miR-590-3p attenuates atherosclerosis by preventing TNF-α-induced proliferation and migration of VSMCs via NF-κB pathway

doi:10.21203/rs.3.rs-3040904/v1

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miR-590-3p attenuates atherosclerosis by preventing TNF-α-induced proliferation and migration of VSMCs via NF-κB pathway

https://doi.org/10.21203/rs.3.rs-3040904/v1

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miR-590-3p displays protective effects in some cardiovascular diseases. However, the mechanisms by which miR-590-3p regulates vascular smooth muscle cells (VSMCs) remain to be elucidated. In this study, we investigated the role of miR-590-3p/CD40/p21^Cip1pathway in VSMCs. The alterations of miR-590-3p, CD40 and /p21^Cip1 were verified in atherosclerosis (AS) plaque and TNF-α treated VSMCs by quantitative Real-time PCR, EdU staining, wound healing test, and flow cytometry. The expression levels of miR-590-3p and p21^Cip1 were decreased, whereas CD40 was increased in the AS plaques . TNF-α is a critical contributor of atherosclerosis and induces VSMCs proliferation and migration by upregulating CD40 and downregulating p21^Cip1. miR-590-3p inhibits the proliferation and migration of VSMCs through up-regulating the expression of p21^Cip1 and down-regulating the expression of CDK2/cyclin E1 by targeting CD40. The TNF-α-induced alterations were partially reversed by exogenous miR-590-3p mimic.The TNF-α-induced proliferation and migration of VSMCs were also prevented by miR-590-3p mimic . Thus, our study revealed a mechanism by which miR-590-3p attenuates atherosclerosis by preventing TNF-α-induced VSMCs proliferation and migration.

atherosclerosis

VSMCs

miR-590-3p

CD40

p21Cip1

Cardiovascular disease is the leading cause of death around the world (1). Atherosclerosis (AS) is the common pathological basis of many cardiovascular diseases, such as coronary heart disease and peripheral vascular diseases. The main clinical consequences of AS are myocardial infarction and stroke caused by the artery stenosis and acute rupture of unstable plaques. Currently, AS is generally considered as a chronic progressive inflammatory disease and its occurrence is the result of complex interactions between circulating inflammatory factors and relevant cells in vascular walls including vascular smooth muscle cells (VSMCs), vascular endothelial cells (VECs) and monocytes(2, 3).

To date the main treatment of AS relies on targeting systemic risk factors, such as the use of statins to prevent hypercholesterolemia(2, 3). However, many patients do not benefit from lipid therapy in long term, and approximately two-thirds of patients receiving statin therapy still have cardiovascular events within 5 years(3). In addition to the excessive lipids, the changes in local microenvironment are also involved in the development of AS. Simply down-regulate the level of lipids in the blood circulation cannot completely cure even relieve AS, only classify the relationship between various AS stimuli and the altered microenvironment in the vascular system in AS helps to provide a new basis for the treatment of AS.

As the development of cell lineage tracer method, the roles of VSMCs in AS are unraveled. By using this technique, scientists found that more than 80% of VSMCs in the AS plaque lack of classic VSMCs markers, suggesting that the function of VSMCs in the AS process could be far more than our current understanding(3). Under normal condition, VSMCs mainly maintain the vascular tone and vascular diameter by its contraction and exhibit finite proliferation ability. However, under AS condition, VSMCs change from contractile type to synthetic form, which is characteristic of enhanced abilities of proliferation, migration, and secretion(7).

