Coronary heart disease diagnostic agent and application of DYSF gene methylation in preparing coronary heart disease diagnostic agent

Abstract

The present invention relates to the use of a reagent for detecting the methylation level of the DYSF gene promoter region and / or a reagent for detecting the expression level of the DYSF gene in the preparation of a diagnostic reagent for coronary atherosclerotic heart disease (coronary heart disease, CAD). Through the present invention, peripheral blood leukocytes can be used as a detection material to detect the expression level and the methylation level of the promoter region of DYSF, and they are jointly used to diagnose the risk of coronary heart disease in a detection subject.

Description

Technical Field

[0001] The present invention relates to the field of diagnosis and early warning of coronary artery disease (CAD), and more particularly, to the use of a reagent for detecting the methylation level of the DYSF gene promoter region and / or a reagent for detecting the expression level of the DYSF gene in the preparation of a diagnostic reagent for coronary artery disease. Background Art

[0002] The incidence of coronary artery disease continues to rise, but the onset age is decreasing year by year. Traditional risk factors such as hypertension, diabetes, hyperlipidemia, obesity, and smoking have received extensive clinical attention, but microscopic risk factors such as genetic and epigenetic mechanisms have not been fully elucidated. It is widely recognized that vascular aging is caused by the gradual increase in the methylation of the estrogen receptor gene promoter in vascular endothelial cells with age.

[0003] Currently, the diagnosis of coronary artery disease mainly relies on arteriography, which is an invasive method and expensive. However, based solely on the results of blood lipids, blood pressure, and blood glucose, it is impossible to accurately predict the state of coronary atherosclerosis in the tested subjects. In order to increase the positive rate of detection and improve the cost-effectiveness of health economics, it is very important to further screen patients before coronary angiography. Clinically, the markers for early warning of coronary artery disease include blood lipids, blood glucose, CRP, etc. However, in some patients, even with strict control of blood glucose and blood lipids, recurrence of coronary artery disease still occurs, indicating the existence of residual risks, and chronic inflammatory responses may be the main cause of residual risks. CRP is the only generally accepted inflammatory index, but it mainly predicts acute cardiovascular events. Currently, there is still a lack of a marker for predicting the severity of coronary atherosclerosis or diagnosing whether the coronary atherosclerotic plaque has reached the diagnostic criteria for coronary artery disease with more than 50% obstruction.

[0004] DYSF is a gene encoding dysferlin, which is a protein composed of 7 β-sheet-rich C2 domains and mainly plays a role in Ca 2+ -dependent cell membrane regeneration and maintenance. Deficiency or mutation of DYSF will lead to DYSF disease, which is a muscle disease that can cause muscle atrophy and walking dysfunction. Its pathogenesis can be partially explained by the dysfunction of DYSF in maintaining cell membrane integrity, resulting in disorder of intracellular redox homeostasis and release of inflammatory cytokines. Further studies have shown that DYSF is also involved in the cell membrane repair and inflammatory regulation of leukocytes and vascular endothelial cells (VECs). And the expression level of DYSF increases as monocytes differentiate into macrophages. At the same time, the complex formed by DYSF, integrin beta3 (ITGB3), and focal adhesion components has been proven to enhance the adhesion ability of monocytes. However, current research has not used DYSF as a marker for early warning of coronary artery disease, nor is there any report on the methylation of the DYSF gene. Summary of the Invention

[0005] In our research, it was found that the expression level of the DYSF gene is highly correlated with the risk of coronary heart disease, and moreover, the methylation level in the promoter region of the DYSF gene affects the expression level of the DYSF gene.

[0006] Based on the above research, the present invention provides the use of a detection agent for the methylation level of the promoter region of the DYSF gene and / or a detection agent for the expression level of the DYSF gene in the preparation of a diagnostic agent for coronary heart disease.

[0007] In a specific embodiment, the sequence of the promoter region of the DYSF gene is as shown in SEQ ID NO.1.

[0008] In a specific embodiment, the amino acid sequence encoded by the DYSF gene is as shown in SEQ ID NO.2.

[0009] The present invention provides a diagnostic agent for coronary heart disease, comprising a detection agent for the methylation level of the promoter region of the DYSF gene and / or a detection agent for the expression level of the DYSF gene.

[0010] The methylation level of the promoter region of the DYSF gene and / or the expression level of the DYSF gene in peripheral blood mononuclear cells are highly correlated with coronary heart disease, and thus the risk of coronary heart disease can be non-invasively evaluated therefrom.

[0011] The present invention also provides a method for changing the ability of monocytes to phagocytose low-density lipoproteins, comprising the step of changing the methylation level and expression level of the promoter region of the DYSF gene in the monocytes.

[0012] In a specific embodiment, the step of changing the methylation level of the promoter region of the DYSF gene in the monocytes by knocking down or overexpressing the DNMT1 gene.

[0013] In a specific embodiment, the amino acid sequence encoded by the DNMT1 gene is as shown in SEQ ID NO.3. Brief Description of the Drawings

[0014] Figure 1 It is a statistical chart of the mRNA expression levels of the DYSF gene, SELL gene, STAT3 gene, and TMX1 gene for coronary heart disease patients and the control group.

[0015] Figure 2 It is a statistical chart of the methylation level of the promoter region of the DYSF gene for coronary heart disease patients and the control group.

[0016] Figure 3 It is a correlation curve between the methylation level of the promoter region of the DYSF gene and the mRNA expression level of the DYSF gene.

[0017] Figure 4 ROC curve statistical charts of the expression levels of DYSF gene, STAT3 gene and TMX1 gene and their combination and the risk of coronary heart disease.

[0018] Figure 5 ROC curve statistical charts of the expression level of DYSF gene, the methylation level of DYSF gene promoter region and their combination and the risk of coronary heart disease.

[0019] Figure 6 Risk maps of the combined model of DYSF, STAT3, and TMX1 expression levels and the combined model of DYSF expression and promoter region methylation level.

[0020] Figure 7 Statistical chart of DYSF promoter region methylation obtained by qMSP for randomly selected 30 coronary heart disease patients and 30 controls.

