Mettl3 promotes oxLDL‐mediated inflammation through activating STAT1 signaling

Abstract Background Atherosclerosis (AS) is the main cause of cerebrovascular diseases, and macrophages act important roles during the AS pathological process through regulating inflammation. Modification of the novel N(6)‐methyladenine (m6A) RNA is reported to be associated with AS, but its role in AS is largely unknown. The aim of this study was to investigate the role and mechanism of m6A modification in inflammation triggered by oxidized low‐density lipoprotein (oxLDL) in macrophages during AS. Methods RAW264.7 macrophage cells were stimulated with 40 μg/ml ox‐LDL, Dot blot, Immunoprecipitation, western blot, Rip and chip experiments were used in our study. Results We found oxLDL stimulation significantly promoted m6A modification level of mRNA in macrophages and knockdown of Methyltransferase‐Like Protein 3 (Mettl3) inhibited oxLDL‐induced m6A modification and inflammatory response. Mettl3 promoted oxLDL‐induced inflammatory response in macrophages through regulating m6A modification of Signal transducer and activator of transcription 1 (STAT1) mRNA, thereby affecting STAT1 expression and activation. Moreover, oxLDL stimulation enhanced the interaction between Mettl3 and STAT1 protein, promoting STAT1 transcriptional regulation of inflammatory factor expression in macrophages eventually. Conclusions These results indicate that Mettl3 promotes oxLDL‐triggered inflammation through interacting with STAT1 protein and mRNA in RAW264.7 macrophages, suggesting that Mettl3 may be as a potential target for the clinical treatment of AS.


| INTRODUC TI ON
Atherosclerosis (AS), the major cause of cardiovascular diseases with its main complications, in particular stroke and myocardial infarction, is a chronic inflammatory disease with lipid accumulation and vascular injury. 1 Owing to more and more aging population and obesity prevalence, the occurrence rate of atherosclerotic disorders is expected to rise over the next decade, and the death number of patient with cardiovascular diseases is expected to reach 23.6 million by 2030. 2,3 Although significant strides have been made in understanding the underlying mechanism of AS development and treating cardiovascular diseases, 4,5 safe and effective drugs for preventing and treating AS are still lacking. Thus, deeply and fully elucidating the pathological mechanisms of AS is essential for finding novel drugs for AS.
Nowadays, AS is considered as a chronic inflammatory disease of the arterial wall characterized by lipid accumulation, cell necrosis, and local inflammation. 6 Macrophages, the major immune cell population in atherosclerotic lesions, play important roles during AS development, from lesion initiation to plaque rupture. 6,7 In plaque microenvironment, low-density lipoprotein is not only phagocytized by macrophages to induce the formation of foam cells but also stimulates macrophages to release inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα). [8][9][10] Recent studies revealed that signal transducer and activator of transcription 1 (STAT1), a novel therapeutical target for AS, act crucial roles in the pathology of AS through promoting expressions of several pro-inflammatory and pro-atherogenic mediators in macrophages. 11 Targeted inhibition of STATs and IRFs was reported to be a potential treatment strategy in cardiovascular disease. Thus, understanding macrophage inflammation during AS is a prerequisite to develop novel therapeutic strategies for AS.
N6-methyladenosine (m6A) is the most abundant internal modification of RNA in eukaryotic cells, and is proved to regulate multiple aspects of RNA metabolism, including RNA processing, nuclear export, RNA translation, and RNA decay. 12,13 The m6A modification is marked by "writers," including Methyltransferase-Like Protein 3 (Mettl3), Mettl14, and the associated protein Wilms' Tumor 1-Associated Protein (WTAP), and is removed by "erasers," such as fat mass and obesity-associated protein (FTO) and AlkB Homolog 5 (ALKBH5). [14][15][16] The m6A modified RNA is recognized by "readers" to regulate gene expression. 17,18 Recent study finds that m6A modification levels of RNA are differentially regulated in atherosclerotic lesions. 19 Besides, Mettl14 is proved to promote endothelial inflammation and AS development, 20 suggesting that m6A modification is associated with AS development. However, whether m6A modification is involved in inflammation induced by macrophages during AS is still unknown.
In this study, we examined the m6A modification level of mRNA in oxLDL-stimulated macrophages, and the expressions of m6A "writers." We found that oxLDL stimulation significantly promoted m6A modification level of mRNA and Mettl3 expres-

