miR‐195‐3p alleviates homocysteine‐mediated atherosclerosis by targeting IL‐31 through its epigenetics modifications

Abstract Atherosclerosis is a serious age‐related disease, which has a tremendous impact on health care globally. Macrophage inflammation is crucial for the initiation and progression of atherosclerosis, and microRNAs (miRNAs) recently have emerged as potent modulators of inflammation, while the underlying mechanisms of its involvement in homocysteine (Hcy)‐mediated macrophage inflammation of atherosclerosis remain largely unknown. Here, we demonstrated that elevated Hcy inhibits the expression of miR‐195‐3p, which in turn enhances IL‐31 expression and thereby causes the secretion of macrophages pro‐inflammatory factors IL‐1β, IL‐6 and TNF‐α and accelerate atherosclerosis. Furthermore, we identified that Hcy can induce DNA hypermethylation and H3K9 deacetylation of miR‐195‐3p promoter due to the increased the binding of DNMT3a and HDAC11 at its promoter. More importantly, Sp1 interacts with DNMT3a suppressed the binding of HDAC11 at miR‐195‐3p promoter and promoted its transcription. In summary, our results revealed a novel mechanism that transcriptional and epigenetic regulation of miR‐195‐3p inhibits macrophage inflammation through targeting IL‐31, which provides a candidate diagnostic marker and novel therapeutic target in cardiovascular diseases induced by Hcy.


| INTRODUC TI ON
With the increase of an aging population, aging-related cardiovascular diseases confer a heavy economic burden on society. The incidence of atherosclerosis increases dramatically with advancing age and constitutes the main cause of cardiovascular diseases mortality and morbidity in the elderly (Hopkins, 2013). Atherosclerosis is a chronic progressive inflammatory disease, recent insights into the pathogenesis of atherosclerosis emphasize the importance of vascular inflammation in the initiation and development of atherosclerotic plaque (Wolf & Ley, 2019). Homocysteine (Hcy) is well recognized to be an independent risk factor for atherosclerosis, in which macrophages play essential roles in the development of atherosclerosis through formation of foam cells and production of inflammatory factors. However, the mechanisms involving in Hcy-related inflammation in macrophages during atherosclerotic plaque formation is still a matter of debate (McCully, 2015b;Wang et al., 2017).
It has been widely accepted that aberrant expression of genes and cytokines contribute to the onset of atherosclerosis, and the focus on the origin and progression of atherosclerosis has shifted from genetic to epigenetic regulation in recent studies (Kuznetsova et al., 2020;Rizzacasa et al., 2019). microRNAs (miRNAs) are a kind of single-stranded endogenous small non-coding RNAs containing 19 to 25 nucleotides and has been now recognized as one of the major epigenetic modulators (Lu & Rothenberg, 2018). There is growing evidence that miRNAs could be able to regulate atherosclerosis physiological and pathological processes through destabilizing target mRNAs and/or inhibiting translation. For example, promotes the secretion of inflammatory cytokines and macrophages intrusion into the endothelial layer, promoting the progression of atherosclerotic lesion . In addition, miR-181b decreases atherosclerotic plaque formation through upregulation of macrophages TIMP-3 expression, meaning miR-181b therapy may serve as another atheroprotective therapeutic strategy (Di Gregoli et al., 2017). These findings suggest that exploring miRNAs effects on atherosclerosis may allow us to exploit them as novel therapeutics or clinical biomarkers thus lead to better management of atherosclerosis.
Of note, as a part of epigenetic machinery, miRNAs are also epigenetically modified by DNA methylation and histone modification like any other protein-coding genes (Yao et al., 2019). Our previous study revealed that DNMT3b-mediated hypomethylation of miR-30a exerts a protection effect of hypoxia postconditioning on aged cardiomyocytes hypoxia/reoxygenation injury .
Hcy is an important intermediate metabolite in the methionine cycle, which has been reported to alter DNA methyltransferase activity, and influence histone deacetylase through the interference of methyl group transferring metabolism (Jakubowski, 2019;Li et al., 2017). As a part of the epigenome, studies have shown that Hcy induces cardiac hypertrophy by promoting MEF2C-HDAC1 complex formation (Kesherwani et al., 2015). Numerous studies demonstrate that in addition to epigenetic mechanisms, regulatory proteins (transcription factors) also exert an essential and fundamental effect on the transcriptional regulation of specific gene or miRNA (Kim et al., 2019). Sp1 is a sequence-specific transcription factor, which binds to the GC and GT boxes to initiate and activate a wide range of viral and cellular genes transcription (Vizcaíno et al., 2015). Sp1 could inhibit the DNA methylation of POLD1 gene promoter by binding and the suppressing DNMT1 activities in breast cancer (Zhang et al., 2016).
Eukaryotes express genes in incredible diversity manner, which is largely dependent on the interaction between transcription factors and epigenetic regulation. For instance, Sp1 could also recruit repressor complexes, such as the HDAC1-HDAC2-mSin3a complex to repress gene transcription (Zhang & Dufau, 2003).
Herein, we identified miR-195-3p as a novel miRNA which was associated with macrophage inflammation and atherosclerosis induced by Hcy. Mechanistically, Sp1 interacts with DNMT3a to suppress HDAC11 binding to miR-195-3p promoter to increase its expression. Our findings shed new light on the involvement of miR-195-3p in macrophage inflammation, which might provide a potential therapeutic strategy for atherosclerosis induced by Hcy.