[0021] Figure 8 Heat map of the expression of DYSF gene, SELL gene, STAT3 gene, CD4 gene, ABR gene, RPS27 gene and TMX1 gene in peripheral blood leukocytes of coronary heart disease patients.

[0022] Figure 9 Oil red O staining photos of cross-sections of aortic sinuses of wild-type mice (WT) and Apoe- / - mice on normal diet (ND) and high-fat diet (HD).

[0023] Figure 10 Statistical charts of the methylation level and gene expression level of the DYSF gene promoter region in peripheral blood monocytes of wild-type mice (WT) and Apoe- / - mice on normal diet (ND) and high-fat diet (HD).

[0024] Figure 11 Statistical chart of the expression level of DYSF gene in DYSF knockdown (DYSF KD) and overexpression (DYSF OE) THP1 cells.

[0025] Figure 12 Statistical chart of SELL mRNA expression level and immunoblotting photos in DYSF knockdown (DYSF KD) and overexpression (DYSF OE) THP1 cells.

[0026] Figure 13 Oil red O staining photos of control group, DYSF knockdown (DYSF KD) and DYSF overexpression (DYSF OE) THP1 cells, and statistical chart of lipid accumulation obtained from the photos.

[0027] Figure 14Photographs of adhesion assays performed on control group, DYSF knockdown (DYSF KD), and DYSF overexpression (DYSF OE) THP1 cells, and statistical graphs of the relative number of cells in each field of view obtained from the photographs.

[0028] Figure 15 Photographs of transwell assays performed on control group, DYSF knockdown (DYSF KD), and DYSF overexpression (DYSF OE) THP1 cells, and statistical graphs of the relative number of cells in each field of view obtained from the photographs.

[0029] Figure 16 Statistical graphs of the expression of DYSF gene in THP1 cells after treatment with PMA or PMA + ox-LDL for a certain period of time.

[0030] Figure 17 Statistical graphs of the methylation of DYSF promoter region (A), mRNA levels of DYSF gene, and immunoblot photographs in DNMT1 gene knockout strains (DNMT1 KD1 and DNMT1 KD2) and DNMT1 gene overexpression strains. Detailed implementation manners

[0031] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0032] 1. DYSF is hypermethylated and highly expressed in patients with coronary heart disease

[0033] We found in the study that there are differences in the expression and methylation of DYSF gene (the amino acid sequence is shown in SEQ ID NO.2, and the promoter region sequence is shown in SEQ ID NO.1) between patients with coronary heart disease and healthy people.

[0034] During the period from December 2018 to July 2019, 139 patients with coronary heart disease were recruited from Zhongnan Hospital of Wuhan University. The diagnosis was based on the presence of more than 50% stenosis lesions in at least one coronary artery in coronary angiography. At the same time, 146 normal people without a history of cardiovascular events were selected as controls. Peripheral blood was collected from 139 patients and 146 controls.

[0035] The mRNA expression levels of DYSF and related core genes were verified in PBL of 120 patients with coronary heart disease and 136 controls. The results are as Figure 1 shown. DYSF (P < 0.0001), SELL (the amino acid sequence is shown in SEQ ID NO.4) (P < 0.0001), and STAT3 (P < 0.0001) were all significantly up-regulated in patients with coronary heart disease, while TMX1 (P < 0.0001) was significantly down-regulated.

[0036] To investigate the potential regulatory mechanisms of DYSF expression, we detected the methylation status of the DYSF promoter region by MDRE-qPCR. The results are as Figure 2 shown, the promoter of DYSF was hypermethylated in patients with coronary heart disease (P<0.0001).

[0037] Analysis of the promoter methylation level of DYSF and its expression level found that the promoter methylation status of DYSF was positively correlated with its expression level, indicating a potential link between the two (r = 0.190, P = 0.0069)( Figure 3 )

[0038] The above experimental evidence shows that the expression level and methylation level of DYSF are likely to be used as diagnostic criteria for coronary heart disease.

[0039] 2. DYSF is an independent risk factor for coronary heart disease

[0040] Binary logistic regression analysis was used to explore the clinical significance of core genes and traditional indicators. During the study of the above 139 patients and 146 controls, through univariate analysis, many variables were identified as risk factors or protective factors for coronary heart disease, including HP history, DM history, TG, HDL-C, FPG, the number and percentage of monocytes, neutrophils and lymphocytes, DYSF methylation level, and the expression levels of DYSF, STAT3, TMX1, CD4, ABR, SELL and RPS27.

[0041] To further explore which variables are independent risk or independent protective factors for coronary heart disease, we included all the above variables in a multivariate analysis. After adjustment by multivariate analysis, 9 variables were obtained. Except for HDL-C, the remaining 8 variables were statistically significant. Among them, TG (OR = 5.302, P = 0.0030), FPG (OR = 1.558, P = 0.0372), monocyte percentage (OR = 2.013, P = 0.0008), DYSF expression level (OR = 4.192, P = 0.0001), STAT3 expression level (OR = 1.570, P = 0.0174), and DYSF methylation level (OR = 5.382, P = 0.0009) were identified as independent risk factors for coronary heart disease. In addition, lymphocyte count (OR = 0.127, P = 0.0006) and TMX1 expression level (OR = 0.184, P = 0.0002) were identified as independent protective factors for coronary heart disease.

[0042] Binary logistic regression analysis was further stratified by gender, and the results showed that the expression and methylation levels of DYSF were independent risk factors in both male and female patients with coronary heart disease.

[0043] ROC curves were plotted and AUCs were calculated to evaluate the potential of core genes as biomarkers for coronary heart disease. The expression levels of DYSF (AUC = 0.770, P < 0.0001), TMX1 (AUC = 0.743, P < 0.0001), and STAT3 (AUC = 0.710, P < 0.0001) and the DYSF methylation level (AUC = 0.724, P < 0.0001) all had high diagnostic ability for coronary heart disease. To further improve the diagnostic ability, we constructed two combined models. As Figure 4 and 5 shown, combining the expression levels of DYSF, STAT3, and TMX1 (AUC = 0.875, P < 0.0001), or combining the DYSF expression with the DYSF methylation level (AUC = 0.888, P < 0.0001) significantly improved the diagnostic ability. The coronary heart disease risk maps of the two combined models are shown in Figure 6 respectively.

