Circ-UBR4 regulates the proliferation, migration, inflammation, and apoptosis in ox-LDL-induced vascular smooth muscle cells via miR-515-5p/IGF2 axis

Abstract The aim of our study is to disclose the role and underlying molecular mechanisms of circular RNA ubiquitin protein ligase E3 component n-recognin 4 (circ-UBR4) in atherosclerosis (AS). Our data showed that circ-UBR4 expression was upregulated in AS patients and oxidized low-density lipoprotein (ox-LDL)-induced vascular smooth muscle cells (VSMCs) compared with healthy volunteer and untreated VSMCs. In addition, ox-LDL stimulated proliferation, migration, and inflammation but decreased apoptosis in VSMCs, which were overturned by the inhibition of circ-UBR4. miR-515-5p was sponged by circ-UBR4, and its inhibitor reversed the inhibitory effect of circ-UBR4 knockdown on proliferation, migration, and inflammation in ox-LDL-induced VSMCs. Insulin-like growth factor2 (IGF2) was a functional target of miR-515-5p, and overexpression of IGF2 reversed the suppressive effect of miR-515-5p on ox-LDL-stimulated VSMCs proliferation, migration, and inflammation. Collectively, circ-UBR4 knockdown decreased proliferation, migration, and inflammation but stimulated apoptosis in ox-LDL-induced VSMCs by targeting the miR-515-5p/IGF2 axis.


Introduction
Atherosclerosis (AS), an inflammatory disease, is a crucial pathogenic factor of most cardiovascular and cerebrovascular diseases, characterized by lipid metabolism disorder and chronic inflammation [1,2].AS is a multistep disease and can be induced by various risk factors [3].The dysfunction and inflammation of vascular smooth muscle cells (VSMCs) are closely related to AS progression [4], and oxidized low-density lipoprotein (ox-LDL)-induced VSMCs' injury are often used to construct AS models in vitro [5,6].Therefore, elucidation of the molecular mechanism affecting ox-LDL induced VSMCs injury is expected to provide potential molecular targets for the treatment of AS.
Circular RNAs (circRNAs) are novel group of endogenous non-coding RNAs with a circular structure generated by back-splicing [7].Previous research confirmed that circRNAs played significant functions in AS progression and served as independent markers for AS diagnosis [8].For example, the inhibition of circCHFR impeded the cell growth and mobility of VSMCs through microRNA (miRNA)-mRNA network [9].Hsa_circ_0010283 is derived from the ubiquitin protein ligase E3 component n-recognin 4 (UBR4) gene, also named as circ-UBR4.Circ-UBR4 was initially manifested to be highly expressed in ox-LDL-treated VSMCs by circRNA microarray analysis [9], and the functional effects of circ-UBR4 on ox-LDL-treated VSMC activities were partly elucidated [10,11].However, the regulatory mechanisms of circ-UBR4 in AS progression are complex and have not been fully uncovered.Therefore, circ-UBR4 was selected as a target in our study to explore its role and molecular mechanism in ox-LDL-induced VSMCs injury, providing more evidence for it to be a potential target in AS therapy.
Insulin-like growth factor2 (IGF2) is a member of insulin-like growth factor (IGF) family [17].The potential association between IGF family and hematological malignancies was revealed in a previous report [18].Interestingly, Sun et al. also revealed that ox-LDL-induced upregulation of IGF2 could activate downstream IGF-related pathways, thereby enhancing the proliferation of VSMCs [19].Therefore, the roles and functional effects of IGF2 were explored in AS progression.
In this research, ox-LDL-stimulated VSMCs were used as cell models of AS.Through bioinformatics analysis, we found that circ-UBR4 had complementary binding sites for miR-515-5p, and miR-515-5p could target IGF2.Therefore, we determined the role of circ-UBR4 and investigated the interplays among circ-UBR4, miR-515-5p, and IGF2 in ox-LDL-induced VSMCs.

Serum samples
The serum samples were collected from 31 AS patients and 25 healthy normal controls in Shanghai Shidong hospital.The written informed consent was acquired from each patient and volunteer, and our study was approved by the Ethics Committee of Shanghai Shidong hospital.