CD40 is a 50-kDa type I transmembrane glycoprotein receptor belongs to tumor necrosis factor receptor (TNFR) superfamily and is initially found in B lymphocytes. The ligand of CD40 is a type II transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily which known as CD40L(8, 9). Dysfunction of CD40/CD40L signaling the is mainly involved in the pathogenesis of atherosclerosis, myocardial infarction, and Kawasaki disease by affecting the activity of NF-κB signaling pathway (10–12). In our previous research, our team found that in AS stimuli induced VSMCs proliferation model, CD40 can promote the phenotypic switch and proliferation of VSMCs, suggesting that CD40 may be involved in the progress of AS by regulating the VSMCs function(10–12). microRNAs (miRNAs) are a kind of highly conserved small non-coding RNA with about 19 to 25 nucleotides at length. By binding to the 3’-untranslating region (3’-UTR) of target mRNA, microRNA can inhibit the transcription or induce the degradation of target mRNA, thus playing a negative regulation on gene expression(15). miR-590 is located at the proximal end of the long arm of human genome 7, belonging to intron miRNA. Studies have found that miR-590 plays a protective role in cardiovascular and cerebrovascular diseases(16).

In this study, we found that miR-590-3p regulates CD40 and attenuates TNF-α induced VSMCs phenotype switch.

2.1 Cell culture

VSMCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 10% fetal bovine serum (FBS) in a 5% CO₂ incubator at 37 ºC. When the cell reached the density at 80%, sterilized PBS was used to wash the cells twice, and 0.25% trypsin was used to digest the cells. When the edge of the cells was narrowed, 10% FBS DMEM was added to stop the digestion. Gently blew the cell layer with the pasteurization straw until the cells were blown off, the dropped cells were collected to the centrifugal tube by centrifugation (1000 r/min, 5 min). After centrifugation, the cells were suspended in complete medium with 10% FBS for further experiments.

2.2 Reverse transcriptase (RT)-PCR and real-time quantitative PCR analysis

Total RNA was extracted and used in reverse transcription by using a First Strand cDNA Synthesis Kit (Thermo, America) as described(13). The levels of the miR590-3p were quantitatively analyzed by using the ViiA 7 Dx Real-Time PCR Systems with specific primers, probes and software (Life Technologies, America) with 2 ^-△△CT method, small nuclear RNA (snRNA) U6 was used as the endogenous control.

2.3 Western blot

Proteins in the lysate of VSMCs were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime Biotech, China) and then transferred onto a polyvinylidene difluoride (PVDF) (Pell, USA) membrane after electrophoresis. The membranes were immunoblotted with Abs against CD40 (human monoclonal antibody, Sigma, America), p21^Cip1 (human polyclonal antibody, CST, America), cyclin E1 (human polyclonal antibody, CST, America), CDK2 (human polyclonal antibody, CST, America) or GAPDH (Abcam, UK) followed by a horseradish peroxidase-conjugated secondary antibody. The immunoblots were detected by a Bio-Rad Calibrated Densitometer.

2.4 Flow cytometry

After different treatments, VSMCs were washed twice with PBS and digested with trypsin without EDTA. Complete cell culture medium was added to stop digestion and the cells were transferred to the centrifuge tube by centrifugation (1000 r/min, 5 min).VSMCs were re-suspended with 1.5 mL pre-cooled PBS and centrifuged at 1000 rpm for 5 min and repeated twice. The supernatant was discarded and 70% ethanol was used to re-suspend the cells, which were fixed for 1 h at 4 ºC. After centrifugation for 5 min at 1000 rpm, the cells were re-suspended with PBS (repeated twice). After cell counting, 1×10⁵ − 4×10⁵ cells were collected in a 1.5 mL centrifuge tube, and the supernatant was discarded after centrifugation at 1000 rpm for 5 min. 200 µL PBS was used to re-suspend the cells and PI working solution with a final concentration at 50 µg/mL was added to the solution. After staining at room temperature for 15 min without light, the prepared samples were detected by flow cytometry.

2.5 EdU assay

Prepare EdU work solution with a final concentration at 50 µM to incubate VSMCs for 2 h in a 5% CO₂ incubator at 37 ºC. Polyformaldehyde was used to fix cells for 30 min. cell rupture: PBS solution containing 0.5% Tritonx-100 was added to the cell culture plate which was then incubated in a shaker for 10 min. The cells were incubated with Apollo® staining solution in a shaker for 30 min without light. Then PBS solution containing 0.5% Tritonx-100 was used to break the cell membrane. After washing by methyl alcohol, Hoechst33342 was used to incubate the cells for 30 min without light. Finally, the samples were detected by Operetta High Content Cytometers (Perkin Elmer, America): Blue fluorescence indicated the total number of cells, red fluorescence indicated the number of newly proliferated cells, and the proportion of EdU positive cells was calculated as follows: cells with red fluorescence /cells with blue fluorescence number × 100%.