[0044] In addition, qMSP was also performed on 30 randomly selected coronary heart disease patients and 30 controls in the above case - control study. As Figure 7 shown, consistent with the results of MDRE - qPCR, qMSP also showed that the DYSF promoter methylation level in coronary heart disease patients was higher than that in the control group (P = 0.0021).

[0045] 3. Functional study of DYSF in peripheral blood leukocytes (PBL) of coronary heart disease

[0046] We also recruited another 12 coronary heart disease patients and 12 controls for transcriptome sequencing. 4 mL of peripheral blood was collected from each subject, and monocytes were isolated using Ficoll - Hypaque solution (Sigma - Aldrich, Germany) and CD14 microbeads (Miltenyi, Germany). Approximately 2×10 6Monocytes were isolated, and approximately 1 μg of total RNA was extracted (Omega, USA). mRNA was purified from total RNA using poly-T oligonucleotide magnetic beads, and then an RNA-seq library was constructed using the NEBNext Multiplex mRNA Library Prep Set for Illumina kit (New England Biolabs, USA). Finally, sequencing was performed on the Illumina NovaSeq 6000 platform. The sequencing data was aligned to the reference genome using Hisat2 (v2.0.5). FeatureCounts (v1.5.0-p3) was used to calculate the sequencing data mapped to each gene. The DESeq2 R package (v1.34.0) was used for differential expression analysis between patients with coronary heart disease and the control group, with a screening threshold of P < 0.05.

[0047] The expression of each gene in PBL was statistically analyzed, and the results are as Figure 8 shown. The expressions of DYSF (P = 0.0441), STAT3 (P = 0.0104), CD4 (P = 0.0056), and ABR (P = 0.0033) were significantly upregulated in the PBL of patients with coronary heart disease.

[0048] 4. DYSF is hypermethylated and highly expressed in Apoe- / - mice

[0049] As Figure 9 shown, there was almost no lipid accumulation and atherosclerotic plaques in wild-type mice, but significant atherosclerotic features could be observed in all Apoe - / - mice. In particular, the induction of a high-fat diet significantly exacerbated the formation of atherosclerotic plaques. Compared with wild-type mice, the methylation level and expression level of the Dysf promoter were both increased in Apoe - / - mice, and the increase was more obvious in Apoe - / - mice fed a high-fat diet ( Figure 10 ).

[0050] The above experiments are sufficient to prove that the expression level and promoter region methylation level of DYSF are highly correlated with the risk of coronary heart disease and can be used as a diagnostic factor for coronary heart disease.

[0051] 5. Cell transgenic experimental study

[0052] Genetic manipulation was performed on THP1 cells to construct a DYSF gene knockout strain (DYSF KD) and an overexpression strain (DYSF OE) ( Figure 11 ), and the expression levels of each gene were detected. The results are as Figure 12As shown, the changes in the mRNA and protein expression levels of SELL were consistent with those of DYSF, indicating that SELL might be a downstream target of DYSF and was regulated by DYSF expression.

[0053] As Figure 11 shown, the ability of THP1 cells to phagocytose oxidized low-density lipoprotein (ox-LDL) was weakened with the knockdown of DYSF and could be significantly enhanced by overexpressing DYSF ( Figure 13 ). Meanwhile, the adhesion and migration abilities of THP1 cells were also enhanced by the overexpression of DYSF ( Figure 14 and 15 ).

[0054] As Figure 16 shown, THP1 cells were treated with PMA and ox-LDL to transform THP1 cells into macrophages or foam cells. The expression level of DYSF increased in macrophages and further increased in foam cells, and similar results were also observed in SELL, STAT3, and ABR.

[0055] THP1 cells with knockdown and overexpression of DNMT1 (amino acid sequence as shown in SEQ ID NO.3) were constructed to change the methylation status of genomic DNA in the cells. The methylation level and expression level of DYSF at different methylation levels were detected. The results were as Figure 17 shown, the DYSF promoter was hypomethylated in DNMT1 knockdown cells and hypermethylated in DNMT1 overexpressing cells. Meanwhile, the expression of DYSF decreased in DNMT1 knockdown cells and increased in DNMT1 overexpressing cells. The above results indicated that the expression of DYSF might be regulated by the methylation of the DYSF promoter. Thus, an increase in the methylation level of the DYSF promoter region could increase the expression of DYSF.

[0056] The above experiments showed that by knocking down or overexpressing the DYSF gene, the ability of cells to transport low-density lipoprotein could be affected. By changing the methylation level of the promoter region of the DYSF gene, the expression level of the DYSF gene could also be changed, thereby affecting the ability of cells to transport low-density lipoprotein.

