Research on the correlation between methyltransferase METTL7B and atherosclersosis

doi:10.21203/rs.3.rs-2755788/v1

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Research Article

Research on the correlation between methyltransferase METTL7B and atherosclersosis

https://doi.org/10.21203/rs.3.rs-2755788/v1

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Background

Atherosclerosis (As) is a serious threat to human health. Although glucose balance, lipid metabolism, inflammation and hypertension have been found to be closely related to AS, it is crucially unclear, whether methyltransferase (METTL) family members are involved in the occurrence and development of AS.

Methods

The differentially expressed genes of METTLs in AS and normal blood vessels in GSE43292 and GSE100927 databases were analyzed. Random forest screening was used to screen marker genes, and the intersection genes in two databases were selected, while GSE28829/GSE41571 and clinical tissue samples were used for verification. The databases were further used to analyze the tissue and cell localization of marker genes and their correlation with lipid metabolism and immune cell infiltration.

Results

There were 7 and 17 differentially expressed METTL genes in GSE43292 and GSE100927 databases, respectively. 7 and 10 AS risk genes were selected by random forest screening. METTL7B and METTL5 were verified as the intersection marker genes using GSE28829, GSE41571 database and clinical AS samples. Compared with the control group, the expression of METTL7B in advanced AS, AS rupture plaque and clinical heavy load plaque tissue significantly increased, and the ROC curve analysis showed that the AUC of METTL7B in GSE28829 and GSE41571 was greater than 0.9. In addition, it was found that METTL7B was significantly correlated with lipid metabolism-related genes, and METTL7B could promote the formation of lipid droplets. METTL7B was positively correlated with immune cell infiltration and macrophage-mediated efferocytosis. Drugs such as FAD, Pralatrexate and Indinavir can act on METTL7B. This study reveals a new mechanism for the occurrence and development of AS thereby providing a potential target for the treatment of AS.

Conclusion

METTL7B can be used as a predictor and therapeutic target for AS.

atherosclerosis

methyltransferase

immune cell infiltration

lipid

efferocytosis

The METTL-like protein forms a 7β chain methyltransferase family, composed of 33 members. It has S-adenosylmethionine binding domain and can mediate the methylation modification of DNA, RNA and protein [1-2]. With the rise of RNA methylation study in recent years, the research of METTL family members has also received more and more attention. Methylation of METTL protein contributes to epigenetics and, in the case of RNA modification, contributes to the apparent transcription regulation of various biological processes. Although the importance of METTLs has been recognized, their function has not been fully elucidated. At present, there are few studies on METTLs family, mainly focusing on tumor [3-5]. Therefore, it is of great significance to further study the functions of METTL family members.

The formation of atherosclerotic plaque is closely related to hypertension, hyperlipidemia, hyperglycemia and other factors. Hypertension, hyperlipidemia and diabetes will cause certain damage to the blood vessel wall of patients, leading to deformation, necrosis and shedding of vascular endothelial cells. After the vascular endothelium is damaged, triglycerides, cholesterol and other substances in the blood will enter the vascular wall through the damaged endothelium and deposit under the vascular endothelium, destructing endothelial structure, making the vascular endothelium thickened and hardened. At the same time, platelets that play the hemostatic function will also quickly adhere and gather at the damaged area, thus gradually forming atherosclerotic plaque [6-7]. More and more evidences show that epigenetics is closely related to AS [8-9], and METTL, as a new member of methylation modification, has attracted more and more attention in methylation modification, especially in RNA methylation modification. This study analyzes the correlation between METTL family members and AS, and explores its role and possible mechanism in AS.

GEO database data downloading and processing

Filter AS-related chip data in GEO database, and download GSE43292 [10] and GSE100927 [11]. The pathological number, chip platform and sample information contained in each database are shown in Table 1. In order to eliminate the differences between batches, we use the "SVA" package in R software for standardization. The specific workflow is shown in Fig.1.

