Atherosclerosis is a complex process involving endothelial and smooth muscle cell activation, monocyte recruitment, and lipid accumulation in the artery walls. It starts with the retention of certain lipoproteins (LPs) in the artery lining, leading to inflammation and dysfunction (1-4). This early inflammation is a key risk factor for atherosclerosis development (5, 6). Inflammatory cells release molecules that attract immune cells, like macrophages, which contribute to plaque formation. Macrophages can take on different roles (M1 for inflammation, M2 for anti-inflammatory effects), to influence disease progression (7-10). Studies show that macrophages can switch between these roles, potentially impacting the development of atherosclerosis.

The AP-1 protein complex, a widely distributed dimeric structure consisting of Jun (e.g., JUN) and Fos (e.g., FOS) subunits, has been recognized for its pivotal roles in various inflammatory pathways. Growing evidence indicates that AP-1 transcription factors, such as c-Jun, JUNB, and JUND, can modulate the expression of crucial inflammatory molecules, thereby influencing the inflammatory response. A study highlighted the essential role of JUNB in modulating interleukin (IL)1β expression during classical inflammation in macrophages (11). Furthermore, c-Jun has been associated with Sirt6-mediated liver inflammation through the regulation of key pro-inflammatory factors, like IL6 and MCP1 (12).

MicroRNAs (miRNAs), a subset of non-coding RNAs approximately 22 nucleotides (nt) in length, play a crucial role in the regulation of RNA silencing and the post-transcriptional modulation of gene expression by binding to the 3’-UTR regions of specific targets (13-17). Comprehensive studies have shown that miRNAs typically recognize their targets through a (6-8) nt site that aligns with their seed region. Each miRNA has the capacity to silence hundreds of genes, while individual genes may be targeted by multiple miRNAs. Mounting evidence suggests that miRNAs participate in various cellular processes, such as inflammation, apoptosis, and cell cycle regulation, by directly or indirectly influencing key factors (18-22).

In this study, we first identified miR-328-5p as a novel regulator in early inflammation and advanced atherosclerosis, possibly through directly targeting JUNB to control its expression. Inhibition of miR-328-5p augments monocyte adhesion and transendothelial migration. Expressions of central inflammatory factors are significantly enhanced when exposed to miR-328-5p inhibitor treatment. Moreover, we find that miR-328-5p plays an inhibitory role on the M1 polarization of macrophages. Taken together, these findings suggest a suppressing function of miR-328-5p for inflammatory atherosclerosis.

miR-328-5p negatively regulates endothelial inflammation

Previous studies have indicated that miR-328 is involved in regulating endothelial cells injury induced by oxidized low-density lipoprotein (23). However, the specific role of miR-328-5p in inflammatory atherosclerosis has not been fully elucidated. To verify the functional involvement of miR-328-5p in regulating endothelial inflammation, we initially treated human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor α (TNFα), a well-known pro-inflammatory inducer, to assess the expression changes of miR-328-5p in response to inflammatory injury. Notably, the qRT-PCR analysis revealed a down-regulation of miR-328-5p expression after TNFα treatment (Fig. 1A, left panel). Moreover, we established a high-fat diet atherosclerosis model to further confirm the decreased expression of miR-328-5p in response to inflammatory atherosclerosis in vivo (Fig. 1A, right panel). These findings suggest a potential role of miR-328-5p in regulating endothelial inflammation and the advanced progression of atherosclerosis.