[0057] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. Sequence Listing <110> Wodepeike (Wuhan) Medical Technology Co., Ltd. <120> Coronary Heart Disease Diagnostic Agent and Application of DYSF Gene Methylation in the Preparation of Coronary Heart Disease Diagnostic Agent <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 250 <212> DNA <213> Homo sapiens <400> 1 cgggttgagt tctggagaga ctgctccaat ccccgaggcg gaaggaggca accgatttgg 60 cgcagcactc agccaggggg tagaagctca ggggaggagc cgagcctttc tcctgtccaa 120 gagcgagatc tgggctacgc cgggcgcccg gagccctagt ccagcccccg gccatcgcgg 180 ccgccgccca gccaggtgca aaatgccgtg tcattgggag actccgcagc cggagcatta 240 gattacagct 250 <210> 2 <211> 2080 <212> PRT <213> Homo sapiens <400> 2 Met Leu Arg Val Phe Ile Leu Tyr Ala Glu Asn Val His Thr Pro Asp 1 5 10 15 Thr Asp Ile Ser Asp Ala Tyr Cys Ser Ala Val Phe Ala Gly Val Lys 20 25 30 Lys Arg Thr Lys Val Ile Lys Asn Ser Val Asn Pro Val Trp Asn Glu 35 40 45 Gly Phe Glu Trp Asp Leu Lys Gly Ile Pro Leu Asp Gln Gly Ser Glu 50 55 60 Leu His Val Val Val Lys Asp His Glu Thr Met 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Glu Asp 690 695 700 Val Asp Ser Leu Val Ala Gln Leu Thr Asp Glu Leu Ile Ala Gly Cys 705 710 715 720 Ser Gln Pro Leu Gly Asp Ile His Glu Thr Pro Ser Ala Thr His Leu 725 730 735 Asp Gln Tyr Leu Tyr Gln Leu Arg Thr His His Leu Ser Gln Ile Thr 740 745 750 Glu Ala Ala Leu Ala Leu Lys Leu Gly His Ser Glu Leu Pro Ala Ala 755 760 765 Leu Glu Gln Ala Glu Asp Trp Leu Leu Arg Leu Arg Ala Leu Ala Glu 770 775 780 Glu Pro Gln Asn Ser Leu Pro Asp Ile Val Ile Trp Met Leu Gln Gly 785 790 795 800 Asp Lys Arg Val Ala Tyr Gln Arg Val Pro Ala His Gln Val Leu Phe 805 810 815 Ser Arg Arg Gly Ala Asn Tyr Cys Gly Lys Asn Cys Gly Lys Leu Gln 820 825 830 Thr Ile Phe Leu Lys Tyr Pro Met Glu Lys Val Pro Gly Ala Arg Met 835 840 845 Pro Val Gln Ile Arg Val Lys Leu Trp Phe Gly Leu Ser Val Asp Glu 850 855 860 Lys Glu Phe Asn Gln Phe Ala Glu Gly Lys Leu Ser Val Phe Ala Glu 865 870 875 880 Thr Tyr Glu Asn Glu Thr Lys Leu Ala Leu Val Gly Asn Trp Gly Thr 885 890 895 Thr Gly Leu Thr Tyr Pro Lys Phe Ser Asp Val Thr Gly Lys Ile Lys 900 905 910 Leu Pro Lys Asp Ser Phe Arg Pro Ser Ala Gly Trp Thr Trp Ala Gly 915 920 925 Asp Trp Phe Val Cys Pro Glu Lys Thr Leu Leu His Asp Met Asp Ala 930 935 940 Gly His Leu Ser Phe Val Glu Glu Val Phe Glu Asn Gln Thr Arg Leu 945 950 955 960 Pro Gly Gly Gln Trp Ile Tyr Met Ser Asp Asn Tyr Thr Asp Val Asn 965 970 975 Gly Glu Lys Val Leu Pro Lys Asp Asp Ile Glu Cys Pro Leu Gly Trp 980 985 990 Lys Trp Glu Asp Glu Glu Trp Ser Thr Asp Leu Asn Arg Ala Val Asp 995 1000 1005 Glu Gln Gly Trp Glu Tyr Ser Ile Thr Ile Pro Pro Glu Arg Lys Pro 1010 1015 1020 Lys His Trp Val Pro Ala Glu Lys Met Tyr Tyr Thr His Arg Arg Arg 1025 1030 1035 1040 Arg Trp Val Arg Leu Arg Arg Arg Asp Leu Ser Gln Met Glu Ala Leu 1045 1050 1055 Lys Arg His Arg Gln Ala Glu Ala Glu Gly Glu Gly Trp Glu Tyr Ala 1060 1065 1070 Ser Leu Phe Gly Trp Lys Phe His Leu Glu Tyr Arg Lys Thr Asp Ala 1075 1080 1085 Phe Arg Arg Arg Arg Trp Arg Arg Arg Met Glu Pro Leu Glu Lys Thr 1090 1095 1100 Gly Pro Ala Ala Val Phe Ala Leu Glu Gly Ala Leu Gly Gly Val Met 1105 1110 1115 1120 Asp Asp Lys Ser Glu Asp Ser Met Ser Val Ser Thr Leu Ser Phe Gly 1125 1130 1135 Val Asn Arg Pro Thr Ile Ser Cys Ile Phe Asp Tyr Gly Asn Arg Tyr 1140 1145 1150 His Leu Arg Cys Tyr Met Tyr Gln Ala Arg Asp Leu Ala Ala Met Asp 1155 1160 1165 Lys Asp Ser Phe Ser Asp Pro Tyr Ala Ile Val Ser Phe Leu His Gln 1170 1175 1180 Ser Gln Lys Thr Val Val Val Lys Asn Thr Leu Asn Pro Thr Trp Asp 1185 1190 1195 1200 Gln Thr Leu Ile Phe Tyr Glu Ile Glu Ile Phe Gly Glu Pro Ala Thr 1205 1210 1215 Val Ala Glu Gln Pro Pro Ser Ile Val Val Glu Leu Tyr Asp His Asp 1220 1225 1230 Thr Tyr Gly Ala Asp Glu Phe Met Gly Arg Cys Ile Cys Gln Pro Ser 1235 1240 1245 Leu