Peripheral blood RNA specific site m6A modified molecular marker combination for detecting ASCVD dangerous population and application of peripheral blood RNA specific site m6A modified molecular marker combination

Abstract

The invention discloses a molecular marker combination for detecting peripheral blood RNA (Ribonucleic Acid) specific site m6A modification of dangerous people suffering from atherosclerotic cardiovascular diseases and application of the molecular marker combination. A specific site m6A modification gene is selected from one or more of RCAN1, VLDLR, C5AR1 and THBD. The invention belongs to the field of molecular biological detection, and further provides a kit for detecting a peripheral blood RNA specific site m6A modification marker combination of people suffering from atherosclerotic cardiovascular diseases. And a new application direction is provided for the marker of the peripheral blood RNA specific site m6A modified molecule and the combination thereof in the aspects of auxiliary screening and risk assessment of ASCVD dangerous people. The specimen is a peripheral blood sample of the subject, and the specimen is easy to obtain, high in clinical operability and noninvasive to the subject.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a combination of peripheral blood RNA specific site m 6 A modification molecular markers for detecting at-risk populations of atherosclerotic cardiovascular disease and its application. Background Art

[0002] Atherosclerotic cardiovascular disease (ASCVD) mainly refers to a series of circulatory system diseases with atherosclerosis as the pathophysiological basis, and is the main cause of global morbidity and mortality. With the continuous aggravation of population aging in China, the incidence of ASCVD continues to rise, and it has long ranked first among the total causes of death in the Chinese population, far higher than tumors and other diseases. At present, there are many limitations in the clinical indicators for evaluating and screening ASCVD. Common clinical ASCVD risk factors, such as total cholesterol (TC), lipoprotein (a) [Lp(a)], and low-density lipoprotein cholesterol (LDL-C), etc., have limitations such as inability to balance sensitivity and specificity, or inability to accurately reflect the severity of the lesion and the progression of the disease, including insufficient predictive ability for acute adverse cardiovascular events and unclear relationship with patient outcomes. At the same time, imaging examination technologies such as coronary angiography are costly and difficult to achieve general examination, screening, and residual risk assessment and prognosis monitoring for ASCVD patients and their at-risk populations. Therefore, a biomarker with higher sensitivity, specificity, and better early discrimination effect is beneficial for the diagnosis, disease course monitoring, and prognosis evaluation of this disease.

[0003] In eukaryotes, N6-methyladenosine (m 6 A) refers to the dynamic and reversible methylation modification occurring at the N6 position of RNA adenine. As the most abundant chemical modification on mRNA, it is mainly enriched near the stop codon and the 3'-untranslated region (3'-UTR). The low abundance of m 6 A modification in mRNA and lncRNA, the reaction inertness of the m 6 A methyl group, and the interference of the RNA structure near m 6 A may affect the recognition, function, and detection of m 6 A. Existing m 6 A detection techniques (such as MeRIP-seq) may not be able to comprehensively capture specific m 6A modification, which may lead to the loss of important information in low-abundance RNA samples. The implementation of some highly sensitive detection methods (such as mass spectrometry) is complex and costly, making it difficult to perform routine detection in a large number of samples.

[0004] Therefore, detecting specific m 6 A modification sites in a large number of peripheral blood samples may provide new risk factors related to ASCVD risk and with better clinical application value, which plays a huge role in further improving the accuracy of the ASCVD prediction model and improving the existing ASCVD risk stratification and residual risk assessment system. Summary of the Invention

[0005] Aiming at the various factors that are difficult to solve by existing detection technologies, such as the low abundance of mRNA / lncRNA carrying m 6 A, the reaction inertness of the m 6 A methyl group, and the interference of the RNA structure near m 6 A, the purpose of the present invention is to provide a combination of m 6 A modification molecular markers at specific sites of peripheral blood RNA for detecting ASCVD high-risk populations and its application. The detection method based on the said markers can non-invasively, quickly and accurately assist in screening ASCVD high-risk populations, and has important application value in ASCVD risk assessment research.

[0006] To achieve the purpose of this invention, the following technical solutions are adopted by the present invention:

[0007] In the first aspect, the present invention provides a solution for detecting the m 6 A modification level at specific sites of peripheral blood RNA for detecting ASCVD high-risk populations; the m 6 A modification genes at the specific sites are selected from RCAN1, VLDLR, C5AR1 and THBD or a combination thereof.