Next, we conducted a monocyte adhesion assay to investigate the impact of miR-328-5p on endothelial inflammation using a synthetic inhibitor and mimic of miR-328-5p. Notably, inhibiting miR-328-5p in HUVECs significantly increased the adhesive capacity of monocytes to HUVECs (Fig. 1B). To validate this discovery, we investigated whether enforced miR-328-5p could mitigate the induced monocyte association with HUVECs triggered by TNFα, a well-known pro-inflammatory stimulus that induces the secretion of adhesion molecules (Fig. 1C). Consistently, the overexpression of miR-328-5p mimic markedly impeded TNFα-induced monocyte adhesion with endothelial cells (Fig. 1D). Furthermore, we employed transendothelial migration analysis to validate the negative role of miR-328-5p in endothelial inflammation. The results showed that overexpressing miR-328-5p mimic in HUVECs inhibited monocyte transmigration through endothelial monolayers (Fig. 1E). To explore the downstream signaling pathway mediated by miR-328-5p, we then established expression profiles in HUVECs with or without miR-328-5p mimic overexpression using high-throughput mRNA sequencing. As expected, a variety of inflammatory pathways were enriched after miR-328-5p aberrant overexpression (Fig. 1F). Collectively, these findings demonstrate the possible inhibitory role of miR-328-5p in regulating endothelial inflammation.

miR-328-5p attenuates the expressions of pro-inflammatory factors

Numerous pro-inflammatory factors are induced and play regulatory roles for the formation of an inflammatory microenvironment when endothelial cells are susceptible to pro-inflammatory stimuli. To examine the effect of miR-328-5p on the expression of these inflammatory genes, we analyzed the expression profiles of the mRNA sequencing data above, and found that 238 and 395 genes were up- and down-regulated, respectively, after miR-328-5p mimic overexpression in HUVECs (Fig. 2A). Further analysis suggested that multiple inflammatory factors involved in the regulation of early endothelial inflammation were significantly suppressed in HUVECs with enforced miR-328-5p overexpression (Fig. 2B). Moreover, qRT-PCR assay further confirmed the increased expressions of vascular cell adhesion molecule 1 (VCAM1), IL6, IL8, and IL1β after miR-328-5p inhibitor treatment (Fig. 2C). In parallel, protein expression changes in miR-328-5p-depleted HUVECs were determined by immunoblot analysis that showed that miR-328-5p attenuation significantly augmented the protein levels of these inflammatory genes (Fig. 2D). Consistent with this result, ELISA analysis further verified this inhibitory effect of miR-328-5p on pro-inflammatory factor secretion (Fig. 2E, F). To verify this regulatory role of miR-328-5p for these inflammatory factors, we overexpressed the mimic of miR-328-5p, then examined its effect on the expressions of pro-inflammatory factors. As expected, enforced overexpression of miR-328-5p greatly abrogated the expression levels of these inflammatory factors (Fig. 2G, H). Overall, these observations suggest that miR-328-5p negatively regulates the expressions of canonical pro-inflammatory factors.

Inhibition of miR-328-5p promotes the conversion of inflammatory macrophages to M1 phenotype

It is widely accepted that when suffering from inflammatory injury, macrophages were able to switch between M1 and M2 states. We therefore asked whether miR-328-5p plays a role in the modulation of macrophage polarization. To prove this hypothesis, we silenced miR-328-5p in THP-1 monocytes, which were then subjected to PMA induction for macrophage induction. Of note, miR-328-5p inhibition remarkably augmented the expression levels of M1-type marker genes, such as MCP1 and TNFα (Supplementary Fig. 1A). Conversely, attenuation of miR-328-5p suppressed the levels of M2-type marker genes, like Arg1 and IL10 (Supplementary Fig. 1B). To further validate this inhibitory effect of miR-328-5p deficiency on M2-type macrophage polarization, miR-328-5p mimic was re-expressed in monocytes, and then subjected to qRT-PCR assessment to detect the expression changes of these type-specific markers. In line with the above findings, miR-328-5p mimic re-expression significantly facilitated the macrophage from M1- to M2-like type polarization (Supplementary Fig. 1C, D). In summary, these collective results strongly support that miR-328-5p limits the macrophage polarization toward M1-like type.