Glu Arg Met Pro Arg Leu Ala Trp Phe Pro Leu Thr Arg Gly Ser 1250 1255 1260 Gln Pro Ser Gly Glu Leu Leu Ala Ser Phe Glu Leu Ile Gln Arg Glu 1265 1270 1275 1280 Lys Pro Ala Ile His His Ile Pro Gly Phe Glu Val Gln Glu Thr Ser 1285 1290 1295 Arg Ile Leu Asp Glu Ser Glu Asp Thr Asp Leu Pro Tyr Pro Pro Pro 1300 1305 1310 Gln Arg Glu Ala Asn Ile Tyr Met Val Pro Gln Asn Ile Lys Pro Ala 1315 1320 1325 Leu Gln Arg Thr Ala Ile Glu Ile Leu Ala Trp Gly Leu Arg Asn Met 1330 1335 1340 Lys Ser Tyr Gln Leu Ala Asn Ile Ser Ser Pro Ser Leu Val Val Glu 1345 1350 1355 1360 Cys Gly Gly Gln Thr Val Gln Ser Cys Val Ile Arg Asn Leu Arg Lys 1365 1370 1375 Asn Pro Asn Phe Asp Ile Cys Thr Leu Phe Met Glu Val Met Leu Pro 1380 1385 1390 Arg Glu Glu Leu Tyr Cys Pro Pro Ile Thr Val Lys Val Ile Asp Asn 1395 1400 1405 Arg Gln Phe Gly Arg Arg Pro Val Val Gly Gln Cys Thr Ile Arg Ser 1410 1415 1420 Leu Glu Ser Phe Leu Cys Asp Pro Tyr Ser Ala Glu Ser Pro Ser Pro 1425 1430 1435 1440 Gln Gly Gly Pro Asp Asp Val Ser Leu Leu Ser Pro Gly Glu Asp Val 1445 1450 1455 Leu Ile Asp Ile Asp Asp Lys Glu Pro Leu Ile Pro Ile Gln Glu Glu 1460 1465 1470 Glu Phe Ile Asp Trp Trp Ser Lys Phe Phe Ala Ser Ile Gly Glu Arg 1475 1480 1485 Glu Lys Cys Gly Ser Tyr Leu Glu Lys Asp Phe Asp Thr Leu Lys Val 1490 1495 1500 Tyr Asp Thr Gln Leu Glu Asn Val Glu Ala Phe Glu Gly Leu Ser Asp 1505 1510 1515 1520 Phe Cys Asn Thr Phe Lys Leu Tyr Arg Gly Lys Thr Gln Glu Glu Thr 1525 1530 1535 Glu Asp Pro Ser Val Ile Gly Glu Phe Lys Gly Leu Phe Lys Ile Tyr 1540 1545 1550 Pro Leu Pro Glu Asp Pro Ala Ile Pro Met Pro Pro Arg Gln Phe His 1555 1560 1565 Gln Leu Ala Ala Gln Gly Pro Gln Glu Cys Leu Val Arg Ile Tyr Ile 1570 1575 1580 Val Arg Ala Phe Gly Leu Gln Pro Lys Asp Pro Asn Gly Lys Cys Asp 1585 1590 1595 1600 Pro Tyr Ile Lys Ile Ser Ile Gly Lys Lys Ser Val Ser Asp Gln Asp 1605 1610 1615 Asn Tyr Ile Pro Cys Thr Leu Glu Pro Val Phe Gly Lys Met Phe Glu 1620 1625 1630 Leu Thr Cys Thr Leu Pro Leu Glu Lys Asp Leu Lys Ile Thr Leu Tyr 1635 1640 1645 Asp Tyr Asp Leu Leu Ser Lys Asp Glu Lys Ile Gly Glu Thr Val Val 1650 1655 1660 Asp Leu Glu Asn Arg Leu Leu Ser Lys Phe Gly Ala Arg Cys Gly Leu 1665 1670 1675 1680 Pro Gln Thr Tyr Cys Val Ser Gly Pro Asn Gln Trp Arg Asp Gln Leu 1685 1690 1695 Arg Pro Ser Gln Leu Leu His Leu Phe Cys Gln Gln His Arg Val Lys 1700 1705 1710 Ala Pro Val Tyr Arg Thr Asp Arg Val Met Phe Gln Asp Lys Glu Tyr 1715 1720 1725 Ser Ile Glu Glu Ile Glu Ala Gly Arg Ile Pro Asn Pro His Leu Gly 1730 1735 1740 Pro Val Glu Glu Arg Leu Ala Leu His Val Leu Gln Gln Gln Gly Leu 1745 1750 1755 1760 Val Pro Glu His Val Glu Ser Arg Pro Leu Tyr Ser Pro Leu Gln Pro 1765 1770 1775 Asp Ile Glu Gln Gly Lys Leu Gln Met Trp Val Asp Leu Phe Pro Lys 1780 1785 1790 Ala Leu Gly Arg Pro Gly Pro Pro Phe Asn Ile Thr Pro Arg Arg Ala 1795 1800 1805 Arg Arg Phe Phe Leu Arg Cys Ile Ile Trp Asn Thr Arg Asp Val Ile 1810 1815 1820 Leu Asp Asp Leu Ser Leu Thr Gly Glu Lys Met Ser Asp Ile Tyr Val 1825 1830 1835 1840 Lys Gly Trp Met Ile Gly Phe Glu Glu His Lys Gln Lys Thr Asp Val 1845 1850 1855 His Tyr Arg Ser Leu Gly Gly Glu Gly Asn Phe Asn Trp Arg Phe Ile 1860 1865 1870 Phe Pro Phe Asp Tyr Leu Pro Ala Glu Gln Val Cys Thr Ile Ala Lys 1875 1880 1885 Lys Asp Ala Phe Trp Arg Leu Asp Lys Thr Glu Ser Lys Ile Pro Ala 1890 1895 1900 Arg Val Val Phe Gln Ile Trp Asp Asn Asp Lys Phe Ser Phe Asp Asp 1905 1910 1915 1920 Phe Leu Gly Ser Leu Gln Leu Asp Leu Asn Arg Met Pro Lys Pro Ala 1925 1930 1935 Lys Thr Ala Lys Lys Cys Ser Leu Asp Gln Leu Asp Asp Ala Phe His 1940 1945 1950 Pro Glu Trp Phe Val Ser Leu Phe Glu Gln Lys Thr Val Lys Gly Trp 1955 1960 1965 Trp Pro Cys Val Ala Glu Glu Gly Glu Lys Lys Ile Leu Ala Gly Lys 1970 1975 1980 Leu Glu Met Thr Leu Glu Ile Val Ala Glu Ser Glu His Glu Glu Arg 1985 1990 1995 2000 Pro Ala Gly Gln Gly Arg Asp Glu Pro Asn