| Homocysteine accelerates macrophage inflammation during atherosclerotic plaque formation
To get a better insight into the possible mechanism of atherosclerosis induced by Hcy, the ApoE −/− mice were fed with regular diet (NC) and high methionine diet (HMD) as described in experimental procedures. An elevation of the intima-media thickness (IMT) of aortic root and increase of blood flow velocity of the ascending aortic were observed from HMD-fed ApoE −/− mice by ultrasound biomi- Atherosclerosis is a chronic inflammatory disease and recent study indicated that atherosclerosis-related inflammation is mainly mediated by pro-inflammatory cytokines (IL-1β, IL-6 and TNFα) and inflammatory signaling pathways (Gisterå & Hansson, 2017). To explore the role of inflammation in Hcy-induced atherosclerosis, the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNFα were validated by immunofluorescence staining analysis. The number of IL-1β, IL-6 and TNFα puncta in atherosclerotic plaque of the HMD-fed ApoE −/− mice were significantly increased, which was evidenced by the increased co-localization with MOMA-2 in aortic root (Figure 1f,g). Meanwhile, a remarkably increase of mRNA expression of IL-1β, IL-6 and TNFα were observed in HMD-fed ApoE −/− mice ( Figure 1h). In addition, the results also showed that the secretion of IL-1β, IL-6 and TNFα F I G U R E 1 Homocysteine promotes macrophage inflammation and atherosclerosis in ApoE −/− mice. After the ApoE −/− mice were fed with regular diet (NC) or high methionine diet (HMD) for 16 weeks (n = 6), then (a) Representative images and quantification of the intima-media thickness (IMT, yellow arrow) in the aortic root and blood velocity (yellow arrow) in the ascending aorta by ultrasound biomicroscopy (UBM). (b) Photomicrographs of the aortic sinus cross-sections staining with HE staining. The blue arrow indicates the plaque region. Scale bar =500 μm. (c) Representative images of Oil Red O staining of the aortic sinus cross-sections (the blue arrow indicates the plaque region, Scale bar =500 μm) and en face aortas and quantitative analysis of the percentage of atherosclerotic plaque area from ApoE −/− mice. (d) The serum levels of total cholesterol (TC), triglyceride (TG) and free cholesterol (FC) in ApoE −/− mice were measured by automatic biochemistry analyzer. (e) Serum levels of Hcy in ApoE −/− mice were measured by automatic biochemistry analyzer, and the correlation between atherosclerotic plaque area and serum Hcy levels was evaluated by Pearson correlation analysis. (f, g) Representative immunofluorescence images and quantification of IL-1β, IL-6, and TNFα (red) co-localization with MOMA-2 (a marker for macrophages, green) in ApoE −/− mice. Nuclei were stained with DAPI (blue). Scale bar =50 μm. (h) The mRNA expression of IL-1β, IL-6 and TNFα in the aortic from ApoE −/− mice were assessed by qRT-PCR. (i) The IL-1β, IL-6, and TNFα secretion in supernatant and mRNA expression in macrophages treated with 100 μmol/L Hcy were detected by ELISA and qRT-PCR, respectively. Data were presented as mean ±SD. *p < 0.05, **p < 0.01 in supernatant of macrophages were increased after Hcy treatment, as well as the mRNA expression of IL-1β, IL-6 and TNFα ( Figure 1i).
Together, these data demonstrated that Hcy facilitated macrophage inflammation in atherosclerosis in ApoE −/− mice.

| miR-195-3p attenuates inflammation and atherosclerotic plaque formation by targeting IL-31
To screen miRNAs that are involved in inflammation and atherosclero-