2.6 Statistical analysis

SPSS software (Version 13.0) was used for the statistical analysis. The data were expressed as the mean ± SD. The differences among the groups were compared using a one-way ANOVA. P < 0.05 was considered to be statistically significant.

3.1 The expression levels of miR-590-3p and p21 ^Cip1 are decreased in AS plaques

To explore the potential role of miR-590-3p and its relevant signal pathway in AS process, we collected AS plaque and corresponding adjacent normal tissues and used Real-time PCR and Western blot to determine the expressions of miR-590-3p, CD40 and p21^Cip1. We found miR-590-3p was significantly decreased in AS plaque than that in adjacent tissues (Fig. 1A). Similarly, AS plaque also showed a decrease in p21^Cip1 expression compared with the adjacent tissues (Figs. 1B, C). More importantly, the decreased expressions of miR-590-3p and p21^Cip1 in AS plaque was accompanied by dramatic increases in CD40 expression (Fig. 1B, D), indicating miR-590-3p/ CD40/ p21^Cip1 pathway may participate in the process of AS.

3.2 The effect of miR-590-3p on CD40 expression and NF-κB signaling.

By analyzing the miRNA database (Targetscan 7.0 and RNA22) and the inverse correlation between miR-590-3p and CD40 in AS plaque, we can infer that CD40 may be a target gene of miR-590-3p. In our previous research, we have confirmed that CD40 is a target gene of miR-590-3p in ECs(17). In this study, we used Real-time PCR and Western blots to test whether CD40 is also the target of miR-590-3p in VSMCs. As shown in Fig. 2A, we first chose miR-590-3p mimic at 100 nM as the optimal concentration for subsequent experiments. Then we found treatment of VSMCs with 100 nM miR-590-3p mimic for 24 h significantly decreased the expressions of CD40 at both transcriptional and protein levels (Fig. 2C, D). These results demonstrate that CD40 is also a target gene of miR-590-3p in VSMCs. As expected, in addition to inhibiting CD40 expression, the phosphorylation levels of NF-κB p65 is decreased after miR-590-3p mimic administration (Fig. 2E), further confirming that miR-590-3p regulates the activation of NF-κB signaling pathway through CD40, thereby affecting downstream genes and cell behavior.

3.3 miR-590-3p prevents TNF-α-induced proliferation of VSMCs .

According to direct cell counting, we found TNF-α induced significant increase in VSMCs number, while pretreatment of cells with miR-590-3p mimic for 24 h partly blocked this effect. We thus investigated whether the altered cell numbers were dependent on changed levels of apoptosis or proliferation. The results from flow cytometry showed no significant difference in the proportion of apoptotic cells among different treatment groups (Fig. 3A). TNF-α administration caused an increase in EdU incorporation, which was blocked by miR-590-3p pretreatment. Conversely, inhibiting miR-590-3p in VSMCs enhanced TNF-α-induced EdU incorporation (Fig. 3B, C). Cell cycle analysis showed that miR-590-3p mimic reduces the proportion of cells in S phase and increase the proportion of cells in G1 phase, while miR-590-3p inhibitor increases the proportion of cells in S phase and reduce the proportion of cells in G1 phase under both basal and TNF-α condition (Fig. 3D, E). Together, these results suggest miR-590-3p inhibits VSMCs proliferation .

Besides, miR-590-3p also prevents TNF-α-induced VSMCs migration. The results of the wound healing assays showed that TNF-α increased the migration ability of VSMCs after scratching, while overexpression of miR-590-3p repress the repair of VSMCs compared with the TNF-α treated group (Fig. 3F).