Differential gene analysis

The difference METTL gene in GSE43292 and GSE100927 was analyzed using the limma package in R4.2.1 [2], and the screening criteria was P<0.05. Use the pheatmap package to draw the heat map, and use the ggboxplot package to draw the differential gene box map.

Functional enrichment analysis

The function enrichment analysis of the differential METTL gene in GSE43292 and GSE100927 was performed using the clusterProfiler and DOSE package in R4.2.1.

Screening and validation of critical gene signatures

In this research, two algorithms were used to screen the new key signals of atherosclerosis. They were random forest (RF), minimum absolute contraction and selection operator (LASSO) logical regression. RF uses random Forest package, and LASSO uses glmnet package. Use R software pROC software package to calculate ROC curve and area under curve (AUC), evaluate the diagnostic value of signal for atherosclerosis, and use rmda package to draw nomogram.

Marker gene verification

Leverage VENN (https://bioinfogp.cnb.csic.es/tools/venny/index.html). The VENN diagram is drawn for the overlapping genes of the two algorithms. Use Graphpad prime 8.0 to analyze the expression and AUC of marker genes in GSE28829, GSE41571 and GSE97320.

Main expression and distribution of METTL7B in human tissues and cells

Utilize BioGPS (http://biogps.org/m/gene/71664/) to analyze the expression and distribution of METTL7B in human organs, and further use HPA online analysis website (https://www.proteinatlas.org/) to analyze the cell distribution of METTL7B in various organs.

Correlation analysis of genes related to lipid metabolism

From the gene enrichment analysis website (GSEA, http://www.gsea-msigdb.org/gsea/msig-db/index.jsp), download 174 genes related to lipid balance (GOBP_LIPID_HOMEOSTASIS), screen the differentially expressed genes in GSE42392 and GSE100927 using the limma package in R4.2.1. Draw the heat map using the pheatmap package, draw the Venn map of the intersection gene using VennDiagram, and analyze the correlation between METTL7B and the intersection differential lipid metabolism gene using reshape2.

Immunocyte infiltration analysis

CIBERSORT was used to detect the differentially expressed genes in GSEA42392 and GSE100927 (https://cibersort.stanford.edu/). The expression difference of immune cells in normal blood vessels and atherosclerotic plaque blood vessels was analyzed. The correlation between METTL7B and differential immune genes was analyzed by Spearman's rank in R software.

Correlation analysis of cell burial related genes

According to the literature [11], select the genes related to cell burial (Table 2), screen the differentially expressed genes in GSE42392 and GSE100927 using the limma package in R4.2.1, draw the heat map using the pheatmap package, draw the Venn map of the intersection gene using VennDiagram, and use reshape2 to analyze the correlation between METTL7B and the intersection differential lipid metabolism gene.

Screening of small molecular compounds

Use Discovery Studio 2019 software for molecular docking. Through PDB database (https://www.rcsb.org/), download the 3D structure of METTL7B. Using zinc small molecular structure database (https://zinc.docking.org/)to download the small molecular structure approved at home and abroad to obtain the drug molecular data set. Then use Discovery Studio 2019 simulation software to carry out routine pretreatment of molecular structure, use DOCK ligand (libDock) to conduct batch rigid molecular docking, and screen the small molecular structure with the highest binding score with METTL7B protein.

Detection of the effect of METTL7B on lipid droplet synthesis by oil red O staining

LO2 hepatocytes in logarithmic growth phase (1×10⁵/well) were inoculated into the 6-well plate, and on the next day, the interference group was transfected with siNC and siMETTL7B with 50nM/well respectively. The METTL7B overexpression group was added with 0 and 1 ul of METTL7B-GFP virus respectively. After 48 hours, 0.5mM oleic acid treatment for 12 hours, oil red O staining was used to detect the effect of METTL7B on fat synthesis.