| Blood sample collections
In all subjects, blood samples were collected in the morning under fasting state. Human MNCs were isolated from peripheral blood from 16 patients with angiographically proven CAD and 8 healthy donors from Ningbo First Hospital. The study was approved by the research ethics review committee of Ningbo First Hospital, and subjects gave written informed consent before entering the study.
Human monocytes were isolated from the peripheral blood by density gradient centrifugation with Histopaque-1077 (1.077 g/ ml, Sigma, USA). The monocytes were cultured in RPMI1640 medium with 10% FBS at atmosphere of 5% CO 2 in a 37°C humidified incubator.

| Total RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
After treatment, total RNA of RAW264.7 macrophage cells was extracted using the Trizol reagent (Invitrogen, USA), and then reverse-transcribed into cDNA using the PrimeScript RT reagent Kit (TaKaRa, Japan). qRT-PCR was performed using SYBR-green

| Western blot
After treatment, total cellular proteins were lysed by RIPA buffer, overnight. After washes, the membrane was incubated with HRPconjugated secondary antibody, treated with ECL substrate and developed using film.

| Enzyme-linked immunosorbent assay
After treatments, the protein expressions of IL-6 or TNFα in the

| Cell viability assay
After treatments, cell viability of macrophages was detected by the Cell Counting Kit-8 (Beyotime, China) according to the manufacturer's instruction.

| Cell apoptosis assay
Cell apoptosis was measured using the TUNNEL staining kit (Beyotime, China) according to the manufacturer's instructions.

| Cell senescence analysis
Cell senescence was measured using the Fluorescein di-beta-Dgalactopyranoside kit (Biolite, China) by fluorescence microscope according to the manufacturer's instructions.

| RNA immunoprecipitation
RNA immunoprecipitation (RIP) assay was performed with Magna RIP Kit (Millipore, USA) following the manufacturer's instructions.
In brief, magnetic beads were mixed with 5 μg anti-m6A antibody before the addition of cell lysates (approximately 2 × 10 7 cells for each sample). After the treatment of proteinase K, interested RNAs were eluted from immunoprecipitated complex and purified for further analysis using qPCR. Immunoprecipitated RNA was analyzed through qRT-PCR.

| Co-Immunoprecipitation
Co-Immunoprecipitation (Co-IP) assay was performed using Dynabeads™ Co-Immunoprecipitation Kit (ThermoFisher USA), according to the manufacturer's instruction. Briefly, the protein extracted with IP Lysis Buffer was subject to beads premixed with anti-Mettl3 antibody (Abcam, USA) or IgG. The immunoprecipitated protein complex was separated from beads after several washes, followed by the identification for partners of Mettl3 by immunoblots.

| Statistical analysis
Data were analyzed using SPSS version 19.0 and presented as means ± standard deviation (SD). Student's t-test was performed to analyze differences between the two groups and ANOVA (parametric) was performed to analyze differences among multiply groups. p < 0.05 was considered to indicate statistical significance.

