates were incubated with magnetic beads coated with anti-Ago2 or anti- immunoglobulin G. The measurement of KLF6 and miR-122-5p enriched in the beads was done by Rt-PCR.

Western blot

The cell protein extraction was carried out using radio-immunoprecipitation assay lysis buffer consisting of 1 mM phenylmethylsulfonyl fluoridenone with the appropriate volume. Subsequently, the application of bicinchoninic acid (Beyotime) was applied to draw a standard curve to determine protein concentration. The protein separation was performed through 10% polyacrylamide gel, electroblotting was performed onto a polyvinylidene fluoride membrane, and then block with 5% skim milk powder was implemented. After incubation with primary antibody, secondary antibody labeled with horseradish peroxidase (1:10 000; ab6721; Abcam), final conduction of the strong chemiluminescence method was for quantification (ChemiDoc-It415 Imager, upland), and Image J software was applied for quantitative analysis. Primary antibody KLF6 (1:1000; sc-365633; Santa Cruz Biotechnology) was conducted.

Statistical analysis

The adoption of SPSS 22.0 was for statistical analysis of the data and the expression of the results was by mean ± standard deviation. Conduction of sample comparison between two groups and multiple groups was done by independent sample t test as well as ANOVA analysis of variance. For P<0.05, differences was considered as statistically significant.

Results

Strengthening circ_FOXO3 and KLF6 and reduced
miR-122-5p are testified in IA tissue

Detection of factors in IA and the control manifested the elevation of ET-1, vWF, circ_FOXO3, and KLF6, and the repression of miR-122-5p in IA (Fig. 1A–F).

Hunt-Hess grade and degree of endothelial injury with smoking history were implicated in circ_FOXO3 in IA tissue

Based on the mean expression of circ_FOXO3, the assignment of the patients was made to reduced and elevated groups. Analysis of the relationship of circ_FOXO3 expression with the clinicopathological characteristics of IA patients affirmed that Hunt-Hess grade, endothelial injury degree with smoking history were involved in circ_FOXO3 (P<0.05), while age, sex with operation method had no correlation with circ_FOXO3 expression.

Repressive circ_FOXO3 attenuates H2O2-induced HBVSMC damage

For analysis of the participation of circ_FOXO3 in IA, HBVSMC were applied to establish the H2O2-induced cell model. As manifested in Fig. 2A, H2O2 stimulation apparently reduced cell proliferation dose-dependently, suggesting that the in vitro model was successfully established. Furthermore, it was found that the abundance of circ_FOXO3 in HBVSMC was obviously elevated after H2O2 treatment (Fig. 2C). For further exploration of the character of circ_FOXO3 in the H2O2 induction model, the introduction of HBVSMC was with transfection vectors or elevation/depression vectors of circ_FOXO3 before H2O2 stimulation (Fig. 2D). It was clarified that repressive circ_FOXO3 alleviated H2O2-induced cell proliferation reduction (Fig. 2E, F) and H2O2-induced apoptosis of HBVSMC (Fig. 2G), while strengthening circ_FOXO3 was the opposite. Briefly, knockout circ_FOXO3 reduces H2O2-induced HBVSMC damage.

MiR-122-5p combines with circ_FOXO3

Via online analysis software was predicted the specific binding region of circ_FOXO3 with miR-122-5p (Fig. 3A). The impairment of luciferase activity in the circ_FOXO3-WT + miR-122-5p mimic was clarified. However, no apparent difference in luciferase activity was observed in the circ_FOXO3-MUT + miR-122-5p mimic, indicating that miR-122-5p specifically binds to circ_FOXO3 (Fig. 3B). The results affirmed that vs Bio-miR-NC, the enrichment of circ_FOXO3 in Bio-miR-122-5p-WT was strengthened and not clearly different in Bio-miR-122-5p-MUT (Fig. 3C). Moreover, detection of miR-122-5p was conducted in the H2O2-induced cell model after the regulation of circ_FOXO3. It was found that up-regulation of circ_FOXO3 reduced miR-122-5p, while the decrease in circ_FOXO3 was contrast (Fig. 3D).

MiR-122-5p mitigates H2O2-induced HBVSMC damage

For the study of the miR-122-5p characters in HBVSMC damage, NC, miR-122-5p, and in-miR-122-5p were transfected before exposure to H2O2 exposure (Fig. 4A). A series of assays testified that elevated miR-122-5p facilitated H2O2-induced cell proliferation (Fig. 4B, C) and decreased apoptosis (Fig. 4D), while reduced miR-122-5p was opposite. In the short term, miR-122-5p is available to attenuate H2O2-induced HBVSMC damage.