Immunohistochemistry

Collect tissue samples of normal blood vessels and autopsy blood vessels with atherosclerosis, and detect the expression of METTL7B by immunohistochemistry, referring to our previous method, METTL7B (Protein, 17001-1-AP, 1:200). The human tissue specimens involved in this article are from the Forensic Judicial Appraisal Institute of Hubei University of Medicine and have been reviewed by the Ethics Committee of Hubei University of Medicine(2022-RE-030).

ELISA

Collect blood samples from patients with hyperlipidemia and healthy people, and detect the content of METTL7B in blood by ELISA. The patients were divided into control group and hyperlipidemia group according to the blood lipid level. At the same time, clinical data were collected, and the correlation between METTL7B and clinical indicators was analyzed with Pierson.

Western blot detection

The use of animals and all animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Hubei University of Medicine. Extract the total protein from the main organs of 8-week-old FVB mice, including heart, liver, spleen, lung and kidney, use SDS-PAGE to isolate the protein, transfer the protein to PVDF membrane, and then seal it with 5% BSA at room temperature for 30 minutes, incubate the METTL7B polyclonal antibody (Protentech, 17001-1-AP, 1:500) overnight at 4 ℃, incubate the HRP-labeled sheep anti-rabbit second antibody (Beyotime, A0208, 1:1000) at room temperature for 2 hours followed by colorization with ECL chemiluminescence kit (Beyotime, P0018FM).

Statistical analysis

R software (version 4.2.1) was used for statistical analyses and the visualization of results. A p-value < 0.05 was considered to indicate statistical significance. Correlation coefficients with an absolute value greater than 0.2 and p < 0.05 were deemed to be significant.

1. Differential gene expression and function analysis of METTL in AS plaque

Seven and 17 differentially expressed METTL genes were screened from GE43292 and GSE100927, respectively. Among them, there were 2 differentially up-regulated genes and 5 down-regulated genes in GSE43292, 8 differentially up-regulated genes and 9 down-regulated genes in GSE100927. The differential gene thermogram and boxplot were shown in Figure 2A-D. Correlation analysis showed that the difference METTL gene in GE43292 and GSE100927 had good correlation, with correlation coefficient between 0.1-0.6 (Figure 2E-F). GO enrichment analysis showed that the differential genes were mainly involved in the methylation biological processes of RNA, ncRNA, mRNA, macromolecules, proteins, and so on. They mainly existed in the complex with mRNA modification, methyltransferase complex and lipid droplets, and mainly played a variety of methylation transfer activities (Fig. 3A-B). KEGG was not enriched in differential gene-related signal pathway.

2. Screening of critical signatures

Utilize RF algorithm and LASSO logistic regression algorithm analysis to screen the critical marker genes. The results showed that 7 genes were identified with RF algorithm in GSE43292 (Fig. 4A-B). ROC curve showed that the AUC value of the model was 0.812 (Fig. 4C), and the AUC values of the 7 marker genes were between 0.664-0.784. Fig. 4D&E showed the nomograms of the model. Because GSE100927 has a large number of samples, we first use machine learning to screen the differential METTL marker genes. The results showed that RF model has a better evaluation function. The AUC values of the 4 models were 0.990, 0.970, 0.965 and 0.970 for RF, SVM, XGB and GLM respectively (Fig 4A-C). The top 10 METTL genes in RF model were METTL13, METTL10, METTL21A, METTL12, METTL11A, METTL8,METTL5, METTL17, METTL7B and METTL21C (Fig 4D). METTL7B and METTL5 were overlapping marker genes of GSE43292 and GSE100927 (Fig 4E).