Cell culture
T/G HA-VSMCs (derived from Aorta) were purchased from ATCC (catalogue: PCS-100-012; Manassas, VA, USA) and cultured in Dulbecco's modified eagle medium (GIBCO BRL, Grand Island, NY, USA) containing 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Carlsbad, CA, USA) at 37°C with 5% CO 2 .The medium was replaced for every 2 or 3 days.Cells between passages 3 and 6 were employed in this study.To establish cell models of AS, VSMCs were treated with ox-LDL (Thermo Fisher Scientific) with different concentrations for 24 h or 50 μg/mL for different times.In function experiments, VSMCs were treated with 50 μg/mL of ox-LDL for 24 h after transfection for 24 h.

RNase R treatment and nuclearcytoplasmic fractionation
Purified RNAs were incubated with RNase R (Epicentre Technologies, Madison, USA), followed by purification with Trizol (Invitrogen).
In addition, Cytoplasmic and Nuclear RNA Purification Kit was used for nuclear-cytoplasmic fractionation assay.Briefly, ice-cold lysis buffer J was added into culture plate to lyse VSMCs.After centrifuging, supernatant was collected as cytoplasmic RNA.The precipitate containing the nuclear RNA was incubated with buffer SK.Finally, cytoplasmic RNA and nuclear RNA were eluted with elution buffer E.

MTT assay
VSMCs were planted into 96-well plates (5,000 cells per well) and cultured at 37°C with 5% CO 2 .After incubation for 24 h, 20 μL of MTT solution (5 mg/mL; Thermo Fisher Scientific) was added into each well at 37°C, and cells were allowed to incubate for another 4 h.The formatted crystal formazan was dissolved by dimethyl sulfoxide (Thermo Fisher Scientific).Microplate reader was used to assess optical density value at 490 nm.

EdU assay
Cell proliferation was also evaluated by EdU incorporation assay using an EdU Apollo DNA in vitro kit (RiboBio, Guangzhou, China).Briefly, cells after transfection were incubated in 96-well plates (5,000 cells per well) for 24 h at 37°C and next subjected with 100 μL of 50 μM EdU per well.After culturing cells with EdU for 12 h, cells were fixed and then counterstained with DAPI.The EdU staining was observed via a fluorescence microscopy (Mshot, Guangdong, China).

Flow cytometry assay
Apoptosis of VSMCs was assessed by Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, Franklin Lakes, NJ, USA).After incubation for 24 h, transfected VSMCs were harvested as a single cell suspension (1 × 10 6 /mL) by trypsin digestion.The staining buffer containing Annexin V-FITC and propidium iodide was added to incubate VSMCs at 4°C for 30 min.The Flow Cytometer (Beckman Coulter, Miami, FL, USA) was used for apoptosis assay.

Transwell assay
For in vitro cell migration assay, 24-well transwell chamber (BD Pharmingen, San Jose, CA, USA) was used.VSMCs resuspended in 200 µL medium without FBS (5 × 10 4 cells/ well) were seeded into the upper compartment, while complete medium was used as nutrients to induce cell migration.After 24 h, the remaining culture medium and the cells that did not migrate were removed carefully, while the migrated cells were fixed and then stained with 0.1% crystal violet (Thermo Fisher Scientific).The migrated cells were imaged under a microscope (100× amplification; Mshot).The number of migrated cells was counted in five randomly selected regions.

Statistical analysis
For each experiment, we set three duplications in adjacent three wells of 96-or 24-well plates.A total of three independent experiments were performed at few days interval using the same batch of frozen cells at passages 3-6.All quantitative data were displayed as mean value ± standard deviation.SPSS 21.0 software (IBM, Somers, NY, USA) was used to process data and analyze differences.The statistical differences in different groups were analyzed by Student's t-test or analysis of variance (followed by Tukey's post-hoc test).P-value less than 0.05 was considered to be statistically significant.

circ-UBR4 was overexpressed in ox-LDLstimulated VSMCs
Schematic diagram in Figure 1a manifested the information of circ-UBR4, showing that circ-UBR4 was produced from UBR4 gene (NM_020765), with 3358 bp in length.We detected circ-UBR4 expression in the serum of AS patients or healthy normal controls and confirmed that circ-UBR4 was overexpressed in AS patients (Figure A1).In order to analyze the potential role of circ-UBR4 in AS progression, we used ox-LDL-stimulated VSMCs as cell models of AS in vitro.As presented in Figure 1b, circ-UBR4 was significantly and dosedependently increased in ox-LDL-stimulated VSMCs.Also, treatment with 50 μg/mL of ox-LDL significantly enhanced the expression of circ-UBR4 in VSMCs in a time-dependent manner (Figure 1c).RT-qPCR assay suggested the predominant cytoplasmic distribution of circ-UBR4 in VSMCs (Figure 1d).Furthermore, circ-UBR4 could resist the digestion of RNase R when compared with linear-UBR4 (Figure 1e).Therefore, the function of circ-UBR4 was explored in ox-LDL-stimulated VSMCs.