miR-328-5p directly targets JUNB to suppress endothelial inflammation

To investigate the precise molecular mechanism by which miR-328-5p regulates inflammatory atherosclerosis, we first analyzed the potential targets of miR-328-5p using the predictor “miRDB”. JUNB, an inflammatory activator, was found to comprise a potential seed region (Fig. 3A). To examine whether this seed region is a specific target site of miR-328-5p, we determined the effect of miR-328-5p on JUNB expression. The qRT-PCR data showed that miR-328-5p inhibition led to a marked increase in both JUNB mRNA and protein expression level (Fig. 3B, C). In line with this, miR-328-5p-mimic overexpression had an obvious inhibitory effect on JUNB expression levels (Fig. 3D, E). To further confirm the direct binding of miR-328-5p with this seed region, we generated a SV40 promoter-driven luciferase reporter carrying this seed region (JUNB-3’UTR-WT) or the seed region-mutated form (JUNB-3’UTR-Mut), then performed luciferase assay. As expected, miR-328-5p mimic overexpression significantly restricted the luciferase activity of JUNB-3’UTR-WT, whereas had no effect on JUNB-3’UTR-Mut activity (Fig. 3F), indicating that JUNB is a direct target of miR-328-5p. Next, we examined the functional involvement of JUNB in the miR-328-5p-mediated suppression of endothelial inflammation. As anticipated, knockdown of JUNB significantly reduced the elevated expression levels of pro-inflammatory factors induced by the absence of miR-328-5p (Fig. 3G-I), as evidenced by qRT-PCR, ELISA, and western blot assays. Conversely, the protein levels of anti-atherosclerotic regulators were notably increased following JUNB depletion (Fig. 3I). Monocyte adhesion assay further validated the critical inhibitory effect of JUNB depletion on endothelial inflammation activated by miR-328-5p deficiency (Fig. 3J). Collectively, these observations suggest that miR-328-5p negatively regulates endothelial inflammation through the post-transcriptional degradation of JUNB.

miR-328-5p negatively regulates atherosclerotic plaque formation

Early endothelial inflammation is a central risk factor for later atherosclerotic plaque development. Hence, we next investigated the role of miR-328-5p in atherogenesis. Intriguingly, silencing miR-328-5p obviously inhibited the levels of the anti-atherosclerotic regulator kruppel-like factor 4 (KLF4), as well as the nitric oxide synthase 3 (NOS3) and the anti-thrombotic regulator thrombomodulin (THBD) (Fig. 4A, B), exhibiting a negative regulatory role of miR-328-5p in the formation of atherogenesis. By contrast, miR-328-5p mimic overexpression dramatically enhanced both the RNA and protein expression levels of these athero-protective regulators (Fig. 4C, D). To further define the regulatory role of miR-328-5p in advanced atherosclerosis, we established the high-fat diet Apoe^−/− mice, a widely used atherosclerosis model, which were then transduced with adeno-associated virus (AAV) overexpressing miR-328-5p or its combination with JUNB. Fluorescent microscopy analysis suggested the high infection efficiency of AAV (Fig. 4E). Notably, Oil Red O staining analysis demonstrated that JUNB reintroduction significantly limited the reduced atherosclerotic plaques caused by miR-328-5p overexpression (Fig. 4F). Furthermore, CD68 immunostaining assay further confirmed that the reduced inflammatory infiltration of macrophages triggered by miR-328-5p mimic overexpression was rescued by JUNB reintroduction (Fig. 4G). Taken together, these collective findings lead us to conclude that miR-328-5p acts as a critical repressor in the pathological development of advanced atherosclerosis, at least in part, by regulating JUNB abundance.

Previous research has shown that miR-328-5p regulates oxidized low-density lipoprotein-induced endothelial cell injury (23). This study explores the role of miR-328 in inflammatory atherosclerosis and its molecular mechanisms. The findings suggest that miR-328-5p acts as a key negative regulator in atherosclerosis development. Reduced miR-328-5p enhances monocyte adhesion to endothelial cells, while increased miR-328-5p inhibits monocyte migration through endothelial monolayers. These results were confirmed in an Apoe^−/− mouse model, showing decreased advanced atherosclerosis with miR-328-5p overexpression. This study establishes miR-328-5p as a critical suppressor of inflammatory atherosclerosis.