| Homocysteine facilitates DNA hypermethylation and H3K9 deacetylation of miR-195-3p promoter
Transcriptional activity is the key to gene expression, which is susceptible to paradigmatic epigenetic marker, such as DNA F I G U R E 2 miR-195-3p protects against atherosclerosis in ApoE −/− mice by targeting IL-31 to exert anti-inflammatory effect. (a) The heatmap shows differential expression of miRNAs in the aorta of ApoE −/− mice with fold change≥2.0, p < 0.05. The expression values are represented by a color scale, red indicated high relative expression, blue indicated low relative expression, and white indicated no change. (b) Differential expressed miRNAs (miR-144, miR-149, miR-193b, miR-3107, miR-34c, miR-378, miR-378b, miR-451, miR-1949, miR-195-3p, miR-342-5p, miR-466c-5p) from Microarray analysis data were confirmed using qRT-PCR in the aortic from ApoE −/− mice fed with regular diet (NC) or high methionine diet (HMD) (n = 6). (c) The expression of miR-195-3p in macrophages treated with 100 μmol/L Hcy was determined by qRT-PCR. (d) The miR-195-3p binding site in the 3'-UTR region of IL-31 was highly conserved among several species. The red box indicated the conserved sequence of IL-31 via targeting to miR-195-3p, and the red letters showed the mutation of the miR-195-3p target in IL-31. (e) The reporter constructs containing 3'-UTR regions of the wild-type (WT) and mutant-type (Mut) IL-31 were co-transfected with miRNA negative control (miR-NC) or miR-195-3p mimic. Relative luciferase activities are normalized by the ratio of firefly and renilla luciferase activities. (f) The expression of IL-31 was detected by qRT-PCR in macrophages transfected with miR-NC or miR-195-3p mimic. (g) The mRNA expression of IL-1β, IL-6 and TNFα were detected in macrophages transfected with miR-NC, miR-195-3p mimic, adenovirus expressing IL-31 (Ad-IL-31) and Ad-GFP solely or collectively in presence of Hcy. (h-j) The expression of miR-195-3p, IMT (yellow arrow) of the aortic root, blood velocity (yellow arrow) of the ascending aorta, and atherosclerosis lesion area were detected by qRT-PCR, UBM and Oil Red O staining after the HMD-fed ApoE −/− mice were injected with PBS, Lv-miR-neg and Lv-miR-195-3p. (k) IL-31, IL-1β, IL-6 and TNFα contents in serum and mRNA levels in the aorta from HMD-fed ApoE −/− mice injected with PBS, Lv-miR-neg and Lv-miR-195-3p were measured by ELISA and qRT-PCR, respectively. Data were presented as mean ±SD. *p < 0.05, **p < 0.01 methylation . We first analyzed the promoter sequence of miR-195-3p using MethPrimer (http://www.uroge ne.org/ methp rimer/), and a CpG island between −2,904 bp to −2,384 bp was observed in miR-195-3p promoter ( Figure 3a). Meanwhile, several fragments (−4,000/+1,045, −1,623/+1,045, −746/+1,045, −614/+1,045) of miR-195-3p promoter were inserted into the firefly F I G U R E 3 Homocysteine induces DNA hypermethylation and H3K9 deacetylation of miR-195-3p promoter in macrophages. (a) A schematic diagram of CpG islands in the miR-195-3p promoter by using the online accessible software MethPrimer. Vertical red lines denoted CpG sites, and CpG islands shown as light blue areas. (b) The promoter activity of miR-195-3p was evaluated by Dual-Luciferase reporter assay. Different deletion fragments of the miR-195-3p promoter (−614/+1045, −746/+1045, −1623/+1045, and −4000/+1045) cotransfected into HEK293 cells with renilla luciferase vector (internal control), and the results were represented as firefly luciferase activity normalized to renilla luciferase activity. (c, d) The methylation status of miR-195-3p promoter were evaluated by bisulfite sequencing PCR (BSP) in the aorta from HMD-fed ApoE −/− mice and macrophages treated with Hcy. The cloned BSP was performed on the miR-195-3p promoter region (−2904 to −2384) relative to the transcription start site (TSS). The white cycle indicates unmethylated CpG dinucleotides whereas the black cycle represents methylated CpG dinucleotides. The percentage of methylation on each CpG dinucleotide was calculated by the number of methylated clones in each CpG site divided by the total number of clones in the same CpG site and shown in the right panel. (e) Chromatin immunoprecipitation (ChIP) assay of H3K9ac and H3K27ac occupancy at miR-195-3p promoter in macrophages treated with Hcy. Chromatin amplified with DNA fragment without antibody precipitation after sonication was used as Input, and IgG was amplified with DNA fragment precipitated by normal mouse IgG. The results were standardized with input DNA, and the data are the percentages of input chromatin. (f) Representative images and quantification of MOMA-2 and H3K9ac levels by immunofluorescence staining in atherosclerotic plaque from ApoE −/− mice. Nuclei were stained with DAPI (blue). Scale bar =50 μm. Data were presented as mean ±SD. **p < 0.01 luciferase vector pGL3, and luciferase reporter assay showed that the fragment (−4,000/−1,623) had the highest transcriptional activity ( Figure 3b), meaning the fragment (−4,000/−1,623) is the miR-195-3p basic core regulation region, which provides the structural basis for methylation modification. Therefore, miR-195-3p methylation status of 42 CpGs from −2,904 bp to −2,384 bp relative to the transcription start site (TSS) was characterized by bisulfite genomic sequencing. The results showed that miR-195-3p promoter has a general hypermethylation status in the aorta of HMD-fed ApoE −/− mice ( Figure 3c). Likewise, hypermethylation of miR-195-3p promoter was also observed in macrophages treated with Hcy ( Figure 3d). These results indicated that Hcy-induced hypermethylation of miR-195-3p promoter. In addition to DNA methylation, a possible mechanism to explain gene transcription regulation is histone acetylation (Martin et al., 2021). We next analyzed an association of the potentially chromatin modification patterns of miR-195-3p by using UCSC database (https://genome.ucsc.edu/) and found that H3K9ac and H3K27ac may occur at 5′ upstream regions of miR-195-3p ( Figure   S2). ChIP assay was applied to measure the enrichment of H3K9ac and H3K27ac at miR-195-3p promoter, and the results indicated that