Knockdown of circ-UBR4 reversed ox-LDL-stimulated VSMCs functions
As circ-UBR4 was overexpressed in ox-LDL-stimulated VSMCs, we next investigated the functional effects of circ-UBR4 inhibition in ox-LDL-stimulated VSMCs.Treatment with 50 μg/mL of ox-LDL enhanced the expression of circ-UBR4 in VSMCs, which was abolished by transfection with si-circ-UBR4 (Figure 2a).The results of MTT assay revealed that cell viability increased by ox-LDL was obviously inhibited by transfection with si-circ-UBR4 (Figure 2b).Similarly, knockdown of circ-UBR4 suppressed the inflammation in ox-LDL-stimulated VSMCs by reducing TNF-α, IL-6, and IL-1β levels (Figure 2c-e).
Besides, ox-LDL-aggravated EdU incorporation and the number of migrated cells in VSMCs were largely repressed by circ-UBR4 downregulation (Figure 2f and g).
Treatment with ox-LDL could inhibit cell apoptosis, while this effect was reversed by the knockdown of circ-UBR4 (Figure 2h).The results of western blot assay indicated that MMP9, CyclinD1, and Bcl-2 were upregulated, while Bax was downregulated in ox-LDL-stimulated VSMCs.However, the alterations induced by ox-LDL were all reversed by knockdown of circ-UBR4 (Figure 2i).Therefore, knockdown of circ-UBR4 inhibited cell proliferation, migration, and inflammation but promoted apoptosis in ox-LDL-stimulated VSMCs.

miR-515-5p was a direct target of circ-UBR4
The circRNA interactome software predicted that circ-UBR4 had more targeted miRNAs.Through literature research, four miRNAs (miR-144-3p, miR-326, miR-370-3p, and miR-515-5p) with low expression in AS and having an inhibition on proliferation and metastasis of VSMCs were selected for RT-qPCR analysis.The results show that circ-UBR4 knockdown could affect the expression of multiple miRNAs, but si-circ-UBR4 had the most obvious promotion effect on miR-515-5p expression (Figure A2).Therefore, miR-515-5p was selected as the target of circ-UBR4 for this study.
The possible complementary sequences between circ-UBR4 and miR-515-5p are presented in Figure 3a.The upregulation of miR-515-5p led to great loss of luciferase activity in WT-circ-UBR4 group, while luciferase activity in MUT-circ-UBR4 group was not affected by miR-515-5p overexpression (Figure 3b).In RIP assay, the immunopurification of Ago2 can be performed to confirm the interaction between miRNA and target genes by detecting RNA enrichment.The results of RIP assay revealed that circ-UBR4 and miR-515-5p were all enriched by Ago2 (Figure 3c).Additionally, pull-down assay showed that high abundance of circ-UBR4 could be pulled down by Bio-miR-515-5p probe (Figure 3d).The above data confirmed the interaction between circ-UBR4 and miR-515-5p.MiR-515-5p expression was inhibited by ox-LDL in VSMCs in a dose-dependent manner and time-dependent manner (Figure 3e-f).

IGF2 was a functional gene of miR-515-5p
The online software Starbase was used to predict the target genes of miR-515-5p.We found that miR-515-5p had putative binding regions in 3′UTR of IGF2 mRNA (Figure 5a).Besides, the overexpression of miR-515-5p reduced the luciferase activity of WT-IGF2 3′UTR group but not MUT-IGF2 3′UTR group (Figure 5b).RIP assay also suggested that miR-515-5p and IGF2 were enriched in Ago2-immunoprecipitated complex when compared with control group, revealing the association between miR-515-5p and IGF2 (Figure 5c).Treatment with ox-LDL increased the mRNA and protein expression levels of IGF2 in VSMCs in dose-dependent manner and time-dependent manner (Figure 5d-g).Transfection with miR-515-5p mimic increased the expression of miR-515-5p in VSMCs, while miR-515-5p decreased in VSMCs after transfection with anti-miR-515-5p (Figure 5h).More importantly, IGF2 was substantially increased in the presence of anti-miR-515-5p in VSMCs but decreased in miR-515-5p-transfected cells, suggesting that IGF2 was negatively regulated by miR-515-5p (Figure 5i and j).These results together suggested that IGF2 was a direct target of miR-515-5p.