3.4 CD40-mediated TNFα-induced VSMCs proliferation and migration . After determining the regulation of miR-590-3p on CD40, then we want to know whether CD40 expression is crucial for the effects of miR-590-3p on VSMCs function. As shown in Fig. 4A and 4B, we first used three siRNA primers targeting CD40 to achieve effective knockdown, then we chose the most effective siRNA primer with a silencing efficiency at about 60% for the subsequent experiment. Treatment of VSMCs with 50 ng/mL TNF-α for 24 h induced increased protein levels of CD40, cyclin E1 and CDK2 accompanied by decreased protein levels of p21^Cip1 (Fig. 5A-E). Interestingly, knockdown of CD40 by siRNA restored the expression of p21^Cip1, and significantly inhibited TNF-α induced expressions of CD40, cyclin E1 and CDK2 (Fig. 5A-E).

Then we investigated the effects of CD40 knockdown on VSMCs proliferation. As shown in Fig. 6, tdeletion of CD40 inVSMCs significantly decreased the EdU positive cell numbers compared to the TNF-α treated group. Flow cytometry also showed that knockdown of CD40 by siRNA reduces the proportion of cells in S phase induced by TNF-α (Fig. 7A, B). These results suggest that TNF-α upregulates CD40 to promote VSMCs proliferation.

CD40 mediates TNF-α-induced VSMCs migration. The results of the wound healing test showed that TNF-α increased the migration ability of VSMCs after scratching, while knockdown of CD40 repress the repair of VSMCs compared with the TNF-α treated group (Fig. 7C).

3.5 p21 ^Cip1 is a downstream regulatory target of miR-590 to control the function of VSMCs. Since p21^Cip1 plays a key role in the regulation of cyclins, we investigated whether the effect of mir-590-3p on the function of VSMCs is exerted by p21^Cip1 through RNA interference of p21^Cip1. We used three siRNA primer targeting p21^Cip1 to achieve effective knockdown, and then we chose the most effective siRNA primer with a silencing efficiency at about 90% for the subsequent experiment (Fig. 8). In the TNF-α induced VSMCs proliferation model, the miR-590-3p mimic up-regulated the expression of p21^Cip1 and down-regulated the expression of cyclin E1 and CDK2 compared with TNF-α treated group. Knockdown of p21^Cip1 by siRNA can partially reverse the expressions of cyclin E1 and CDK2 down-regulated by miR-590-3p under TNF-α stimulation (Fig. 9A-D).

For VSMCs function, we observed that inhibition of p21^Cip1 by siRNA was able to partially reverse the effect of the miR-590-3p mimic reflected by increased EdU positive cells, suggesting p21^Cip1 is a downstream set point of miR-590-3p/CD40/ p21^Cip1 pathway (Fig. 10A, B). Similarly, miR-590-3p mimics reduced the proportion of cells in S phase after TNF-α treatment, while siRNA- p21^Cip1 partially reversed the effect of the miR-590-3p mimics, increasing the proportion of cells in S phase after TNF-α treatment (Fig. 10C, D).

AS is a chronic inflammatory disease and the onset of many cardiovascular and cerebrovascular diseases. During AS process, VSMCs in the vascular wall transformed from contractile type to secreting type with impaired contractile ability and enhanced migration ability and proliferation ability at the same time. After migrating to the vascular intima, a large amount of fibrous tissue synthesized by the VSMCs, along with recruited mononuclear cells, damaged cell fragments, deposited lipids and extracellular matrix proteins, co-mediate the formation of AS plaques. In order to identify the changed microenvironment of vascular system in response to AS stimuli, we collected the mRNA and protein from AS plaque and its adjacent normal tissues and examined relevant expressions of miRNAs and proteins. Consistent with our previous research, we observed an increase in CD40 expression in AS plaque compared with adjacent normal tissues. Further comparison of the expression levels of miR-590-3p in AS plaque and corresponding normal tissues showed miR-590-3p is down-regulated in AS plaque. More importantly, we have observed an inverse correlation between miR-590-3p and CD40 levels in AS plaque, suggesting that miR-590-3p participate in the process of AS by targeting at CD40. The effects of CD40/CD40L system on AS has been well studied. CD40/CD40L system exhibits the pro-AS function mainly through activation of NF-κB pathway and subsequent release of various inflammatory cytokines as well as regulating the phenotypes of VSMCs and VECs(19, 20). Though the anti-AS effects of miR-590-3p has been proposed, the previous researches mainly focused on the protective effects of miR-590-3p on AS induced VECs apoptosis, and the underlying mechanism was attributed to its regulation on lipoprotein lipase, oxidized low density lipoprotein receptor 1 (OLR1, also known as LOX-1) and hypoxia-inducible factor 1α (HIF-1α). However, limited research demonstrates the involvement of miR-590-3p in AS via regulating CD40 expression. It is generally recognized that miRNAs regulate the expression of target mRNA by complete or uncomplete match with 3’-UTR of target mRNA, so miR-590-3p may display its anti-AS effects by targeting different mRNAs. Our current study reveals a mechanism by whichmiR-590-3p exerts atheroprotective effects