| Homocysteine downregulation of miR-195-3p expression via synergy of DNMT3a and HDAC11
As the above data suggested that the DNA methylation and H3K9 deacetylation of miR-195-3p promoter support miR-195-3p transcription regulation, we next examined the key enzymes involved in Hcy-mediated DNA methylation and histone acetylation. As shown in Figure  This prompted us to study whether DNMT3a and HDAC11 could improve macrophage inflammation in atherosclerotic plaque formation. The macrophage inflammation was correspondingly promoted as evidenced by the marked increase of secretion and mRNA expression of IL-1β, IL-6, TNFα and IL-31 ( Figure 4f). Similar trends were found after macrophages transfected with siRNAs against DNMT3a (si-DNMT3a) and siRNAs against HDAC11 (si-HDAC11) ( Figure   S3a,b). The results showed that inhibition of DNMT3a and HDAC11 could attenuate the levels including DNA methylation, H3K9 deacetylation in miR-195-3p promoter and macrophage inflammation, while increased miR-195-3p expression (Figure 4g-j). In addition, a stronger effect was found in macrophages after treatment with si-DNMT3a and si-HDAC11 collectively. These results indicated that DNMT3a and HDAC11 at miR-195-3p promoter play a synergistic role in the suppression of miR-195-3p expression, thereby improve macrophage inflammation induced by Hcy.

| Sp1 enhances miR-195-3p expression by direct binding to its promoter
Sp1, a prototypic C2H2-type zinc finger protein, has been reported to activate or repress gene transcription in response to physiologic or pathologic stimuli (Vizcaíno et al., 2015). To examine whether Sp1 operates by a similar mechanism in macrophages during atherosclerotic plaque formation, we first analyzed miR-195-3p promoter sequence using the Searching Transcription Factor Binding Sites

| Sp1 interacts with DNMT3a to suppress HDAC11 binding to miR-195-3p promoter
Given that transcription activation of miR-195-3p is dependent on Sp1, we proceeded to explore the effect of Sp1 on the binding ability of DNMT3a and HDAC11 to miR-195-3p promoter. ChIP assay showed that silencing Sp1 in macrophages resulted in a significant increase binding of DNMT3a and HDAC11 to the miR-195-3p promoter after Hcy treatment (Figure 6a). Then, ChIP and Re-ChIP assays were applied to explore the potential link between Sp1, DNMT3a, and HDAC11 in macrophages. ChIP assay shown that both Sp1, DNMT3a and HDAC11 bound to the miR-195-3p promoter after Hcy treatment (Figure 6b, left). Re-ChIP assay further indicated that the enrichment of DNMT3a at miR-195-3p promoter was decreased, whereas no obvious change was observed for HDAC11 enrichment, meaning a close relationship between DNMT3a and Sp1 (Figure 6b, right). This prompted us to further examine the relationship between DNMT3a and Sp1 using Co-IP analyses. As shown in Figure 6c Collectively, these data strongly suggested that the amino acids from 496 to 610 of Sp1 was important for the interaction between DNMT3a and Sp1 to suppress HDAC11 at miR-195-3p promoter thus facilitate miR-195-3p transcription in macrophages.

| DISCUSS ION
The development of atherosclerosis and the incidence of its risk factors and clinical manifestations increase dramatically with age and are responsible for most cardiovascular morbidity and mortality in the elderly. The concept that inflammation has deleterious and additive effects in the onset and the progression of atherosclerosis and its complications has gained considerable attention (Gisterå & Hansson, 2017). Over the past decade, Hcy-mediated pro-inflammatory cytokines production and lipid accumulation in macrophages have been reported to promote the progression of atherosclerotic plaque (McCully, 2015a). Despite a wealthy of evidence indicated the potential benefits of treatment of inflammation in atherosclerosis, it remains a great threat to human health worldwide. The present study was aimed at investigating the effect of miR-195-3p on the inflammation of macrophages and atherosclerosis, and to elucidate the mechanisms by which miR-195-3p downregulation mediated by Hcy was regulated by both epigenetic modification and transcription mechanism. Collectively, these findings will provide a perspective on therapeutic opportunities for miRNAs in hyperhomocysteinemia (HHcy)-associated cardiovascular diseases. F I G U R E 4 DNMT3a and HDAC11 play a pivotal role in the inhibition of miR-195-3p expression induced by Homocysteine. (a) The change of miR-195-3p in macrophages treated with DNMTs specific inhibitor or HDACs specific inhibitor in presence of Hcy. Among them, DC_05, Theaflavin-3, 3′-digallate (TFD) and Nanaomycin A (NA) are specific inhibitor of DNMT1, DNMT3a and DNMT3b, respectively. Butyrate (BTT), Nicotinamide (NAM) and JB3 are specific inhibitor of HDAC I/II, HDAC III and HDAC11, respectively. (b) The expression of DNMT3a and HDAC11 was measured in the aorta from ApoE −/− mice by qRT-PCR and western blot, respectively. (c) DNA methylation status of miR-195-3p promoter was performed by BSP in macrophages treated with TFD and JB3 in presence of Hcy. (d) The H3K9ac occupancy at miR-195-3p promoter in macrophages treated with TFD and JB3 in presence of Hcy was determined by ChIP assay. (e) The expression of miR-195-3p in macrophages treated with TFD and JB3 in presence of Hcy were determined by qRT-PCR. (f) The secretion and mRNA expression of IL-1β, IL-6, TNFα and IL-31 were evaluated by ELISA and qRT-PCR, respectively. (g) Methylation status of miR-195-3p promoter in macrophages transfected with siRNAs against DNMT3a (si-DNMT3a) and HDAC11 (si-HDAC11) in presence of Hcy was evaluated by BSP. (h) ChIP analysis of H3K9ac at miR-195-3p promoter by using anti-H3K9ac in macrophages transfected with si-DNMT3a and si-HDAC11 in presence of Hcy. (i) qRT-PCR was performed to determine the miR-195-3p expression in macrophages transfected with si-DNMT3a and si-HDAC11 in presence of Hcy. (j) ELISA and qRT-PCR were used to determine the secretion and mRNA expressions of IL-1β, IL-6, TNFα and IL-31 in macrophages transfected with si-DNMT3a and si-HDAC11 in presence of Hcy. Data were presented as mean ±SD. *p < 0.05, **p < 0.01; # p < 0.05, ## p < 0.01 | 11 of 17