3. Verification of METTL7B and METTL5 in atherosclerosis

To verify whether METTL7B and METTL5 play a role in the development of atherosclerosis, we first analyzed their expressions in GSE28829 and GSE41571. The results showed that the expression of METTL7B was significantly higher in both advanced AS (GSE28829) and ruptured plaque (GSE41571) than that in the control groups (Fig 5A, p<0.05), but there was no significant difference in the expression of METTL5 (Fig 5A, p>0.05). ROC curves showed the AUC for METTL7B in GSE28829 and GSE41571 were 0.9372 and 0.9333 respectively (Fig 5B). Figure 5C shows the role of METTL7B in the development of AS. Therefore, in the follow-up, we will focus on analyzing the functions of METTL7B.

For further support, we measured the expression of METTL7B protein in human atherosclerosis. Immunohistochemistry showed that the expression of METTL7B in AS was significantly higher than that in normal group, and it was mainly located in foam cells (Fig 6A-B). Immunoblotting showed that the expression of METTL7B in vascular endothelial cells in ox-LDS-induced foaming was significantly higher than that in the control group (Fig. 6C-D). To sum up, the results further support that METTL7B, as a potential diagnostic marker, was located in macrophages in atherosclerosis.

4. Correlation analysis between METTL7B and lipid metabolism

Firstly, the bioGPS database was used to analyze the organ distribution of METTL7B mRNA in human tissues. It was found that the distribution of METTL7B mRNA in the coronary artery was very low, while the distribution of METTL7B mRNA in the liver was the highest (Figure 7A). Further use of The human protein atlas database (https://www.proteinatlas.org/) to analyze the protein expression of METL7B in human tissues, we found that it was the most expressed in the liver (Fig. 7B). After isolating various tissues and organs of mice, Western detection found that METTL7B was mainly expressed in the liver, but very low or no expression in other organs, such as kidneys, skin, etc. (Fig. 7C). In addition, we also used Human protein atlas to analyze the expression of METTL7B in various liver cells. It was found that METTL7B was mainly expressed in hepatocytes, and only a small amount was expressed in kupffer cells and immune cells (Fig. 7D).

Since AS is closely related to lipid metabolism, and liver is the most important organ for lipid metabolism, and GO analysis also shows that METTL7B is located in lipid, it is speculated that METTL7B may participate in lipid metabolism balance. Therefore, we analyzed the differential expression of lipid metabolism genes in GSEA database in GSE43292 and GSE28829 (Fig. 7D), and selected 50 differentially expressed genes related to lipid balance. Correlation analysis shows that METTL7B has a good correlation with the above 50 genes related to lipid metabolism, among which there was a significant correlation with APOE, ADCK1, ABCG1, CAV1, FUNDC2, SCARB1, POLD1, PPARG, PPKAA1, MED13, etc. (Figure 8).

In this study, we also selected blood samples from people with hyperlipidemia and normal blood lipids, and found that the blood level of METTL7B in people with hyperlipidemia was significantly higher than that in the normal control group, and METTL7B was positively correlated with blood lipidsTG and HDL-C (Figure 9A). In addition, we also interfered and over-expressed the LO2 METTL7B gene in human hepatocytes and used oleic acid to induce the formation of fat droplets. It was found that the over-expression of METTL7B could promote the formation of fat droplets compared with the wild-type group, while its knockdown could inhibit the formation of fat droplets (Fig. 9B), indicating that METTL7B could affect fat metabolism.

We collected three AS and three normal blood vessel specimens respectively. Immunohistochemical detection showed that METTL7B expression in plaque vessels was significantly higher than that in normal vessels, and it was mainly distributed in vascular endothelium and foam areas (Figure 1C). This result suggests that METTL7B may be closely related to AS, so we have downloaded the data of different progression stages of AS from the GEO database, namely GSE28829 (including 13 early plaque and 16 late plaque tissue chips), GSE41571 (including 6 stable plaque and 5 ruptured plaque tissue chips), GSE41571 (including 6 non-atherosclerotic and 5 atherosclerotic blood sample chips), and analyzed the expression of METTL7B. It was found that METTL7B was more expressed in advanced plaque and ruptured plaque. In addition, METTL7B was also found to be more expressed in the blood of patients with AS (Fig. 2). These results indicate that METTL7B may participate in the occurrence and development of AS.