Met Asn Pro Lys Leu Glu 2005 2010 2015 Asp Pro Arg Arg Pro Asp Thr Ser Phe Leu Trp Phe Thr Ser Pro Tyr 2020 2025 2030 Lys Thr Met Lys Phe Ile Leu Trp Arg Arg Phe Arg Trp Ala Ile Ile 2035 2040 2045 Leu Phe Ile Ile Leu Phe Ile Leu Leu Leu Phe Leu Ala Ile Phe Ile 2050 2055 2060 Tyr Ala Phe Pro Asn Tyr Ala Ala Met Lys Leu Val Lys Pro Phe Ser 2065 2070 2075 2080 <210> 3 <211> 1616 <212> PRT <213> Homo sapiens <400> 3 Met Pro Ala Arg Thr Ala Pro Ala Arg Val Pro Thr Leu Ala Val Pro 1 5 10 15 Ala Ile Ser Leu Pro Asp Asp Val Arg Arg Arg Leu Lys Asp Leu Glu 20 25 30 Arg Asp Ser Leu Thr Glu Lys Glu Cys Val Lys Glu Lys Leu Asn Leu 35 40 45 Leu His Glu Phe Leu Gln Thr Glu Ile Lys Asn Gln Leu Cys Asp Leu 50 55 60 Glu Thr Lys Leu Arg Lys Glu Glu Leu Ser Glu Glu Gly Tyr Leu Ala 65 70 75 80 Lys Val Lys Ser Leu Leu Asn Lys Asp Leu Ser Leu Glu Asn Gly Ala 85 90 95 His Ala Tyr Asn Arg Glu Val Asn Gly Arg Leu Glu Asn Gly Asn Gln 100 105 110 Ala Arg Ser Glu Ala Arg Arg Val Gly Met Ala Asp Ala Asn Ser Pro 115 120 125 Pro Lys Pro Leu Ser Lys Pro Arg Thr Pro Arg Arg Ser Lys Ser Asp 130 135 140 Gly Glu Ala Lys Pro Glu Pro Ser Pro Ser Pro Arg Ile Thr Arg Lys 145 150 155 160 Ser Thr Arg Gln Thr Thr Ile Thr Ser His Phe Ala Lys Gly Pro Ala 165 170 175 Lys Arg Lys Pro Gln Glu Glu Ser Glu Arg Ala Lys Ser Asp Glu Ser 180 185 190 Ile Lys Glu Glu Asp Lys Asp Gln Asp Glu Lys Arg Arg Arg Val Thr 195 200 205 Ser Arg Glu Arg Val Ala Arg Pro Leu Pro Ala Glu Glu Pro Glu Arg 210 215 220 Ala Lys Ser Gly Thr Arg Thr Glu Lys Glu Glu Glu Arg Asp Glu Lys 225 230 235 240 Glu Glu Lys Arg Leu Arg Ser Gln Thr Lys Glu Pro Thr Pro Lys Gln 245 250 255 Lys Leu Lys Glu Glu Pro Asp Arg Glu Ala Arg Ala Gly Val Gln Ala 260 265 270 Asp Glu Asp Glu Asp Gly Asp Glu Lys Asp Glu Lys Lys His Arg Ser 275 280 285 Gln Pro Lys Asp Leu Ala Ala Lys Arg Arg Pro Glu Glu Lys Glu Pro 290 295 300 Glu Lys Val Asn Pro Gln Ile Ser Asp Glu Lys Asp Glu Asp Glu Lys 305 310 315 320 Glu Glu Lys Arg Arg Lys Thr Thr Pro Lys Glu Pro Thr Glu Lys Lys 325 330 335 Met Ala Arg Ala Lys Thr Val Met Asn Ser Lys Thr His Pro Pro Lys 340 345 350 Cys Ile Gln Cys Gly Gln Tyr Leu Asp Asp Pro Asp Leu Lys Tyr Gly 355 360 365 Gln His Pro Pro Asp Ala Val Asp Glu Pro Gln Met Leu Thr Asn Glu 370 375 380 Lys Leu Ser Ile Phe Asp Ala Asn Glu Ser Gly Phe Glu Ser Tyr Glu 385 390 395 400 Ala Leu Pro Gln His Lys Leu Thr Cys Phe Ser Val Tyr Cys Lys His 405 410 415 Gly His Leu Cys Pro Ile Asp Thr Gly Leu Ile Glu Lys Asn Ile Glu 420 425 430 Leu Phe Phe Ser Gly Ser Ala Lys Pro Ile Tyr Asp Asp Asp Pro Ser 435 440 445 Leu Glu Gly Gly Val Asn Gly Lys Asn Leu Gly Pro Ile Asn Glu Trp 450 455 460 Trp Ile Thr Gly Phe Asp Gly Gly Glu Lys Ala Leu Ile Gly Phe Ser 465 470 475 480 Thr Ser Phe Ala Glu Tyr Ile Leu Met Asp Pro Ser Pro Glu Tyr Ala 485 490 495 Pro Ile Phe Gly Leu Met Gln Glu Lys Ile Tyr Ile Ser Lys Ile Val 500 505 510 Val Glu Phe Leu Gln Ser Asn Ser Asp Ser Thr Tyr Glu Asp Leu Ile 515 520 525 Asn Lys Ile Glu Thr Thr Val Pro Pro Ser Gly Leu Asn Leu Asn Arg 530 535 540 Phe Thr Glu Asp Ser Leu Leu Arg His Ala Gln Phe Val Val Glu Gln 545 550 555 560 Val Glu Ser Tyr Asp Glu Ala Gly Asp Ser Asp Glu Gln Pro Ile Phe 565 570 575 Leu Thr Pro Cys Met Arg Asp Leu Ile Lys Leu Ala Gly Val Thr Leu 580 585 590 Gly Gln Arg Arg Ala Gln Ala Arg Arg Gln Thr Ile Arg His Ser Thr 595 600 605 Arg Glu Lys Asp Arg Gly Pro Thr Lys Ala Thr Thr Thr Lys Leu Val 610 615 620 Tyr Gln Ile Phe Asp Thr Phe Phe Ala Glu Gln Ile Glu Lys Asp Asp 625 630 635 640 Arg Glu Asp Lys Glu Asn Ala Phe Lys Arg Arg Arg Cys Gly Val Cys 645 650 655 Glu Val Cys Gln Gln Pro Glu Cys Gly Lys Cys Lys Ala Cys Lys Asp 660 665 670 Met Val Lys Phe Gly Gly Ser Gly Arg Ser Lys Gln Ala Cys Gln Glu 675 680 685 Arg Arg Cys Pro Asn Met Ala Met Lys Glu Ala Asp Asp Asp Glu Glu 690 695 700 Val Asp Asp Asn Ile Pro Glu