Discussion
AS is a common risk factor for cardiovascular and cerebrovascular diseases all over the world [20].Although majority of basic and clinical research focusing on AS, the pathogenesis mechanism of AS is not fully addressed.Our data suggested that the suppression of circ-UBR4 decreased proliferation, migration, and inflammation, while it increased the apoptosis in ox-LDL-stimulated VSMCs by targeting the miR-515-5p/IGF2 axis.As we all know, ox-LDL could stimulate migration and proliferation of VSMCs [21].Under pathological conditions, migrated and proliferated VSMCs in intimal layer of artery were found to participate in the early AS formation [22].Besides, it was identified that ox-LDL stimulated cell adhesion molecules expression and thus induced a series of pathological changes, including inflammatory reactions and injury [23].Therefore, ox-LDL-induced VSMCs were widely used as cell models of AS.Not surprisingly, inflammatory reaction was also associated with the pathogenesis of AS [24].The pro-inflammatory cytokines, including TNFα and IL-6, could induce dysfunction of VSMCs and promote AS progression [25].We also confirmed that the productions of TNF-α, IL-6, and IL-1β were increased in ox-LDL-induced VSMCs.
Recently, circRNAs function as competitive endogenous RNAs to sponge miRNAs and then suppress their functions, which has been confirmed in the development of AS [26].For example, Guo et al. reported a series of differentially expressed circRNAs in ox-LDL-induced VSMCs, including circ-UBR4 [27].Recently, Ding et al. reported that the circ-UBR4/miR-370-3p/high mobility group box 1 networks mediated cell proliferation of ox-LDL-treated VSMCs, suggesting the important roles of circ-UBR4 in AS [10].Our results suggested that miR-515-5p was involved in the regulatory mechanism of circ-UBR4 in AS progression.
The previous research confirmed that miR-515-5p was implicated in the development of human diseases by targeting key mRNAs, such as chromobox homolog 4 [28], IL-6 [29], and YES proto-oncogene 1 [30].In addition, miR-515-5p was confirmed to play an important role in AS progression, and long noncoding RNA LOXL1 antisense RNA 1 could sponge miR-515-5p to facilitate the development of AS [31].Similar to the above conclusion, we found that miR-515-5p regulated proliferation, migration, inflammation, and apoptosis of ox-LDL-treated VSMCs by targeting IGF2.
Taken together, circ-UBR4 regulated the proliferation, migration, inflammation, and apoptosis of ox-LDL-induced VSMCs via miR-515-5p/IGF2 axis.Our study enriched the role of circ-UBR4 in ox-LDL-induced VSMCs and thus provided a new perspective to understand AS pathogenesis.However, some limitations existed in our present study.We only determined the role of circ-UBR4 in cell models of AS in vitro, and animal models of AS were not provided in our present study.Therefore, VSMCs from various resources and animal models should be used to further validate our present findings in future work.

Conclusion
In summary, treatment with ox-LDL increased the expression of circ-UBR4 in VSMCs.We also demonstrated that the downregulation of circ-UBR4 could effectively repress proliferation, migration, and inflammation but increase apoptosis of ox-LDL-induced VSMCs via miR-515-5p/IGF2 axis, hinting that circ-UBR4 might be a new diagnostic marker for AS patients.

Figure 1 :
Figure 1: The expression level of circ-UBR4 in ox-LDL-stimulated VSMCs.(a) The information of circ-UBR4 structure and formation.(b) and (c) The relative expression level of circ-UBR4 was evaluated by RT-qPCR in ox-LDL-stimulated VSMCs.(d) The expression levels of circ-UBR4, GAPDH, and U6 were assessed by RT-qPCR in cytoplasmic and nuclear fraction RNAs.(e) RT-qPCR was used to show the expression of linear-UBR4 and circ-UBR4 in VSMCs after treatment with RNase R. ***P < 0.001.