Hsa_circ_0001445 inhibits ox-LDL-induced HUVECs inflammation,oxidative stress and apoptosis by regulating miRNA-640

Abstract

The role of Hsa_circ_0001445 in oxidation Low Lipoprotein (ox-LDL) induced HUVEC inflammatory damage remains poorly characterized. The present study investigated the performance of the circRNA Hsa_circ_0001445 on ox-LDL-induced HUVEC inflammatory damage. ox-LDL was employed to treat HUVECs and the expression of Hsa_circ_0001445 in cells were detected by qRT-PCR. Then, the overexpression plasmid of circ_0001445 was transfected into HUVECs. The Cell Counting Kit-8 assay was performed to detect cell viability, and the expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in treatment cells were measured using ELISAs. Furthermore, the oxidative stress kit was used to detect the levels of malondialdehyde, superoxide dismutase and glutathione peroxidase in treatment cells. Flow cytometry assay was applied to measure cell apoptosis, and the expressions of apoptosis-related protein were measured by western blot. The luciferase reporter assay was applied to confirm the target binding between Hsa_circ_0001445 and micro-RNA-640 (miRNA-640). Next, miRNA-640 mimic was transfected into ox-LDL-induced HUVECs, and then cell proliferation, expression level of inflammatory factors, oxidative stress and apoptosis level in treatment cells were assessed, with the expression of related proteins measured. The results revealed that the expression of Hsa_circ_0001445 was obviously downregulated in ox-LDL-induced HUVECs. Overexpression of Hsa_circ_0001445 promoted cell proliferation, inhibited ox-LDL-induced HUVEC inflammatory response, downregulate the expression of TNF-α, IL-1β and IL-16, overexpression of Hsa_circ_0001445 inhibited cell apoptosis. miRNA-640 was confirmed as a direct target of Hsa_circ_0001445, and miRNA-640 mimic reversed the effects of Hsa_circ_0001445 overexpression on ox-LDL-induced HUVECs. Our findings concluded that Hsa_circ_0001445 inhibits ox-LDL-induced HUVEC inflammation, oxidative stress and apoptosis by regulating miRNA-640.

Keywords

Hsa_circ_0001445 ox-LDL-induced HUVECs inflammation oxidative stress apoptosis miRNA-640

Introduction

Atherosclerosis (AS) is a main pathological basis of cardiovascular and cerebrovascular diseases, such as coronary heart disease, myocardial infarction and cerebral infarction. AS is a chronic cardiovascular disease that endangers human health and is one of the most common causes of death in the elderly.¹ The main lesion of atherosclerosis is lipid deposition in some arteries, accompanied by smooth muscle cells and fibrostromal hyperplasia, which gradually develops into atherosclerotic plaques.^2,3 Its incidence is increasing year by year, and it may cause the serious complication that threatens human health.⁴ The research on the pathogenesis and therapeutic targets of AS has always been the focus of scholars at home and abroad.^5,6 Therefore, screening new drug intervention targets to provide a new theoretical basis for the occurrence and development of AS, and seeking for new therapeutic drugs are two important strategies for the prevention and treatment of AS.

Circular RNAs (circRNA) is a new type of non-coding RNA (ncRNA) that forms a covalently closed ring by combining the 5′-end and 3′-end. It is such a special form that leading to the resistance of circRNA to the digestion of RNase R (Ribonuclease R), making it an ideal biomarker for diagnosis of diseases.^7,8 The structure of circRNA is conserved during evolution, showing high stability, tissue-specific variation and expression levels. Studies have found that circRNA plays an essential role in epigenetic regulation, cell cycle regulation, cell differentiation regulation and other life activities.^9–12 Recent studies have shown that it plays a key role in regulating the occurrence and development of cardiovascular diseases, including myocardial infarction, heart failure and atherosclerosis.^13–15 A strong inverse correlation was reported between plasma Hsa_circ_0001445 and the severity of coronary atherosclerosis, suggesting that Hsa_circ_0001445 may be a promising target of coronary atherosclerosis. It has been reported that circRNAs may bind to miRNAs and thus act as miRNA sponges that regulate gene expression.¹⁶ However, miRNAs are a class of highly conserved non-coding single-stranded RNA that inhibits their translation or promotes their degradation by binding to the 3′ non-coding region of mRNA, and play a role in gene regulation at the post-transcriptional level.¹⁷ Hsa_circ_0001445 can target bind to miRNA-640 by searching the circinteractome website. It has been reported that miRNA-640 is overexpressed in degenerated nucleus pulposus tissues and cells, and the inflammatory environment promotes the expression of miRNA-640 through the NF-κB signaling pathway, suggesting that miRNA-640 mediated inflammatory response that exacerbates inflammation-related diseases.¹⁸ However, the role of ox-LDL-induced HUVEC inflammatory injury has not been reported. In this study, we first investigated the effects and mechanism of Hsa_circ_0001445 in AS.