After determining the regulatory role of miR-590-3p on CD40 and its abnormal decrease in AS process, we tried to investigate the possible mechanism underlying the anti-AS effects of miR-590-3p. With the going deep of the research work on VSMCs function in the process of AS, different phenotypes of VSMCs have been found in AS and usually coexist in AS plaque, which contribute to the formation, development and rupture of the AS plaque(2). In this study, we mainly focused on the role of miR-590-3p in VSMCs function, and our result first provides direct evidence that miR-590-3p also plays a vital role in VSMCs function in AS. Mechanistically, we demonstrate that miR-590-3p targets CD40 to repress the NF-κB pathway and activated p21^Cip1, which restrict VSMCs from rapidly entering the proliferative cycle.

Our research group has been committed to the study of the relationship between CD40 and AS, and has reported the involvement of CD40 in promoting the development of AS by targeting VSMCs and VECs(14, 24). This current study corroborates the role of miR-590-3p in VSMCs function is associated with CD40 and NF-κB signaling. However, the mechanisms remain to be clarified. In this study we tried to explore the downstream of CD40 signaling under AS condition and the regulation of miR-590-3p in this pathway. In each stage of AS, enhanced proliferation of VSMCs is accompanied by the loss of regulation in cell cycle. In response to various harmful stimulants, DNA damage and replication stress may occur in nucleus and induce the activation of cell cycle checkpoints regulation, which may negatively regulate the uncontrolled cell cycle to avoid too much generation of abnormal cells. This negative regulation is mediated by cyclin-dependent-kinase (CDK) and cyclin-dependent kinase inhibitor (CKI), and p21^Cip1 is the first found CKI and can inhibit the formation of multiple CDK-cyclin complexes. Existing studies have shown that many drugs, cytokines and miRNAs are involved in AS though regulating p21^Cip1(25, 26). So it is possible that the effects miR-590-3p/CD40 on VSMCs proliferation may also be associated with p21^Cip1. In fact, Armanious and colleagues have already reported the association of NF-κB with p21^Cip1. ADAM10 inhibits the expression of p21^Cip1 and cyclin D1 and promote the proliferation of mantle cell lymphoma (MCL) cells by activating the NF-κB and TNF-α pathway(27). In ovarian cancer cells, the NF-κB-p21-VEGF was the most activated signal pathway (28). After binding to its ligand, CD40 activates its downstream signaling pathway including NF-κB, which in turn regulates the expression of p21^Cip1. To test this hypothesis, we examined the expressions of CD40 and p21^Cip1 in AS plaque, and found that CD40 was upregulated while p21^Cip1 was down-regulated compared with the adjacent tissues. In order to verify whether the miR-590-3p/CD40/ p21^Cip1 signaling pathway is established, we used siRNA methods to intervene two important points in this pathway. In TNF-α induced proliferation model of VSMCs, silencing CD40 by siRNA significantly restored the expression of p21^Cip1 accompanied by decreased expressions of CDK2 and cyclin E1. The results from flow cytometry also showed that silence of CD40 caused a decreased cell proportion of S phase and increased cell proportion of G1 phase, suggesting lower levels of proliferation. Conversely, knockdown p21^Cip1 by siRNA after miR-590-3p mimic administration reversed the effect of the miR-590-3p mimic, which was reflected in up-regulation of CDK2 and cyclin E1 as well as increased cell proportion of S phase. Together, these data indicate that the miR-590-3p/CD40/ p21^Cip1 pathway plays a pivotal role in TNF-α induced VSMCs proliferation model.