5. Correlation analysis between METTL7B and immune cell infiltration

Because METTL7B is also expressed in blood vessels, and immuno -histochemistry also shows that it is mainly located in foam cells of AS plaque, the single cell sequencing analysis of The human protein atlas database was performed showing that METTL7B was mainly located in macrophages (Figure 10A) in blood vessels. In order to further analyze whether METTL7B is related to immune cell infiltration, it was found that METTL7B was significantly correlated with Macrophages M0, B cell memory, T cells CD4 memory resting, and T cells CD (Fig. 10B-C, P<0.05).

6. Correlation analysis between METTL7B and eferocytosis

There are 5 and 6 differentially expressed genes of 14 eferocytosis related genes in GSE43292 and GSE100927, respectively, including CXCL1, CALR, ICAM3 and MFGE8. Correlation analysis showed that METTL7B had a significant positive correlation with CALR, ICAM3 and CXCL1 in GSE43292 and GSE100927 databases, and a significant negative correlation with MFGE8 in GSE100927 (Figure 11B). These results suggest that METTL7B may be related to macrophage-mediated cell burial.

7. Screening small molecule compound targeting MTTL7B

Through the docking analysis of the 3D structure of METTL7B and the small molecular compounds in the zinc small molecular structure database, we found that the top 10 small molecular compounds with the binding ability of METTL7B were shown in Table 3, namely, ZINC000008215434, ZINC000001536109, ZINC000022448696, ZINC00000200505305, ZINC000038144598, ZINC000001529323, ZINC000002036915, ZINC000053073961, ZINC000004099104 and ZINC000030731084. Their corresponding molecular names are FAD, Pralatrexate, Indinavir, 5-Methyltetrahydrolic acid disodium salt, Alexidine dihydrochloride, Methodrexate, Aminopterin, Antrafenine, SN38 glucuronide, APC.

Coronary atherosclerotic heart disease (referred to as "coronary heart disease" for short) is a kind of heart disease that causes myocardial cell necrosis due to the occurrence of vascular stenosis or occlusion on the basis of coronary atherosclerosis [1]. Coronary heart disease has brought huge health and economic burden to individuals and families. It is a major public health problem that China needs to face. Therefore, it has important social significance and economic benefits for further studying the pathogenesis of coronary heart disease and providing a new direction for the treatment of coronary heart disease.

Epigenetics refers to a biological discipline in which the gene function of the body is reversible and heritable without changing the gene sequence. It mainly includes DNA methylation, histone modification and non-coding RNA. Epigenetics is closely related to the occurrence and development of many diseases, such as malignant tumors, nervous system diseases and metabolic diseases [12–14]. In recent years, more and more studies have shown that RNA methylation also plays an important role in the field of coronary heart disease. Some studies have shown that m6A modification is closely related to lipid metabolism. When analyzing the m6A methyl in the mouse model of fatty liver induced by high-fat diet, it was found that the methylation and hypomethylation genes were mainly enriched in lipid metabolism or metabolism-related processes [15]. After improving the effect of curcumin in diet on lipid metabolism disorder induced by lipopolysaccharide, the expression levels of METTL3, METTL14, ALKBH5, FTO and YTHDF2 were also affected, and the abundance of m6A methylation increased [16]. In addition, some members of m6A modification have also been confirmed to regulate lipid metabolism in an m6A modification dependent manner. For example, PPARα, which is an important member of PPARγ pathway, involved in lipid metabolism, can be directly regulated by METTL3 or YTHDF2 [17]. In addition, FTO not only acts as a splicing regulator of its adipogenesis-related target genes, but also regulates liver fat accumulation through RNA demethylation [18].