Materials and methods

Cell culture and treatment

The HUVECs (INCELL, San Antonio, TX, USA) were cultured in the Eagle’s Minimum Essential Medium (EMEM) medium (#30-2003, A TCC) supplemented with 10% FBS (A TCC® 30-2020™) and 1% penicillin-streptomycin. The cells were maintained in an atmosphere of 5% CO₂, at 37°C.

HUVECs were cultured with various concentrations of ox-LDL (0, 10, 25, and 50 µg/ml) for 0, 24, 48, and 72 h at 37°C.

Transfection

The HUVECs with a density of 1 × 10⁵/well were cultured in a 6-well plate with EMEM containing 10% fetal bovine serum. Lipofectamine™2000 (Invitrogen, USA) was transfected as per manufacturer’s instructions. The overexpression-circRNA-hsa_circ_0001445 (Oe-circ_0001445), overexpression-NC (Oe-NC), and miRNA-640 mimic or vector were purchased from GenePharma (Shanghai GenePharma Co., Ltd). Western blotting and RT-QPCR were used to detect transfection efficiency.

Cell counting kit-8 (CCK-8) assay

The cell viability of HUVECs was detected via CCK-8 reagent (Dojindo Molecular Technologies, Inc.), according to the manufacturer’s protocol. The HUVECS cells were seeded into 96 well plates at destiny of 0.5 × 10³ cells/ml. After the HUVECs were incubated with ox-LDL (50 µg/ml) at 37°C, 10 μl CCK-8 reagent (Roche Diagnostics) was added to each well and incubated for 2 hours at 37°C, and then the optical density was measured at 450 nm using a microplate reader.

Evaluation of TNF-α, IL-1β, and IL-6 level

The levels of TNF-α, IL-1β, and IL-6 in cells were evaluated via the corresponding detection kits (Beyotime Biotechnology, China) with a microplate reader (MULTISKAN MK3, Thermo, San Jose, CA, USA). The cells after treatment were centrifuged and the supernatants in the different group were submitted for detection according to the protocols of corresponding manufacturer. Each sample was measured in triplicate.

Cell apoptosis analysis

Apoptosis was detected by flow cytometry with FITC Annexin V/PI Apoptosis Detection Kit I (Ribobio, Guangzhou, China). After transfection, HUVECs were collected and fixed in pre-cooled 70% ethanol at 4°C overnight. Then, the cells were re-suspended in a binding buffer (300 μL). Annexin V-FITC and Piperidine iodide (PI, 10 mg/mL) were incubated in the dark for 20 minutes at room temperature. Flowjo software (Tree Star, Ashland, OR, USA) was used to analyze the double-stained cell apoptosis. The experiments were independently repeated three times.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and DNA synthesis was performed using a reverse transcription reagent kit (Invitrogen). Thermo Script RT-PCR system (Invitrogen, Basel, Switzerland) was used for QRT-PCR detection. Levels of RNA levels calculated using 2^-ΔΔCt method. Each experiment was independently performed three times.