Upon exposure to various pro-inflammatory stimuli, endothelial cells can release a plethora of inflammatory factors, including classical adhesion molecules that facilitate macrophage recruitment, as well as cytokines and chemokines that contribute to the inflammatory microenvironment. Disruption of miR-328-5p led to a notable increase in pro-inflammatory factors like VCAM1, IL6, and IL8, underscoring the crucial role of miR-328-5p in regulating inflammatory cascades.

Monocyte-derived macrophages are key drivers of atherosclerosis, able to switch between pro-inflammatory M1 and athero-protective M2 states in response to stimuli (8, 24). M1 macrophages promote inflammation, while M2 macrophages have anti-inflammatory effects, which are crucial in advanced atherosclerosis (8, 25). Macrophages influence the inflammatory microenvironment and atherosclerotic lesion development. Our study identifies miR-328-5p as a pivotal regulator in macrophage polarization. Suppressing miR-328-5p promotes M1 polarization, while overexpressing its mimic enhances M2 formation, underscoring its importance in regulating macrophage polarization.

Numerous crucial factors are involved in the regulation of inflammatory atherosclerosis, including the anti-atherosclerotic regulator KLF4 and NOS3, as well as the anti-thrombotic regulator THBD. These factors are widely recognized as central regulators that negatively regulate the progression of atherogenesis (26-28). To investigate the potential connection between miR-328-5p and atherosclerosis, the impact of miR-328-5p on the expression levels of these atherosclerotic regulators was examined. Interestingly, miR-328-5p triggers a marked increase in the levels of these atherosclerotic repressors, which might partially elucidate the inhibitory effect of miR-328-5p on atherosclerosis. As previously documented, AP-1 tightly associates and collaborates with NF-κB signaling to regulate the inflammatory response (29-31). Activation of NF-κB signaling leads to the transcriptional upregulation of pro-inflammatory factors, such as VCAM1, IL6, and IL8, while simultaneously downregulating the expression of KLF4. The decrease in KLF4, a well-established anti-atherosclerotic regulator, may subsequently inhibit its downstream effectors, THBD and NOS3. Additionally, our RNA-seq data demonstrates the involvement of NF-κB signaling in the miR-328-5p-mediated inflammatory response. It is of significant interest to explore and validate these potential molecular mechanisms that are responsible for the regulation of pro-atherosclerotic events by JUNB.

To elucidate the underlying mechanism by which miR-328-5p mediates inflammatory atherosclerosis, it was found that miR-328-5p directly targets and degrades the RNA level of JUNB, resulting in the downregulation of JUNB protein expression. Reintroducing JUNB effectively reversed the atherosclerotic phenotype induced by miR-328-5p overexpression, underscoring the pivotal role of JUNB in the miR-328-5p-mediated suppression of inflammatory atherosclerosis. This study provides novel insights into the functional importance of the miR-328-5p/JUNB axis in atherosclerosis, and highlights a potential therapeutic target for the clinical treatment of inflammatory atherosclerosis.

This work was supported by grants from the National Natural Science Foundation of China (81900392), Henan Outstanding Youth Science Fund (202300410307), Xinxiang Medical University Doctor Support Foundation (300-505307, XYBSKYZZ201902), Graduate Research Innovation Support Program (YJSCX202206Z).

The authors have no conflicting interests.

Additional data and materials may be requested from the corresponding author on reasonable request.

Z.H., X.Z and H.L. designed and conceived the project. Y.Z. and Y.L. performed most of the experiments. Z.H. and Y.L. performed data analysis. X.Z. and H.L. wrote the manuscript. All authors commented on the manuscript.