Besides the potentiated proliferation ability, VSMCs also showed increased migration ability under TNF-α stimulation. Interestingly, pretreatment of VSMCs with miR-590-3p mimic significantly repressed VSMCs repair after wounding, suggesting that miR-590-3p inhibits the migration ability of VSMCs. More importantly, silencing CD40 almost completely reversed the potentiated VSMCs migration induced by TNF-α. Because the activation of NF-κB signaling pathway stimulates the transcription of numerous proinflammatory and proliferative genes, and there is a complex crosstalk between NF-κB and MAPK pathway(29), so it is reasonable to see a definite influence of miR-590-3p/CD40/ p21^Cip1 on VSMCs migration.

In summary, this research demonstrates miR-590-3p/CD40/ p21^Cip1 pathway mediates the VSMCs function under AS condition. The findings in this study indicate a novel pathway in which miR-590, CD40 and p21^Cip1 are involved. Our research may also have extensive implications for the diagnosis and therapy of AS.

AS Atherosclerosis

VSMCs vascular smooth muscle cells

VECs vascular endothelial cells

CD40 Cell differentiation antigen 40

CD40L Cell differentiation antigen 40 ligand

CEA Carotid Endarterectomy

MCP-1 monocyte chemoattractant protein-1

βPDGFR platelet-derived growth factor beta receptor

HAVSMC human aortic smooth muscle cell

cIMT carotid intima media thickness

miRNA microRNA

NF-κB Nuclear factor κB

VCAM-1 vascular cell adhesion molecule 1

ICAM-1 intercellular cell adhesion molecule 1

IL-6 Interleukin 6

IL-1β Interleukin 1β

LPL lipoprotein lipase

ox-LDL oxidized low-density lipoprotein

CDK cyclin-dependent kinase

CKI cyclin-dependent kinase inhibitor

PBS Phosphate-buffered saline

PCR Polymerase chain reaction- restriction

SDS Sodium dodecyl sulfate

PDGF-BB platelet-derived growth factor-BB

TEMED Tetramethylethylenediamine

TNF-α Tumor necrosis factor-α

TRAF6 TNF receptor associated factor 6

3’-UTR 3’-untranslated region 3’

Acknowledgments

We would like to thank our colleagues (Lihua Huang PhD, Shengfeng Wang MD, Yulu Zhou Ph.D.) of the Third Xiangya Hospital for their help to our study.

Author Contributions

RG and RFC conceived this project; HY and XDP collected the samples, did the cell experiments. YH, RFC, XY and YXF did the molecule experiments. YYX, FQN and SW analyzed and interpreted the data. RFC and RG wrote the article. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Availability of data and materials

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

Ethics approval and consent to participate

This study was approved by the Animal Ethics Committee of Central South University. (Permission Number: CSU-2022-0066).

Funding

This project was supported by Hunan Provincial Natural Science Foundation (No.2021JJ31004) and the New Xiangya Talent Project of the Third Xiangya Hosipital of Central South University (JY201520).

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No competing interests reported.

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miR-590-3p attenuates atherosclerosis by preventing TNF-α-induced proliferation and migration of VSMCs via NF-κB pathway

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Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Cell culture

2.2 Reverse transcriptase (RT)-PCR and real-time quantitative PCR analysis

2.3 Western blot

2.4 Flow cytometry

2.5 EdU assay

2.6 Statistical analysis

3 Results

3.2 The effect of miR-590-3p on CD40 expression and NF-κB signaling.

4 Discussion

5 Conclusions

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