Western blot

RIPA buffer (Beyotime, P0013B) was added to the supernatant for lysis, and the protein samples were obtained by centrifugation.10% SDA-PAGE gel was configured for electrophoresis separation of proteins. Then the separated proteins were transferred to PVDF membrane by wet method. Five percent skim milk was sealed at room temperature for 1 hour, and incubated at 4°C overnight. The primary antibodies incubated on the membrane were: Bcl2 (#ab182858, Abcam), Bax (#ab32503, Abcam), cleaved-caspase 3 (#ab49822, Abcam), cleaved-caspase 9 (#ab2324, Abcam), Pro-caspase3 (#ab32499, Abcam), Pro-caspase 9 ( #ab202068, Abcam), MCP-1 (#ab25124, Abcam), NLRP3 (#ab263899, Abcam), (#ab155970, Abcam). Then, the protein signals were then incubated with a secondary antibody (# ab 150077, Abcam) for 2 hours at 37°C and detected with a chemiluminescence kit (GE Healthcare, Chicago, IL, USA). Image J software was used to analyze the gray value of each strip.

Statistical analysis

GraphPad Prism 8 software was used for statistical analysis. The data of each indicator were expressed as mean ± standard deviation (Means ± SD), and one-way analysis of variance (ANOVA) was used for comparison and analysis among multiple Means. Pair-wise comparison between groups was tested by Student’s Test. p < 0.05 was considered as statistically significant. All experiments were independently performed three times.

Results

Expression levels of Hsa_circ_0001445 in OX-LDL induced HUVECS

To explore the roles of Hsa_circ_0001445 in OX-LDL induced HUVECS, we first examined the mRNA expression of Hsa_circ_0001445 in HUVECS after treatment different concentration of OX-LDL (0, 10, 25, and 50 µg/ml) using qRT-PCR. As shown in Figure 1(a), Hsa_circ_0001445 expression was gradually decreased with the increase of OX-LDL concentration. The expression level of Hsa_circ_0001445 in HUVECs cells treated with 50 g/ml OX-LDL at different time (0 hour, 24 hours, 48 hours, and 72 hours) was detected by qRT-PCR. As shown in Figure 1(b), Hsa_circ_0001445 expression was gradually decreased with the prolongation of OX-LDL induction. To further investigate the effect of Hsa_circ_0001445, qRT-PCR was used to determine its expression after altering expression of Hsa_circ_0001445 with overexpression Hsa_circ_0001445 (Oe-circ_0001445). The overexpression of Hsa_circ_0001445 upregulated Hsa_circ_0001445 expression significantly (Figure 1(c)).

Overexprssion of Hsa_circ_0001445 promoted proliferation and alleviated inflammatory response of ox-LDL—induced HUVECs

To further elucidate the role of Hsa_circ_0001445 in proliferation and inflammatory response, ox-LDL-induced HUVECs were divided into the control group, ox-LDL group, ox-LDL+Oe-NC group and ox-LDL+Oe-circ_0001445 group. Firstly, CCK-8 assay was used to detect the cell ability of ox-LDL-induced HUVECs to prolifetare. We found that Oe-circ_0001445 accelerated the proliferation of ox-LDL-induced HUVECs when compared with Oe-NC group, but had no influence between control and ox-LDL+Oe-NC group (Figure 2(a)). As shown in Figure 2(b), compared with the ox-LDL+Oe-NC group, much less expression of inflammation factors (TNF-α, IL-1β and IL-6) was found in ox-LDL+Oe-circ_0001445 group, suggesting that Overexprssion of Hsa_circ_0001445 alleviated inflammatory response of ox-LDL—induced HUVECs. Moreover, the protein levels of the monocyte chemotactic protein (MCP), the NLR family Pyrin domain protein 3 (NLPR3) and adaptor protein (ASC) were measured by western blot, showing that overexpression of Hsa_circ_0001445 remarkably decreased MCP, NLPR3 and ASC expressions (Figure 2(c)). As shown in Figure 2(d), the expression levels of superoxide dismutase (SOD), malondialdehyde (MDA) and Lactic dehydrogenase (LDH) were measured by ELISA, the results demostrated that overexpression of Hsa_circ_0001445 remarkably decreased MDA, LDH and increased SOD expressions.