Met Pro Ser Pro Lys Lys Met His Gln 705 710 715 720 Gly Lys Lys Lys Lys Gln Asn Lys Asn Arg Ile Ser Trp Val Gly Glu 725 730 735 Ala Val Lys Thr Asp Gly Lys Lys Ser Tyr Tyr Lys Lys Val Cys Ile 740 745 750 Asp Ala Glu Thr Leu Glu Val Gly Asp Cys Val Ser Val Ile Pro Asp 755 760 765 Asp Ser Ser Lys Pro Leu Tyr Leu Ala Arg Val Thr Ala Leu Trp Glu 770 775 780 Asp Ser Ser Asn Gly Gln Met Phe His Ala His Trp Phe Cys Ala Gly 785 790 795 800 Thr Asp Thr Val Leu Gly Ala Thr Ser Asp Pro Leu Glu Leu Phe Leu 805 810 815 Val Asp Glu Cys Glu Asp Met Gln Leu Ser Tyr Ile His Ser Lys Val 820 825 830 Lys Val Ile Tyr Lys Ala Pro Ser Glu Asn Trp Ala Met Glu Gly Gly 835 840 845 Met Asp Pro Glu Ser Leu Leu Glu Gly Asp Asp Gly Lys Thr Tyr Phe 850 855 860 Tyr Gln Leu Trp Tyr Asp Gln Asp Tyr Ala Arg Phe Glu Ser Pro Pro 865 870 875 880 Lys Thr Gln Pro Thr Glu Asp Asn Lys Phe Lys Phe Cys Val Ser Cys 885 890 895 Ala Arg Leu Ala Glu Met Arg Gln Lys Glu Ile Pro Arg Val Leu Glu 900 905 910 Gln Leu Glu Asp Leu Asp Ser Arg Val Leu Tyr Tyr Ser Ala Thr Lys 915 920 925 Asn Gly Ile Leu Tyr Arg Val Gly Asp Gly Val Tyr Leu Pro Pro Glu 930 935 940 Ala Phe Thr Phe Asn Ile Lys Leu Ser Ser Pro Val Lys Arg Pro Arg 945 950 955 960 Lys Glu Pro Val Asp Glu Asp Leu Tyr Pro Glu His Tyr Arg Lys Tyr 965 970 975 Ser Asp Tyr Ile Lys Gly Ser Asn Leu Asp Ala Pro Glu Pro Tyr Arg 980 985 990 Ile Gly Arg Ile Lys Glu Ile Phe Cys Pro Lys Lys Ser Asn Gly Arg 995 1000 1005 Pro Asn Glu Thr Asp Ile Lys Ile Arg Val Asn Lys Phe Tyr Arg Pro 1010 1015 1020 Glu Asn Thr His Lys Ser Thr Pro Ala Ser Tyr His Ala Asp Ile Asn 1025 1030 1035 1040 Leu Leu Tyr Trp Ser Asp Glu Glu Ala Val Val Asp Phe Lys Ala Val 1045 1050 1055 Gln Gly Arg Cys Thr Val Glu Tyr Gly Glu Asp Leu Pro Glu Cys Val 1060 1065 1070 Gln Val Tyr Ser Met Gly Gly Pro Asn Arg Phe Tyr Phe Leu Glu Ala 1075 1080 1085 Tyr Asn Ala Lys Ser Lys Ser Phe Glu Asp Pro Pro Asn His Ala Arg 1090 1095 1100 Ser Pro Gly Asn Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly Lys Pro 1105 1110 1115 1120 Lys Ser Gln Ala Cys Glu Pro Ser Glu Pro Glu Ile Glu Ile Lys Leu 1125 1130 1135 Pro Lys Leu Arg Thr Leu Asp Val Phe Ser Gly Cys Gly Gly Leu Ser 1140 1145 1150 Glu Gly Phe His Gln Ala Gly Ile Ser Asp Thr Leu Trp Ala Ile Glu 1155 1160 1165 Met Trp Asp Pro Ala Ala Gln Ala Phe Arg Leu Asn Asn Pro Gly Ser 1170 1175 1180 Thr Val Phe Thr Glu Asp Cys Asn Ile Leu Leu Lys Leu Val Met Ala 1185 1190 1195 1200 Gly Glu Thr Thr Asn Ser Arg Gly Gln Arg Leu Pro Gln Lys Gly Asp 1205 1210 1215 Val Glu Met Leu Cys Gly Gly Pro Pro Cys Gln Gly Phe Ser Gly Met 1220 1225 1230 Asn Arg Phe Asn Ser Arg Thr Tyr Ser Lys Phe Lys Asn Ser Leu Val 1235 1240 1245 Val Ser Phe Leu Ser Tyr Cys Asp Tyr Tyr Arg Pro Arg Phe Phe Leu 1250 1255 1260 Leu Glu Asn Val Arg Asn Phe Val Ser Phe Lys Arg Ser Met Val Leu 1265 1270 1275 1280 Lys Leu Thr Leu Arg Cys Leu Val Arg Met Gly Tyr Gln Cys Thr Phe 1285 1290 1295 Gly Val Leu Gln Ala Gly Gln Tyr Gly Val Ala Gln Thr Arg Arg Arg 1300 1305 1310 Ala Ile Ile Leu Ala Ala Ala Pro Gly Glu Lys Leu Pro Leu Phe Pro 1315 1320 1325 Glu Pro Leu His Val Phe Ala Pro Arg Ala Cys Gln Leu Ser Val Val 1330 1335 1340 Val Asp Asp Lys Lys Phe Val Ser Asn Ile Thr Arg Leu Ser Ser Gly 1345 1350 1355 1360 Pro Phe Arg Thr Ile Thr Val Arg Asp Thr Met Ser Asp Leu Pro Glu 1365 1370 1375 Val Arg Asn Gly Ala Ser Ala Leu Glu Ile Ser Tyr Asn Gly Glu Pro 1380 1385 1390 Gln Ser Trp Phe Gln Arg Gln Leu Arg Gly Ala Gln Tyr Gln Pro Ile 1395 1400 1405 Leu Arg Asp His Ile Cys Lys Asp Met Ser Ala Leu Val Ala Ala Arg 1410 1415 1420 Met Arg His Ile Pro Leu Ala Pro Gly Ser Asp Trp Arg Asp Leu Pro 1425 1430 1435 1440 Asn Ile Glu Val Arg Leu Ser Asp Gly Thr Met Ala Arg Lys Leu Arg 1445 1450 1455 Tyr Thr His His Asp Arg Lys Asn Gly Arg Ser Ser Ser Gly Ala Leu 1460 1465 1470 Arg Gly Val Cys Ser Cys Val Glu