Overexpression of miR-29a-3p Suppresses Proliferation, Migration, and Invasion of Vascular Smooth Muscle Cells in Atherosclerosis via Targeting TNFRSF1A

Objective Increasing evidence highlights the significance of microRNAs (miRNAs) in the progression of atherosclerosis (AS). Our aim was to probe out the role and regulatory mechanism of miR-29a-3p in AS. Methods An in vivo model of AS was conducted by high-fat diet ApoE−/− mice. Oxidized low-density lipoprotein- (ox-LDL-) exposed vascular smooth muscle cells (VSMCs) were utilized as an in vitro of AS. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were detected. Hematoxylin and eosin (H&E) and Masson's staining was presented to investigate the pathological changes. miR-29a-3p and TNFRSF1A expression was detected by RT-qPCR. Proliferative, migrated, and invaded abilities of VSMCs were determined via a series of assays. The interaction between miR-29a-3p and TNFRSF1A was verified through luciferase reporter assay. Results Upregulated miR-29a-3p and downregulated TNFRSF1A were found both in vitro and in vivo models of AS. miR-29a-3p mimic distinctly decreased the serum levels of TC, TG, and LDL-C and increased serum HDL-C levels. Moreover, its overexpression could ameliorate plaque formation of AS mice. In ox-LDL-induced VSMCs, miR-29a-3p overexpression notably decreased cell proliferation, migration, and invasion, which was reversed by TNFRSF1A overexpression. Also, miR-29a-3p could directly target the 3′UTR of TNFRSF1A. Conclusion miR-29a-3p overexpression ameliorated plaque formation of AS and suppressed proliferation, migration, and invasion of ox-LDL-induced VSMCs via TNFRSF1A, which offered novel insights into the progression of AS.


Introduction
Atherosclerosis (AS) is a chronic vascular inflammatory disease related with endothelial dysfunction [1]. Despite the progress made in lifestyle management and medication, total mortality of AS exceeds 50% in developed countries. Thus, there is an urgent need to clarify the molecular mechanisms of AS and to determine effective treatment options. AS is triggered by various risk factors, including the aberrant proliferation of VSMCs. The pathological proliferation of VSMCs has been shown to accelerate AS progression and restenosis of arteries [2]. VSMC dysfunction plays a crucial function in the thickening of atherosclerotic intima. During AS, abnormal proliferation of VSMCs participates in the formation of atherosclerotic plaques [3]. Thus, it is essential to fully understand the behaviors of VSMCs in AS for determining therapeutic targets for the prevention and treatment of AS.
Ox-LDL is a common risk factor for AS, which accelerates the formation of atherosclerotic plaques as well as VSMC migration and proliferation. miRNAs are a class of highly conserved noncoding endogenous RNAs, approximately 22 nucleotides in length. They can bind 3 ′ untranslated region (3 ′ UTR) of a specific-target mRNA sequence, thereby mediating posttranscriptional gene expression [4]. miRNAs may be crucial in affecting the function of VSMCs, driving atherosclerotic plaque formation and cholesterol homeostasis. Studies have confirmed that the abnormal expression patterns of several miRNAs promote the progression of AS, such as miR-144 [5], miR-155 [6], and miR-148b [7]. The function of miR-29a-3p in the pathogenesis of cardiovascular diseases (like myocardial fibrosis [8], cardiac ischemia-reperfusion [9], and cardiac hypertrophy [10]) has attracted wide attention. Nevertheless, the role of miR-29a-3p in AS remains unclear.
TNF receptor-1 (TNFRSF1A) has been confirmed to be in association with the progression of various cardiac diseases, including OS. It can mediate endothelial cell dysfunction and inflammation [11]. TNFRSF1A could accelerate the exacerbation of AS [12]. Clinical trial results demonstrated that high circulating TNFR2 level in patients with stable coronary heart disease is in association with increased risk of cardiovascular events and death [13]. Nevertheless, the regulatory mechanism of TNFRSF1A is not well uncovered. In this study, we investigated the role of miR-29a-3p and TNFRSF1A in vitro and in vivo models of AS. Furthermore, the interactions between miR-29a-3p and TNFRSF1A were identified for AS in this study.

Animal Models.
A total of twenty 8-week-old ApoE -/male mice (SCXK (Su) 2018-0008) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Jiangsu, China). High-fat feed was composed of 78% basic feed, 10% egg yolk powder, 2% cholesterol, and 10% lard (TP28522; Nantong Trophy Feed Technology Co., Ltd., Jiangsu, China). All mice were randomly allocated into four groups. Three groups of mice were fed free high-fat diet for 12 weeks. At the same time, the remaining group of mice was fed basic diet normally. All mice were fed freely and kept in an environment with a day-night cycle of 12 hours, a temperature of 22-25°C, and a humidity of 50-70%. At the 10th week of modeling, 15 mice freely fed high-fat diet were randomly divided into 3 groups (AS group, AS+NC mimic group, and AS+miR-29a-3p mimic group). Briefly, mice were injected with 50 μg adenovirus vector NC mimic and miR-29a-3p mimic (General Biotechnology Co., Ltd., Anhui, China) through the jugular vein every other day for 12 weeks and continued to be fed a high-fat diet. By week 12, all mice were euthanized. Peripheral blood was collected, and smooth muscle cells were isolated. Our research was approved by the Animal Ethics Committee of The First Affiliated Hospital of Soochow University (2019033).