Overexprssion of Hsa_circ_0001445 alleviated apoptosis of ox-LDL-induced HUVECs

To investigate the underlying mechanism of the Hsa_circ_0001445 in ox-LDL-induced HUVECs, flow cytometric analyses were conducted to assess the rate of apoptosis in ox-LDL-induced HUVECs. The increased cell apoptosis was found in ox-LDL-induced group and overexpression circ_0001445 could reduce the cell apoptosis in DSS-induced group compared with ox-LDL+oe-NC group (Figure 3(a)). Furthermore, compared with ox-LDL+oe-NC group, overexprssion of Hsa_circ_0001445 significantly increased Bcl-2, and decreased Bax, cleaved caspase-3 and cleaved caspase-9 expression levels in ox-LDL+Oe-circ_0001445 group, which was consistent with the results of the apoptosis assay (Figure 3(b)).

Hsa_circ_0001445 targets miRNA-640

To deeply investigate the molecular mechanism of Hsa_circ_0001445 in HUVECs, the potential target of Hsa_circ_0001445 was predicted. The biology website available of circinteractome predicted there was a binding site between Hsa_circ_0001445 and miRNA-640 (Figure 4(a)). Furthermore, target binding between miRNA-640 and Hsa_circ_0001445 was verified by a dual-Luciferase reporter gene assay. The luciferase activity of Hsa_circ_0001445-WT in the miRNA-640 mimic group was markedly decreased by the dual luciferase reporter gene assay. As shown in Figure 4(b), miRNA-640 expression was gradually increased in HUVECs treatment with OX-LDL and miRNA-640 expression was gradually increased with the prolongation of OX-LDL induction (Figure 4(c)). In addition, compared with the ox-LDL and ox-LDL-Oe-NC groups, miRNA-640 was obviously decreased in ox-LDL-induced HUVECs transfected Oe-circ_0001445 (Figure 4(d)). These results suggested that circRNA_0010452 could target miR-543 and inhibit its expression.

Overexpression of Hsa_circ_0001445 alleviated inflammation and apoptosis of ox-LDL-induced HUVECs via targeting miRNA-640

Considering the targeting relationship between Hsa_circ_0001445 and miRNA-640, now the focus of our study shifted to investigating the mechanism of miRNA-640 in ox-LDL-induced HUVECs. Following the treatment of miRNA-640 mimic and Oe-circ_0001445, ox-LDL-induced HUVECs exhibited a striking decrease in proliferation in the ox-LDL+Oe-circ_0001445+miRNA-640 mimic group comparing to the ox-LDL+Oe-circ_0001445+mimic-NC group (Figure 5(a)). As shown in Figure 5(B–D), the levels of inflammatory factors, MCP, NLPR3, ASC, MDA and LDH in ox-LDL-induced HUVECs cells were incrased obviously by miRNA-640 mimic. Overexprssion of Hsa_circ_0001445 alleviated inflammation response of ox-LDL-induced HUVECs via targeting miRNA-640. Simultaneously, the flow cytometric analyses results illustrated that apoptotic rate was significantly decreased in ox-LDL+Oe-circ_0001445 group, when compared with those in ox-LDL group (Figure 6(a)). The effects of Oe-circ_0001445 were partly reversed by miRNA-640 mimic, indicating that Overexpression of HSA_circ_0001445 inhibits ox-LDL-induced apoptosis of HUVECs via targeting miRNA-640. Furthermore, the western blotting results indicated that miRNA-640 mimic significantly decreased Bcl-2, and increased Bax, cleaved caspase-3 and cleaved caspase-9 expression levels in ox-LDL+Oe-circ_0001445+miRNA-640 mimic group, which was consistent with the results of the apoptosis assay (Figure 6(b)).

Discussion

Endothelial cell injury, monocyte phagocytosis of ox-LDL gradually forms foamlike cells, and vascular smooth muscle cell proliferation and migration contribute to plaque formation, which is the main step of the occurrence and development of AS. The change of VSMCs is related to the formation of AS, and circRNA plays a key role in regulating the growth and differentiation of VSMCs.