In this Research, we used a series of AS-related databases to analyze the correlation between METTL family genes and AS, which was verified using clinical blood and AS tissue samples. We found that the expression level of METTL7B in vascular tissue of patients with AS was significantly higher than that of normal control group, and higher in advanced AS and ruptured plaque. ROC analysis METTL7B can be used as the evaluation index of AS diagnosis and risk prediction. METTL7B, as an RNA methyltransferase, is still in the initial stage of functional certification. The current research reports are also very limited, and mainly focus on the correlation with tumor. However, it can be predicted that it should have strong biological functions in the body.

The formation of AS plaque is closely related to hypertension, hyperlipidemia, hyperglycemia and other factors. So how does METTL7B respond to the occurrence and development of AS? To understand this problem, first of all, we should clarify the distribution of METTL7B in tissues and cells. Through bioinformatics analysis, we found that its expression level in blood vessels is low, mainly in stem cells in the liver, and liver cells are the most important organ of lipid metabolism. Therefore, it may be related to abnormal lipid metabolism. Isabel Baiges et al [19], found through bioinformatics analysis that according to the liver proteomics of rats fed high-fat food, grape seed proanthocyanidins reduced fat production, and revealed some new targets related to the reversal of symptoms of metabolic syndrome, including METTL7B, SLC30A1, CD36, GFPT1, etc. This result supports our reasoning. Therefore, we further analyzed the correlation between METTL7B and lipid metabolism genes, and found that it was significantly correlated with APOE, ADCK1, ABCG1, CAV1, FUNDC2, SCARB1, POLD1, PPARG, PPKAA1, MED13, etc. It suggests that METTL7B may promote the occurrence and development of AS by regulating these genes.

AS is a lipid-driven chronic inflammatory disease, which usually forms plaques in large and medium-sized arteries, and is the main cause of ischemic heart disease and stroke [20]. The immune system is one of the main regulatory systems in the occurrence and progression of atherosclerosis. HPA single cell sequencing analysis showed that METTL7B was mainly located in macrophages in blood vessels, which was also confirmed by immunohistochemistry in this study. Macrophages play a crucial role in the occurrence and development of AS. They can not only phagocytose lipids to form foam cells thereby promoting the occurrence of AS, but also mediate plaque rupture through inflammatory reaction. In addition, macrophages can also bury the apoptotic cells in the plaque. Therefore, this study further analyzed whether METTL7B was related to immune cell infiltration, and found that METTL7B was significantly correlated with Macrophages M0, B cell memory, T cells CD4 memory resting, and T cells CD. It suggests that METTL7B may participate in the immune inflammatory reaction in the process of AS by regulating the polarization of macrophages.

Effectocytosis refers to the process of phagocytic cells clearing programmed death cells, which is of great significance for maintaining the balance of the body [21]. In most tissues, cell burial is accomplished by professional phagocytes (macrophages and dendritic cells) or non-professional phagocytes (epithelial cells and fibroblasts) near dying cells. Cell burial can prevent the secondary necrosis of dying cells from releasing harmful cell contents (such as oxides and proteases) that may cause inflammation. The dysfunction of macrophage burial function cannot clear the apoptotic cells in time, which can lead to secondary cell necrosis and the release of pro-inflammatory cytokines, thus promoting the development of AS [22]. This study found that multiple cell burial genes were differentially expressed in AS, and METTL7B was significantly positively correlated with CALR, ICAM3 and CXCL1 in GSE43292 and GSE100927 databases, and significantly negatively correlated with MFGE8 in GSE100927. In addition, it was also found that interference with METTL7B expression could promote macrophage-mediated endothelial cell burial. The above results suggest that METTL7B can promote the occurrence and development of AS by inhibiting macrophage-mediated cell burial.