Ala Gly Lys Ala Cys Asp Pro Ala 1475 1480 1485 Ala Arg Gln Phe Asn Thr Leu Ile Pro Trp Cys Leu Pro His Thr Gly 1490 1495 1500 Asn Arg His Asn His Trp Ala Gly Leu Tyr Gly Arg Leu Glu Trp Asp 1505 1510 1515 1520 Gly Phe Phe Ser Thr Thr Val Thr Asn Pro Glu Pro Met Gly Lys Gln 1525 1530 1535 Gly Arg Val Leu His Pro Glu Gln His Arg Val Val Ser Val Arg Glu 1540 1545 1550 Cys Ala Arg Ser Gln Gly Phe Pro Asp Thr Tyr Arg Leu Phe Gly Asn 1555 1560 1565 Ile Leu Asp Lys His Arg Gln Val Gly Asn Ala Val Pro Pro Pro Leu 1570 1575 1580 Ala Lys Ala Ile Gly Leu Glu Ile Lys Leu Cys Met Leu Ala Lys Ala 1585 1590 1595 1600 Arg Glu Ser Ala Ser Ala Lys Ile Lys Glu Glu Glu Ala Ala Lys Asp 1605 1610 1615 <210> 4 <211> 372 <212> PRT <213> Homo sapiens <400> 4 Met Ile Phe Pro Trp Lys Cys Gln Ser Thr Gln Arg Asp Leu Trp Asn 1 5 10 15 Ile Phe Lys Leu Trp Gly Trp Thr Met Leu Cys Cys Asp Phe Leu Ala 20 25 30 His His Gly Thr Asp Cys Trp Thr Tyr His Tyr Ser Glu Lys Pro Met 35 40 45 Asn Trp Gln Arg Ala Arg Arg Phe Cys Arg Asp Asn Tyr Thr Asp Leu 50 55 60 Val Ala Ile Gln Asn Lys Ala Glu Ile Glu Tyr Leu Glu Lys Thr Leu 65 70 75 80 Pro Phe Ser Arg Ser Tyr Tyr Trp Ile Gly Ile Arg Lys Ile Gly Gly 85 90 95 Ile Trp Thr Trp Val Gly Thr Asn Lys Ser Leu Thr Glu Glu Ala Glu 100 105 110 Asn Trp Gly Asp Gly Glu Pro Asn Asn Lys Lys Asn Lys Glu Asp Cys 115 120 125 Val Glu Ile Tyr Ile Lys Arg Asn Lys Asp Ala Gly Lys Trp Asn Asp 130 135 140 Asp Ala Cys His Lys Leu Lys Ala Ala Leu Cys Tyr Thr Ala Ser Cys 145 150 155 160 Gln Pro Trp Ser Cys Ser Gly His Gly Glu Cys Val Glu Ile Ile Asn 165 170 175 Asn Tyr Thr Cys Asn Cys Asp Val Gly Tyr Tyr Gly Pro Gln Cys Gln 180 185 190 Phe Val Ile Gln Cys Glu Pro Leu Glu Ala Pro Glu Leu Gly Thr Met 195 200 205 Asp Cys Thr His Pro Leu Gly Asn Phe Ser Phe Ser Ser Gln Cys Ala 210 215 220 Phe Ser Cys Ser Glu Gly Thr Asn Leu Thr Gly Ile Glu Glu Thr Thr 225 230 235 240 Cys Gly Pro Phe Gly Asn Trp Ser Ser Pro Glu Pro Thr Cys Gln Val 245 250 255 Ile Gln Cys Glu Pro Leu Ser Ala Pro Asp Leu Gly Ile Met Asn Cys 260 265 270 Ser His Pro Leu Ala Ser Phe Ser Phe Thr Ser Ala Cys Thr Phe Ile 275 280 285 Cys Ser Glu Gly Thr Glu Leu Ile Gly Lys Lys Lys Thr Ile Cys Glu 290 295 300 Ser Ser Gly Ile Trp Ser Asn Pro Ser Pro Ile Cys Gln Lys Leu Asp 305 310 315 320 Lys Ser Phe Ser Met Ile Lys Glu Gly Asp Tyr Asn Pro Leu Phe Ile 325 330 335 Pro Val Ala Val Met Val Thr Ala Phe Ser Gly Leu Ala Phe Ile Ile 340 345 350 Trp Leu Ala Arg Arg Leu Lys Lys Gly Lys Lys Ser Lys Arg Ser Met 355 360 365 Asn Asp Pro Tyr 370 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 cgggttgagt tctggagaga 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <400> 6 agctgtaatc taatgctccg g 21

Claims

1. Use of a reagent for detecting the methylation level of the DYSF gene promoter region and / or a reagent for detecting the expression level of the DYSF gene in the preparation of a diagnostic agent for coronary heart disease.

2. The use according to claim 1, wherein, the sequence of the DYSF gene promoter region is as shown in SEQ ID NO.

1.

3. The use according to claim 2, wherein, the amino acid sequence encoded by the DYSF gene is as shown in SEQ ID NO.

2.

4. A method for changing the ability of monocytes to phagocytose low-density lipoproteins, wherein, it includes the step of changing the methylation level of the DYSF gene promoter region and / or the gene expression level in the monocytes, and the method is an in vitro method.

5. The method according to claim 4, wherein, the step of changing the methylation level of the DYSF gene promoter region in the monocytes by knocking down or overexpressing the DNMT1 gene.

6. The method according to claim 5, wherein, the amino acid sequence encoded by the DNMT1 gene is as shown in SEQ ID NO.3.

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