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Accumulating evidences demonstrated that inflammation could drive the formation, progression, and rupture of atherosclerotic plaques (Gencer et al., 2021). Macrophages are an important component of vessel wall infiltrates, which exert the pathogenic effects on vascular inflammation mainly through the secretion of soluble factors, such as cytokines, growth factors and enzymes (Koelwyn et al., 2018). Hcy is a sulfur-containing amino acid, and the elevated level of Hcy is considered toxic as the excess of which may be exported out from cells into systemic circulation (Azzini et al., 2020). Macrophages polarize into M1 and M2 distinct functional subpopulations exposed to environmental signals like Hcy (Winchester et al., 2015). Notably, M1 macrophages contribute to Th1 responses, and mediate inflammatory and tissue disruptive reactions through an armamentarium of pro-inflammatory cytokines, including IL-1β, IL-6 and TNFα (Rigoni et al., 2020). The uncontrolled inflammation could hamper macrophages cholesterol efflux and efferocytosis directly, which result in the accumulation of debris and enlargement of necrotic cores in atherosclerotic plaque (Baardman et al., 2020). Moreover, inflammatory mediators released by plaque macrophages aggravate local tissue damage, which in turn arouses more inflammation and forms a vicious circle . In this study, our results showed that Hcy promotes the production of pro-inflammatory cytokines IL-1β, IL-6 and TNFα in macrophages during the formation of atherosclerotic plaques, suggesting that macrophage inflammation might be a critical target of the atherogenic effect of Hcy.
It is widely accepted that epigenetic and genetic alterations are responsible for the initiation and progression of atherosclerosis (Xu et al., 2018). In recent years, miRNAs as one of an epigenetics modifications have attracted attention for its role in atherosclerosis. In addition, it has been reported that miRNAs modulate gene expression at the post-transcriptional level either by inhibiting messenger RNA (mRNA) translation or by promoting mRNA degradation (Michlewski & Cáceres, 2019). Previous studies have reported that miR-126 could inhibit the production of inflammatory cytokines through MAPK signaling pathway to prevent atherosclerosis progression and development (Hao & Fan, 2017). miR-195, a member of the miR-15 family, was often downregulated in various types of cancers, such as breast cancer and non-small cell lung cancer However, more investigations are needed to fully uncover the detailed underlying mechanism. Similar to protein-coding genes, miRNAs expression is also controlled by epigenetic modifications, including DNA methylation, histone modifications, and chromatin remodeling, which are susceptible to the environment factors (Yao et al., 2019). DNA methylation is one of the most well characterized epigenetic phenomena, which is typically mediated by DNA methyltransferases (DNMTs, including DNMT1, DNMT3a and DNMT3b) and requires the presence of Sadenosylmethionine (SAM), a methyl donating compound that delivers methyl groups to maintain other metabolic reactions in mammals (Horvath & Raj, 2018). Hcy is an intermediate product released during methionine cycle. Methionine combines with adenosine to produce SAM, which is converted to SAH and further hydrolyzed to Hcy and adenosine after releasing its methyl group, suggesting the critical importance for Hcy-methionine cycle in the maintenance of a balanced amount of SAM, and normal DNMTs activity (Greco et al., 2020). We sought to understand the miR-195-3p regulation mechanism in Hcyinduced atherosclerosis, we found that the promoter of miR-195-3p contains one CpG island and 42 CpG sites. Moreover, promoter DNA hypermethylation has been detected due to the increased binding of DNMT3a at its promoter after Hcy treatment. This point was supported by our previous study that Hcy-induced DNA hypermethylation could trigger autophagy by suppressing CFTR expression in the liver of ApoE −/− mice (Yang et al., 2018).
Interestingly, Hcy has been reported to induce differential levels of DNA methylation, range from hyper-to hypomethylation, which could be explained by other mechanisms in regulating DNA F I G U R E 5 Sp1 increases the transcription of miR-195-3p by directly binding to its promoter. (a) The expression of Sp1 in the aorta of ApoE −/− mice were measured by qRT-PCR and western blot. (b) qRT-PCR and western blot determined the expression of Sp1 in macrophages treated with Hcy or/and mithramycin A (MTM, Sp1 specific inhibitor). (c) The promoter activity of miR-195-3p, the expression of miR-195-3p and pri-miR-195-3p were measured by luciferase reporter assay and qRT-PCR after cells treated with Hcy or/and MTM. (d) The levels of miR-195-3p methylation in promoter was analyzed by BSP, and (e) the levels of H3K9ac at miR-195-3p promoter were detected by ChIP, and (f) the promoter activity of miR-195-3p, the expression of miR-195-3p and pri-miR-195-3p were measured by luciferase reporter assay and qRT-PCR, and (g) the secretion and mRNA expression of IL-1β, IL-6, TNFα and IL-31 were determined by ELISA and qRT-PCR in macrophages transfected with the adenovirus expressing Sp1 (Ad-Sp1) or Ad-GFP in presence of Hcy. (h) The methylation status of the miR-195-3p promoter, and (i) H3K9ac occupancy at miR-195-3p promoter were detected by ChIP, and (j) the promoter activity of miR-195-3p, the expression of miR-195-3p and pri-miR-195-3p were measured by luciferase reporter assay and qRT-PCR, and (k) the IL-1β, IL-6, TNFα and IL-31 secretion and mRNA expression were determined by ELISA and qRT-PCR in macrophages transfected with siRNAs against Sp1 (si-Sp1) or control siRNA (si-NC) in presence of Hcy. (l) Sp1 binding at miR-195-3p promoter in macrophages was assessed by ChIP using an anti-Sp1 antibody. The Sp1-enriched miR-195-3p promoter containing the putative Sp1 binding sites was amplified by PCR. (m) Sequential deletion and substitution mutation analyses identified Sp1-responsive regions