Xu et al.¹⁹ carotid artery balloon injury model was established, found circDiaph3 by raising Igf1r expression so as to promote the proliferation and migration of VSMCs. Meanwhile, studies have shown that CIRC-SatB2 can promote proliferation and migration of vascular smooth muscle cells by inhibiting miR-939 and up-regulating STIM1 expression.²⁰ Silencing CIRC-SatB2 or blocking the binding of CIRC-Satb2 to miR-939 may be a new treatment method for AS, and cirC-SatB2 may be a biomarker for the diagnosis of AS.

In this study, we found that the expression of HSA_circ_0001445 in OX-LDL decreased gradually with the increase of OX-LDL concentration in HUVECS.Overexpression of HSA_circ_0001445 inhibits ox-LDL-induced HUVECs inflammation and ox-LDL-induced HUVECs apoptosis. We searched through the circinteractOME website that HSA_circ_0001445 can target bind to miRNA-640. It has been reported that miRNA-640 is overexpressed in denatromedullated nucleus pulpoii tissues and cells, and the inflammatory environment promotes the expression of miRNA-640 through the NF—B signaling pathway,² suggesting that miRNA-640 mediated inflammatory response exacerbates inflammatory disease.

Increased levels of endothelial dysfunction and/or inflammatory markers were observed in atherosclerosis and cardiovascular events.²¹ People at high risk of ACS can be identified based on plaque, blood and myocardial vulnerability markers.²²These markers are associated with atherosclerotic disease processes and are closely associated with immune system and inflammatory processes.Inflammatory factors play a role in arterial lipid deposition and the proliferation and migration of smooth muscle cells to the intima.In addition, the immune system can gradually influence the progression of atherosclerosis through its association with known cardiovascular risk factors such as hypertension, smoking, overwork, dyslipidemia, and obesity. The vicious cycle of inflammation may be caused by both direct and indirect mechanisms, such as those involved in lipid regulation and further inflammatory progression.²³ Arterial damage initially causes some beneficial inflammatory response, but the persistence of stimuli may lead to a persistent inflammatory response that alters the initial recovery of the injury response.Subsequent destruction of the damaged areas may eventually reduce arterial function, and inflammatory processes may eventually become part of the progression of CHD.²³ In addition, accumulated evidence suggests that endothelial dysfunction is a precursor to atherosclerosis, that it exists in the preclinical stage and can be detected before angiographic or ultrasound shows changes in vessel wall structure.²⁴ Many of the risk factors for atherosclerosis may also contribute to endothelial dysfunction.Endothelial cells, as the main regulator of vascular homeostasis, play a number of vascular protective roles.Such as maintaining the balance between vasodilation and vasoconstriction, inhibiting and stimulating smooth muscle cell proliferation and migration, as well as thrombosis and vasolysis.²⁵ When this balance is disrupted, endothelial dysfunction occurs, leading to increased endothelial permeability, platelet aggregation, leukocyte adhesion, and cytokine production. In this syudy, we found that overexpres-sion of HSA_circ_0001445 inhibits ox-LDL-induced HUVECs inflammatory response, and reduces the expression level of inflammatory factors and oxidative stress.

Conclusion

In conclusion, our findings concluded that Hsa_circ_0001445 inhibits ox-LDL-induced HUVEC inflammation, oxidative stress and apoptosis by regulating miRNA-640. However, there are still many deficiencies in this study, further research on which pathway HSA_circ_0001445 affects the expression of mirNA-640 by acting on has not been conducted and ox-LDL affects the expression of inflammatory factors by stimulating multiple pathways, and whether there is any interaction between other pathways and hsa_circ_0001445/miRNA-640 confirmed in this experiment, the influence of Hsa_circ_0001445 over other microRNAs?

Are all issues that need to be resolved in further study. Therefore, we will focus on the influence of Hsa_circ_0001445 over other microRNAs or pathways and the role of Hsa_circ_0001445/ miRNA-640 in animal experiment and clinical research in further studies.

Footnotes

Availability of Data and Materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by “The Top-level Clinical Discipline Project of Shanghai Pudong (PWYgf2018-02)”.

ORCID iD

Chenghui Fan

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