Overexpression of miR-29a-3p Distinctly Reduces Aorta
Plaque Formation for AS Mice. In this study, we conducted an AS model using ApoE -/male mice. In Figures 1(a) and 1(b), miR-29a-3p expression was distinctly reduced in the serum and aorta vascular tissues of the AS model. As expected, miR-29a-3p mimic notably increased its expression in the AS model. We also found that there was an increased mRNA expression level of TNFRSF1A in the AS model compared to controls (Figure 1(c)). However, for AS mice injected with miR-29a-3p mimic, TNFRSF1A mRNA expression was significantly reduced (Figure 1(c)). Similarly, TNFRSF1A protein expression was significantly elevated in the AS model, which was reversed by miR-29a-3p mimic (Figures 1(d) and 1(e)). Biochemical test results demonstrated that there were higher serum levels of TC (Figure 1(f)), TG (Figure 1(g)), and LDL-C (Figure 1(h)) and lower HDL-C (Figure 1(i)) in the AS model compared to controls. However, after treatment with miR-29a-3p mimic, the serum levels of TC (Figure 1(f)), TG (Figure 1(g)), LDL-C (Figure 1(h)), and HDL-C (Figure 1(i)) were prominently reversed in the AS model. As shown in H&E results, the aorta vascular wall was smooth, and there was no atheromatous plaque in the vascular intima for mice in the control group (Figure 1(j)). In converse, there were obvious plaques in the AS mice. For AS mice treated with miR-29a-3p mimic, plaques were distinctly suppressed compared to those with NC mimic. Masson's staining results demonstrated that there was no atheromatous plaque formation in the vascular intima of the control mice ( Figure 1(k)). In the AS model, the intima of the arteries was significantly thickened, and there were fibrous and lipid plaques. Furthermore, the surface of the plaque was covered with collagen fibers, and there were thin fiber cap and few collagen

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fibers for AS mice. Intriguingly, miR-29a-3p mimic injection prominently reduced plaque formation of AS mice.

3.2.
Overexpression miR-29a-3p Suppresses Proliferation of ox-LDL-Induced VSMCs in AS. VSMCs were exposed by different concentrations of ox-LDL to induce AS. CCK-8 was utilized the appropriate concentration of ox-LDL. In Figure 2(a), cell viability of VSMCs was notably increased with a concentration manner. Moreover, the expression of miR-29a-3p was significantly reduced with a concentrationdependent manner (Figure 2(b)). When the concentration of ox-LDL was 100 μg/ml, VSMCs had the highest cell viability, and miR-29a-3p expression had the lowest expression level. Thus, 100 μg/ml ox-LDL was determined for further analysis. When induced by 100 μg/ml ox-LDL, the cell viability of VSMCs was distinctly increased, with a time-dependent manner (Figure 2(c)). Also, as time went by, miR-29a-3p expression was gradually reduced (Figure 2(d)). In ox-LDLinduced VSMCs, its expression was elevated after transfection by miR-29a-3p mimic (Figure 2(e)). CCK-8 results suggested that miR-29a-3p mimic prominently suppressed the viability of VSMCs induced by ox-LDL (Figure 2(e)). Furthermore, colony formation assay results showed that the proliferative ability of VSMCs was induced by ox-LDL, which was reversed by miR-29a-3p mimic (Figures 2(g) and 2(h)). We also detected the expression of proliferationrelated proteins including Ki67 and PCNA in VSMCs. As expected, the expression of Ki67 and PCNA was significantly increased in VSMCs exposed by ox-LDL, which was distinctly reduced by miR-29a-3p mimic (Figures 2(i) and 2(j)).

Overexpression of miR-29a-3p Reduces Migration as Well as Invasion of ox-LDL-Induced VSMCs in AS.
Wound healing assay results demonstrated that ox-LDL-induced VSMCs had significantly higher migrated ability compared to control (Figures 3(a) and 3(b)). However, miR-29a-3p mimic distinctly suppressed the migrated abilities of VSMCs induced by ox-LDL (Figures 3(a) and 3(b)). As shown in Transwell assay results, ox-LDL exposure notably elevated the migrated and invaded abilities of VSMCs, which were reversed by miR-29a-3p overexpression (Figures 3(c)-3(f)). The expression of MMP9 and MMP2 proteins was examined in VSMCs using western blot. In Figures 3(g) and 3(h), their expression was elevated in VSMCs induced by ox-LDL, which was decreased by miR-29a-3p overexpression. The above findings suggested that miR-29a-3p could inhibit migration and invasion of ox-LDL-induced VSMCs in AS.