in miR-195-3p proximal promoter region, the relative luciferase activities of miR-195-3p promoter in macrophages co-transfected with Ad-Sp1 and solely or serially truncated Sp1 binding sites at miR-195-3p promoter were detected using luciferase reporter assay. Data were presented as mean ±SD. *p < 0.05, **p < 0.01; # p < 0.05, ## p < 0.01 methylation (Mandaviya et al., 2014). Recently, it has been shown that DNMT1 recruited HDAC1 to repress gene expression or to form heterochromatin structures (Ma et al., 2021). Some studies supported that DNA methylation can provide binding sites for methyl-binding domain proteins (MBDs), which can interact with HDACs to mediate gene repression (Pandey et al., 2014). Besides, Hcy can induce H3K9ac and inhibit the expression of HDAC1 in foam cells (Zhao et al., 2017). In agreement with these observations, F I G U R E 6 Sp1 inhibits HDAC11 binding to miR-195-3p promoter by binding to DNMT3a. (a) The enrichment of DNMT3a or HDAC11 at miR-195-3p promoter were determined by ChIP in macrophages after transfected with si-NC or si-Sp1. (b) ChIP analysis with anti-Sp1 antibody was eluted and subjected to a Re-ChIP using either the anti-DNMT3a or HDAC11 antibody in macrophages treated with Hcy. Representative gels shown the PCR product performed with primers flanking the Sp1 binding regions at miR-195-3p promoter. (c) The interaction between Sp1 and DNMT3a was analyzed by Co-Immunoprecipitation (Co-IP) in macrophages treated with Hcy. (d) Immunofluorescent images shown the co-localization of Sp1 (green) and DNMT3a (red) in Hcy-treated macrophages using laser confocal microscopy, and nuclei were stained with DAPI (blue). Scale bar =50 μm. (e) Schematic diagram depicting the structure of Sp1 and truncation mutants of the GST-tagged Sp1 subunit. (f) GST pull-down assays were performed using recombinant GST, GST-(261-495), GST-(496-610) and GST-(619-785) to pull-down DNMT3a. Bound and 10% of input DNMT3a were detected by western blot using an anti-HA antibody. Specific GST-Sp1 mutants are indicated by red arrows. (g) The enrichment of HDAC11 and H3K9ac at miR-195-3p promoter in Hcy-treated macrophages infected with wild-type (WT-Sp1) and mutant (Δ496-610) was assessed by ChIP assay using an anti-HDAC11 and H3K9ac antibody. (h) The expression of miR-195-3p in Hcy-treated macrophages infected with wild-type (WT) and mutant (Δ496-610) adenoviral vector expressed Sp1 using qRT-PCR. Data were presented as mean ±SD. *p < 0.05, **p < 0.01; # p < 0.05 our results found that Hcy induces H3K9 deacetylation and HDAC11 binding at miR-195-3p promoter, indicated cooperative interaction between DNA methylation and histone acetylation in the mediation of transcriptional repression. Nevertheless, although the DNA methylation and H3K9ac were found to be involved in the reduction of miR-195-3p in Hcy-mediated atherosclerosis, we cannot completely exclude the possibility that the effects of Hcy on other mechanisms to alter miR-195-3p expression during atherosclerosis.
Therefore, more delicate investigations are needed for a complete understanding of the mechanisms of miR-195-3p in Hcy-mediated atherosclerosis.
Gene expression program is largely regulated by the inducible expression of transcription factors in response to the specific stimuli.
DNA methylation and histone acetylation occurring in a regulatory region could work together to alter the binding profile of transcription factors and the specific gene expression (Cusack et al., 2020).
Sp1 is one of the transcription factors that regulate the inflammationrepair process. Previous study reported that modulation of NF-κB and Sp1 expression may be important targets for the prevention and treatment of atherosclerotic vascular disease (Lee et al., 2013). In this study, we found two putative Sp1 binding sites at miR-195-3p promoter, which could recruit DNMT3a and HDAC11. Meanwhile, our work further demonstrates that Sp1 suppress macrophage inflammation which contribute to the overall decrease in plaque formation.
Since Hcy is involved in the methionine cycle by using as a methyl group carrier, in which the methyl group of Hcy is transferred to not only DNA but also histones and other proteins, the presence of methyl group at a specific CpG dinucleotide site may directly prevent DNA from recognizing and binding to transcription factors (Lai et al., 2020). Furthermore, methylated DNA can prevent the binding of HDACs, which can catalyze histone modification and form chromatin-remodeling complexes, leading to altered structure of chromatin and transcription repression (Pasyukova et al., 2021).
Epigenetic regulation on transcription is initiated by specific transcription factors that bind to the promoters of genes upon altering transcription. These factors promote changes in chromatin structure and binding of RNA polymerase II (RNAPII) to promote future transcriptional reactivation (Álvarez-Errico et al., 2015;Le et al., 2020). These findings correlate with DNA methylation and chromatin changes, this prompted us to study the specific mechanism of Sp1 regulating miR-195-3p transcription. The present study further uncovered that the binding region between Sp1 and DNMT3a are necessary for HDAC11 binding to the miR195-3p promoter. Thus, these results indicated that the therapeutic promises of miR-195-3p in treating Hcy-induced atherosclerosis and clarified the importance of Sp1 in the regulation of DNA methylation and H3K9ac to be involved in Hcy-mediated miR-195-3p downregulation.
In summary, our results suggested that Hcy-mediated miR-195-3p promotes macrophage inflammation and the progress of atherosclerosis by targeting IL-31. More importantly, DNA methylation and H3K9 deacetylation catalyzed by DNMT3a and HDAC11 are responsible for the downregulation of miR-195-3p, and Sp1 interacts with DNMT3a was essential for HDAC11 to bind to miR-195-3p promoter. Given the reversibility of epigenetic changes, our study aimed to restore miR-195-3p expression to be beneficial for cardiovascular patients, particularly for HHcy-related atherosclerosis.