Although great progress has been made in the treatment of AS, AS and its cardiovascular and cerebrovascular diseases are still the top one killer threatening human health. Exploring new molecular mechanisms and exploring new therapeutic directions are still the focus of scientists. In this study, we found that FAD, Pralatrexate, Indinavir, 5-Methyltetrahydrolic acid disodium salt, Alexidine dihydrochloride, Methotrexate, Aminopterin, Antrafenine, SN38 glucuronide, APC, etc. have strong binding force with METTL7B through molecular docking. Most of them are used for clinical treatment of tumors, such as Pralatrexate [23], Alexidine dihydrochloride [24], Methodrexate [25], Aminopterin [26] Indinavir can effectively combat HIV-1 [27]. The main function of APC is antipyretic and analgesic, while Antrafenine [28] can be used to treat acute and chronic gastroenteritis. It is not clear whether these drugs can also target METTL7B to treat AS. A large number of experiments are needed to prove that.

To sum up, this study found that METTL7B was significantly elevated in AS tissues and blood samples, and could be used as a marker of AS and its progress. METTL7B is closely related to lipid metabolism genes and macrophage M0 increasing M1. It is speculated that METTL7B may participate in the occurrence and development of AS by regulating lipid metabolism and immune cell infiltration.

Acknowledgements

We thank Fangcheng Zhu, Hubei University of Medicine, very much, who provided us with blood vessel tissue samples. We also thank Jue Wang, Hubei University of Medicine, very much, who helped us complete immunohistochemistry.

Authors’ contributions

JW, FC conceptualized and wrote the main text and fgures. JX analyzed information and made suggestions for improvement. XYP, YXZ, LNZ, LW and XL revised the overall text to provide accuracy. All authors read and approved the final manuscript.

Funding

This study was supported by grants from the National Natural Science Foundation of China (Grant/Award Number: 82270530), and Hubei Natural Science Foundation Innovation Group Project(Grant/Award Number: 2022-CFA036). Innovative Research Program for Graduates of Hubei University of Medicine (YC2021017, YC2022018, YC2023004, YC2023021). The Scientific and Technological Project of Shiyan City (22Y33). Open Project of Hubei Key Laboratory for Embryonic Stem Cell Research(2022ESOF015).

Availability of data and materials

The datasets supporting the conclusions of this article are available in the GEO (Gene Expression Omnibus) (https://www.ncbi.nlm.nih.gov/geo/) repository. (GSE100927, GSE43292, GSE28829, GSE41571).

Ethical Approval

The human tissue specimens involved in this article are from the Forensic Judicial Appraisal Institute of Hubei University of Medicine and have been reviewed by the Ethics Committee of Hubei University of Medicine(2022-RE-030). The use of animals and all animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Hubei University of Medicine.

Consent for publication

Not applicable.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Table 1.GEO data information

Data	Platform	Sample size	Sample type	Data type
GSE100927	GPL17077	104	AS(69) and control(35) peripheral artery	RNAseq
GSE43292	GPL6244	64	Core(32) and shoulders(32) of the atheroma plaque	Microarray
GSE28829	GPL570	29	early(13) and advanced(16) atherosclerotic plaque	Microarray
GSE41571	GPL570	11	stable(6) and ruptured(5) atherosclerotic plaques	Microarray

Table 2.efferocytosis gene list

LPC

CXCL1

S1P

ATP/UTP

PTDSER

CALR

ICAM3

MFGE8

C1Q

GAS6

TSP1

CD47

C31

CD24

Table 3. The top 10 drug list targeted to METTL7B.

ZINC name	score	Popular Name
ZINC000008215434	189.674	FAD
ZINC000001536109	189.007	Pralatrexate
ZINC000022448696	184.143	Indinavir
ZINC000002005305	183.454	5-Methyltetrahydrofolic acid disodium salt
ZINC000038144598	177.38	Alexidine dihydrochloride
ZINC000001529323	175.815	Methotrexate
ZINC000002036915	175.519	Aminopterin
ZINC000053073961	174.029	Antrafenine
ZINC000004099104	173.01	SN38 glucuronide
ZINC000030731084	171.497	APC

No competing interests reported.

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Research on the correlation between methyltransferase METTL7B and atherosclersosis

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