Discussion
In our study, ApoE -/mice was used to conduct the AS model using high-fat diet [14,15]. miR-29a-3p was prominently lowly expressed in the AS mouse model. Increasing evidence suggests that abnormal expression miR-29a-3p is in association with various heart diseases. For example, its expression is upregulated in the right ventricular outflow tract of congenital heart disease patients [16]. Moreover, it suppresses cardiomyocyte proliferation. It has been found that miR-29a-3p could possess the protective function against cardiac fibrosis via inhibition of macrophage migration [17]. Also, upregulation of miR-29a-3p may protect cardiomyocytes from damage induced by hypoxia [18]. Our biochemical test results suggested that serum levels of TC, TG, and LDL-C were distinctly increased, and serum HDL-C levels were prominently decreased in the AS model. Nevertheless, miR-29a-3p mimic treatment improved serum levels of TC, TG, LDL-C, and HDL-C for the AS model. It has been acknowledged that TC, TG, and LDL-C are risk factors and HDL-C is a protective factor for AS [2]. More importantly, H&E and Masson's staining results confirmed that miR-29a-3p overexpression could prominently reduce arterial wall thickening as well as plaque formation of AS mice.
ox-LDL is a key risk factor for AS progression, which can affect the proliferation, migration, and invasion of VSMCs [19]. Thus, in this study, ox-LDL-induced VSMCs were utilized to construct an in vitro AS model. As expected, miR-29a-3p expression was prominently decreased with a concentration and time-dependent manner. Previous studies have shown that proliferative [20], migrated [21], and 6 BioMed Research International   [22] abilities of VSMCs are closely related to AS progression. After treatment with miR-29a-3p mimic, we found that the cell proliferative ability of VSMCs was suppressed according to CCK-8 and colony formation assays. Also, miR-29a-3p mimic transfection notably decreased the expression of Ki67 and PCNA induced by ox-LDL in VSMCs. Ki67 and PCNA are signs of cell proliferation and mediate DNA replication [23]. Our wound healing and Transwell assay results showed that miR-29a-3p mimic distinctly inhibited the migrated and invaded abilities of VSMCs induced by ox-LDL. Also, miR-29a-3p overexpression suppressed the expression of MMP9 and MMP2 proteins in VSMCs induced by ox-LDL. During the formation of atherosclerotic plaques, VSMCs migrate from the medium to the intima. VSMCs secrete MMPs, a family of zinc-dependent endopeptidases in the intima [24]. MMPs can promote the degradation of the extracellular matrix (ECM) as well as migration of VSMCs [25].
TNFRSF1A mRNA and protein expression was notably elevated both in the AS mouse model and ox-LDL-induced VSMCs, consistent with a previous study [26]. Intriguingly, after treatment with miR-29a-3p mimic, TNFRSF1A expression was remarkably inhibited. Emerging evidence suggests that miRNAs function via mediating the translation or   BioMed Research International stability of target mRNA [27][28][29]. As previous studies, TNFRSF1A could be mediated by microR-29c, thereby reducing poststroke depression [30]. Furthermore, TNFRSF1A regulated by miR-29a promotes AR42J cell apoptosis [31]. Our dual-luciferase report confirmed that TNFRSF1A could be targeted by miR-29a-3p. Further analysis found that TNFRSF1A overexpression could reverse the reduction of cell proliferative, migrated, and invaded abilities by miR-29a-3p overexpression in ox-LDL-induced VSMCs. Also, its upregulation distinctly inhibited the expression of Ki67, PCNA, MMP9, and MMP2 proteins, which were reversed by TNFRSF1A overexpression in ox-LDL-induced VSMCs.
However, several limitations of this study need to be pointed out. AS is a complex disease. Plaque progression involves different stages, from early atherosclerotic lesions to advanced lesions. During this process, the molecular players involved change continuously; the same can be true for potential target genes of miRNAs. Thus, further large-scale studies are needed to in-depth clarify the specific treatment mechanism of AS and transfer these findings to clinical research. Taken together, we conducted an in vivo model of AS by high-fat diet ApoE -/mice and an in vitro model by ox-LDLexposed VSMCs. miR-29a-3p overexpression could improve plaque formation of AS and inhibit proliferation, migration,

Conclusion
In this study, miR-29a-3p was downregulated and TNFRSF1A was upregulated both in the AS mouse model and ox-LDL-induced VSMCs. miR-29a-3p overexpression reduced plaque formation of AS mice. It could directly target the 3′UTR of TNFRSF1A. Moreover, miR-29a-3p overexpression suppressed cell proliferation, migration, and invasion of ox-LDL-induced VSMCs, which was reversed by TNFRSF1A overexpression. Thus, our findings could deepen the understanding about the molecular mechanisms of OS.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.