| Animal treatment
6-week-old apolipoprotein E knockout (ApoE −/− ) male mice purchased from the Laboratory Animal Center of Peking University Health Science Center (Beijing, China) were housed individually at room temperature, provided food and water ad libitum. After 2 weeks adaption, they were randomly divided into NC group (regular diet: 20% protein, 4.5% fat, 55.5% carbohydrate) and HMD group (high methionine diet: 1.7% methionine). Frozen sections of aortic root and the entire aorta were stained with Hematoxylin-eosin (HE) or Oil red O for quantification of the lesion area. Aortic lesion size of each animal was obtained by averaging the lesion areas in three sections from the same mice. Boundary lines were drawn around these regions, and the area measurements were obtained by ImageJ software.  or Lv-miR-neg were injected to HMD fed-ApoE −/− mice via tail vein, and once every 4 weeks. The titer of the virus was 2 × 10 9 TU/ml, and an equal volume of 50 μl phosphate buffered solution (PBS) was used as vehicle control. After 20th weeks, mice were killed and blood was

| Ultrasound biomicroscopy (UBM)
The mice were anesthetized and laid supine. The warm ultrasonic transmission gel was placed on the chest. The mechanical transducer (Vevo 770, Visual Sonics, Toronto, Canada) was set at 40 MHz, and the Real-Time Micro Visualization Scanhead (RMV 704) was used for intima-media thickness (IMT) and blood velocity measuring. All mice were measured at least 3 times, and the measurement data were statistically analyzed.

| Immunofluorescence
Frozen aortic roots sections or macrophages were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.2% Triton X-100 for 15 min. After blocking with 10% goat serum, the tissue sections or macrophages were indicated with primary antibodies at 4°C overnight. After washing with PBS for 3 times, tissue sections or macrophages were incubated with fluorescein-conjugated secondary antibodies (Santa Cruz Biotechnology) for 2 h at room temperature. The cell nuclei were stained by incubation with 4, 6-diamidino-2-phenylindole (DAPI, Bioss, Woburn, MA, USA). The fluorescence was detected and photographed by confocal microscopy (Olympus, Tokyo, Japan). The fluorescence intensity was quantified with ImageJ software, and relative fluorescence intensity was calculated as the targeted fluorescence intensity relative to the DAPI fluorescence intensity. Besides, the co-localization analysis was performed using co-localization plug-in for ImageJ after converted the individual fluorescent channel images to 8-bit grayscale.