MiR-122-5p targets KLF6 that mediates the modulation of the circ_FOXO3/miR-122-5p axis

For further exploration of the regulatory network of miR-122-5p, the analysis of miR-122-5p’s molecular targets was carried out through starBase. KLF6 is a latent target, and the targeting of miR-122-5p on KLF6 was affirmed in Fig. 5A. For confirmation of this association, the construction of KLF6-WT and -MUT carriers was manifested. MiR-122-5p mimic resulted in an apparent reduction in luciferase activity in KLF6-WT, with no influence on the activity of KLF6-MUT (Fig. 5B). The enrichment of a large amount of KLF6 and miR-122-5p was manifested in the AgO2-based complex (Fig. 5C). Furthermore, the determination of the impacts of miR-122-5p on KLF6 was made in HBVSMC introduced with miR-NC, miR-122-5p mimic and in-miR-122-5p. Elevated miR-122-5p apparently decreased KLF6, but the knockdown one was the opposite (Fig. 5D, E). Transfection of the depressive KLF6 vector and the circ_FOXO3 + sh-KLF6 vector was carried out in the H2O2-induced HBVSMC model, and validation was carried out by qPCR (Fig. 5F). In the experiment (Fig. 5G–I) that sh-KLF6 clearly facilitated cell proliferation activity, reduced the rate of apoptosis, and effectively reversed inhibition of cell proliferation and the promotion of apoptosis via elevated circ_FOXO3. Shortly, KLF6 mediates circ_FOXO3/miR-122-5p axis to regulate H2O2-induced HBVSMC damage.

Discussion

IA, named cerebral aneurysm, is a severe cerebrovascular disease resulting from weakness of the cerebral vein or artery wall (Wang et al., 2019; Jin et al., 2019). As neuroimaging technology develops and noninvasive screening methods emerge, such as craniocerebral angiography (CTA) and magnetic resonance angiography (MRA), elevated unruptured IA has been detected. Once IA ruptures, it will make for subarachnoid hemorrhage (SAH), with a surprising fatality rate (Kalaria et al., 2002; Li et al., 2013). Except for surgical cutting or interventional embolization, there is no effective medical treatment. As a new-type gene expression regulator, circRNA has also been confirmed to function pivotally in many diseases. A previous study has confirmed that endogenous competitive RNA networks related to circRNA are involved in IA development (Qin et al., 2021), while functional polymorphisms in miRNA gene promoter regions have been documented to be associated with IA risk (Sima et al., 2017). In this research, clinical data showed that circ_FOXO3 and KLF6 were up-regulated and miR-122-5p was down-regulated in IA tissues, and circ_FOXO3 expression was associated with Hunt-Hess grade, degree of endothelial injury, and smoking history.

As a vital cell type forming medium in intracranial arteries, smooth muscle cells are crucial in the formation and rupture of IA (Starke et al., 2014). With reference to previous studies (Wang et al., 2018), circRNAs are associated with VSMC dysfunction and the presence of IA (Huang et al., 2019). Circ_FOXO3 is a promising cancer-related biomarker (Yang et al., 2021), in bladder cancer (Li et al., 2020) and squamous cell carcinoma of the esophagus (Xing et al., 2020). In this study, transfection of elevated or reduced circ_FOXO3 vectors into the H2O2-stimulated HBVSMC model found that suppressive circ_FOXO3 could promote H2O2-induced HBVSMC cell proliferation and inhibit apoptosis, while overexpressed circ_FOXO3 could do the opposite. It was suggested that knockdown circ_FOXO3 can attenuate H2O2-induced HBVSMC damage.

Next, the regulatory mechanism downstream of circ_FOXO3 was explored. The binding site of circ_FOXO3 with miR-122-5p was predicted through bioinformatics websites, which was verified. Previous studies manifested that miR-122-5p was involved in the regulation of various cells, such as breast cancer (Huang et al., 2021), cervical cancer (Gao et al., 2021), etc. It was confirmed in the study the proliferation-promoting and antiapoptotic effects of miR-122-5p in HBVSMC introduced with H2O2, while the reduction of miR-122-5p was the contrary. The above experiments confirmed that circ_FOXO3 aggravated H2O2-induced HBVSMC damage by repressing miR-122-5p.

Next, further exploration was carried out at the downstream target of miR-122-5p. KLF6, a member of the specific protein 1/Krupel-like transcription factor family (Sp1/KLF), is originally cloned from white blood cells. KLF6 is obviously increased in VSMC of diabetic patients and in VSMC treated with high glucose (Zhou et al., 2020). In the results, KLF6 was recognized as a functional target of miR-122-5p. Furthermore, curbed KLF6 apparently increased cell proliferation and reduced apoptosis. Furthermore, by cotransfection of KLF6 with circ_FOXO3, repressive KLF6 reversed the up-regulated damage of circ_FOXO3 to H2O2-treated HBVSMCs, suggesting that KLF6 is involved in mediating circ_FOXO3.

In summary, the study found that circ_FOXO3 can facilitate the proliferation and induce H2O2-induced HBVSMC apoptosis, which was achieved by regulating miR-122-5p with KLF6 targeting. Later animal models of IA will be applied for further in vivo analysis.

Declarations

Acknowledgements. Not applicable.

Funding Statements. Not applicable.

Declaration of Conflicting Interests. The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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