| oxLDL stimulation significantly increases m6A modification levels in macrophages and knockdown of Mettl3 inhibits oxLDL-induced m6A modification and inflammatory response
Recent study reports that aberrant m6A level exists in different stages of atherosclerotic lesions. 19 To explore whether m6A modification is involved in oxLDL-induced inflammatory response in macrophages, we first examined the global m6A levels in oxLDL-stimulated macrophages. As shown in Figure 1A,

| Mettl3 promoting oxLDL-induced inflammatory response in macrophages depends on its methyltransferase activity
M6A modification of mRNA is related to regulation of gene expression. 21 qRT-PCR analysis showed that oxLDL treatment significantly increased IL-6 or TNFα mRNA expression in macrophages  Figure 2F). In addition, overexpression of Mettl3 or Mettl3 M3d did not affect cell viability or apoptosis ( Figure S1D,E). Collectively, the results suggest that Mettl3 can promote oxLDL-induced inflammatory response in macrophages depends on its methyltransferase activity.

| Mettl3 promotes oxLDL-induced inflammatory response in macrophages through regulating STAT1
Considering that inflammatory response is usually triggered by promoting oxLDL-induced inflammatory response in macrophages is associated with these signaling pathways. As shown in Figure 3A, oxLDL stimulation significantly increased the protein expressions of phosphorylated p38 (p-p38), p-JNK1/2, p-p65, p-STAT1, and p-STAT3, while Mettl3 knockdown obviously suppressed the upregulation of total STAT1 and p-STAT1 protein expressions. Furthermore, we found that oxLDL stimulation significantly enhanced m6A modification of STAT1 mRNA ( Figure 3C). Moreover, knockdown of STAT1 not only obviously inhibited oxLDL-induced IL-6/TNFα mRNA expression in macrophages but also suppressed Mettl3 overexpressioninduced IL-6/TNFα mRNA expression ( Figure 3D,E). Overall, the results indicate that Mettl3 promotes oxLDL-induced inflammatory response in macrophages through regulating STAT1.

| oxLDL stimulation significantly promoted m6A modification of STAT1 mRNA, and enhanced Mettl3 interacting with STAT1 mRNA
Considering that knockdown of Mettl3 inhibit oxLDL-induced activation of STAT1 ( Figure 3A) and Mettl3 has recently been reported to methylate STAT1 mRNA, 27 we further explore whether

| Mettl3 interacts with STAT1 to promote inflammatory factor expression in monocytes from patients with angiographically proven CAD
To further explore the relationship between m6A modification and inflammatory response in AS, we collected blood samples of patients with angiographically proven CAD and healthy donors in clinical, and then detected the m6A modification changes in monocytes. As shown in Figure 6A,B, m6A modification levels of RNA in monocytes from patients with CAD were significantly higher than those in healthy donors, and Mettl3 protein levels were markedly higher than those in healthy donors, which are consistent with our previous in-vitro results ( Figure 1A,C). Furthermore, we found that expressions of pro-inflammatory factors, IL-6 and TNFα from monocytes of patients with CAD were also significantly higher than those from healthy donors, and knockdown of Mettl3 ( Figure 6C) evidently inhibited IL-6 and TNFα expressions ( Figure 6D). Moreover, Co-IP analysis showed that Mettl3 could bind more STAT1 protein in monocytes from patients with CAD than that in healthy donors ( Figure 6E). In addition, Chip analysis showed that more Mettl3 and STAT1 protein bound to IL-6 gene promoter in monocytes from patients with CAD, compared to those in healthy donors ( Figure 6F,G).
Overall, these results indicate Mettl3 interacts with STAT1 to promote inflammatory factor expression in monocytes from angiographically proven CAD patients.

| DISCUSS ION
Atherosclerosis is now generally considered as a chronic inflammatory disorder with the interaction between inflammation and lipids as a major hallmark. 6 Macrophages, as the most abundant immune cell type in atherosclerotic lesions, play essential roles during all stages of the disease. 7 Recent study reports m6A modification levels  interacts with STAT1 to promote STAT1 transcriptional regulation of inflammatory factor expression in RAW264.7 macrophages, which was also demonstrated in the monocytes from patients with angiographically proven CAD, suggesting that Mettl3 may be a potential target for the clinical treatment of AS.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.