| In situ hybridization
For the detection of miR-195-3p in atherosclerotic plaques, frozen aortic roots sections were subjected to in situ hybridization. In brief, denature Denmark) at 90°C and dilute the probe in Exiqon ISH buffer. Then tissue sections were treated with the slides with freshly prepared acetylation buffer for 7 min and washed 3 times in RNAse-free PBS at room temperature. Remove PBS and immediately add 50 μl probe solution to slides and start a preset hybridization program for 1 h at 60°C.

| miRNAs microarray
Total RNA from aorta of ApoE −/− mice were isolated using TRIzol Reagent (Invitrogen, Eugene, OR, USA) and the RNA integrity was examined by electrophoresis. Following labeled with Hy3 by using the miRCURY array labeling kit (Agilent Technologies, Santa Clara, CA, USA) and hybridization to a miRCURY LNA microRNA array (Agilent Technologies, Santa Clara, CA, USA). Microarray images were acquired by the Agilent G2565CA scanner (Agilent Technologies, Santa Clara, CA, USA) and analyzed by Agilent Feature Extraction Software (version 10.7). The differentially expressed miRNAs were screened out with |Log 2 Fold Change|≥2 and p < 0.05 as the thresholds. TargetScan, miRWalk and miRbase were applied to predict the target genes of miRNAs, respectively. Common genes predicted different algorithms were retained as target genes.

| Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from aorta tissue or macrophages and then reverse-transcribed into cDNA by using the Reverse Transcription Kit (Takara, Dalian, China). qRT-PCR analysis was performed using the FTC3000 qRT-PCR detection system (Funglyn Biotech Inc, Canada), and all reactions were performed thrice. Bulge-Loop™  Table 1. Furthermore, the mRNA and miRNA expression were normalized using GAPDH or U6 as reference gene, and the relative gene expression was calculated using the 2 −ΔΔCt method.

| Western blot
Macrophages and aorta tissue were lysed in cold lysis buffer. The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocked with 5% non-fat milk, the membranes were incubated with indicated primary antibodies at 4°C overnight. After 3 times washing, the membranes were incubated with horseradish peroxidase (HRP)-lighted secondary antibodies for 1 h, and the protein expression was then detected by using the chemiluminescence kit (KeyGEN, Nanjing, China).
Optical density of bands was quantified using densitometry and normalized to a β-actin loading control.

| Bisulfite sequencing PCR (BSP)
Genomic DNA was purified from macrophages and tissues using a QIAamp DNA Mini Kits (Qiagen, Hilden, Germany).

| Luciferase reporter assay
Dual-luciferase reporter assay was used to evaluate the activity of miR-195-3p promoter and the direct downstream target IL-31. For promoter activity assay, the different fragments of miR-195-3p promoter were inserted into the pGL3-control plasmid, and the wild-type (WT) and mutant-type (Mut) 3′-UTR of IL-31 were cloned and inserted into pGL3-control vector, respectively. Then, the HEK293T cells were plated in 24-well plates and co-transfected with above reporter constructs, renilla luciferase expression vector, an internal control (pGL3basic) or miR-195-3p mimics. Luciferase activities were measured by using the dual-luciferase reporter assay system (Promega, USA) 48 h after transfection according to the manufacturer's protocol.
Normalized firefly luciferase activity was compared between groups.

| Chromatin immunoprecipitation (ChIP) and
Re-ChIP assay Antibody-chromatin complexes were then pulled down with protein A-sepharose beads that had been blocked with BSA and salmon sperm DNA. After washing, the immunoprecipitated DNA was eluted and resolved electrophoretically on a 2% agarose gel. In Re-ChIP assays, the complexes were eluted from the beads by incubation with 10 mM DTT at 37°C for 30 min after washing the protein-G-Sepharose beads from the primary IP, with a 50-fold dilution with 1× sonication buffer, the eluates were subjected to IP with the second antibody. Subsequently, the immunoprecipitated DNA was subjected to PCR analyses by using primers targeting the promoter regions of miR-195-3p.

| Co-Immunoprecipitation (Co-IP)
Cells washed for 3 times with PBS were lysed in cold lysis buffer followed by centrifugation at 12,000g for 15 min. The cell lysates were incubated with indicated antibodies for 1 h and then incubated with protein G-Dyna beads (Thermo Fisher, 10003D) for 30 min at 4°C. The agarose beads were washed 3 times with cold lysis buffer, followed by elution with 40 μl of protein lysis buffer. Then, 10 μl of 5× loading buffer (Beyotime, Shanghai, China) was added, and the mixture was boiled for 5 min and subjected to western blot.

| Statistical analysis
GraphPad Prism 6.0 software was used for statistical analysis. All data are presented as Mean ±SD from at least three independent experiments. Differences between groups were analyzed by one-way analysis of variance combined with the student's-Newman-Keuls post hoc test or the nonparametric Mann-Whitney U test. Pearson test analyzed the correlation between serum Hcy levels and atherosclerotic lesion area. The values of p < 0.05 was considered as statistically significant.

ACK N OWLED G EM ENTS
This work was supported by the grants from the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest. the study, organized, designed, and wrote the paper. All authors reviewed and approved the final submitted manuscript.

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