[0008] In the early stage of the present invention, methylation RNA immunoprecipitation high-throughput sequencing (m6A-specific methylatedRNA immunoprecipitation with next-generation sequencing, m6A-RIP-seq) was combined with whole transcriptome sequencing (RNA sequencing, RNA-seq) technology to obtain the characteristic RNA m 6 A modification peak map of the macrophage-derived foam cell model, and determined the "m 6Target genes characterized by "low methylation at the A peak and up-regulated mRNA expression"; on the peripheral blood levels of single-sample ASCVD risk populations and healthy controls, the target gene template amount was adjusted using conventional RT-qPCR technology, and after adjustment, the qPCR technology method of single-base extension and ligation was used to perform the re-screening and verification of the specific site m 6 of A modification levels in the peripheral blood of ASCVD risk populations and healthy controls; five specific site m 6 of A modification molecular markers in the peripheral blood RNA of ASCVD risk populations were found.

[0009] The specifically located site m 6 of A-modified genes related to ASCVD risk populations screened by the present invention are selected from RCAN1, VLDLR, C5AR1, and THBD, and their levels in the peripheral blood of at least one ASCVD risk population are different from those in the peripheral blood of at least one healthy control, thus reflecting the differential expression in ASCVD risk populations and healthy controls, and demonstrating high sensitivity and high specificity for the auxiliary screening and risk assessment of ASCVD risk populations.

[0010] In a specific embodiment, the specific m 6 of A modification site sequence of RCAN1 mRNA is as shown in SEQ ID No.1, the specific m 6 of A modification site sequence of VLDLR mRNA is as shown in SEQ ID No.2, the specific m 6 of A modification site sequence of C5AR1 mRNA is as shown in SEQ ID No.3, and the specific m 6 of A modification site sequence of THBD mRNA is as shown in SEQ ID No.4.

[0011] In a second aspect, the present invention provides the application of a system for detecting the specific site m 6 of A modification level in the peripheral blood RNA in products for the auxiliary screening and risk assessment of ASCVD risk populations, and the specifically located site m 6 of A-modified genes are selected from one or a combination of RCAN1, VLDLR, C5AR1, or THBD.

[0012] In a specific embodiment, the specific m 6 of A modification site sequence of RCAN1 mRNA is as shown in SEQ ID No.1, the specific m 6 of A modification site sequence of VLDLR mRNA is as shown in SEQ ID No.2, the specific m 6 of A modification site sequence of C5AR1 mRNA is as shown in SEQ ID No.3, and the specific m6 The A modification site sequence is shown in SEQ ID No. 4. In a specific embodiment, the method for detecting the m 6 A modification level in peripheral blood RNA includes reagents and / or instruments for detecting the m 6 A modification level at specific sites in peripheral blood RNA.

[0013] In a specific embodiment, the product includes a system, the system includes reagents and / or equipment, and the reagents include preparations or kits.

[0014] In a third aspect, the present invention provides a kit for the auxiliary screening and risk assessment of populations at risk of atherosclerotic cardiovascular disease. The kit contains reagents for detecting the m 6 A modification level at specific sites in peripheral blood RNA, and the specific site m 6 A-modified genes are selected from one or a combination of RCAN1, VLDLR, C5AR1, or THBD.

[0015] Preferably, the specific m 6 A modification site sequence of RCAN1 mRNA is shown in SEQ ID No. 1, the specific m 6 A modification site sequence of VLDLR mRNA is shown in SEQ ID No. 2, the specific m 6 A modification site sequence of C5AR1 mRNA is shown in SEQ IDNo. 3, and the specific m 6 A modification site sequence of THBD mRNA is shown in SEQ ID No. 4.

[0016] The kit uses single-base extension and ligation-based qPCR amplification technology to detect the m 6 A modification level at specific sites in peripheral blood RNA; preferably, the principle of the single-base extension and ligation-based qPCR technology is that the ligation activity of the m6A modification ligase in the RNA template inhibits the single-base extension mediated by DNA polymerase. Compared with the product formed by an RNA template without m 6 A modification change, the RNA template containing m 6 A modification change will be significantly reduced;

[0017] Preferably, the single-base extension and ligation-based qPCR technology can achieve simple qPCR-based methods for detecting m 6 A site modification differences.

[0018] Preferably, the single-base extension and ligation-based qPCR technology can complete the m 6 A detection with single-base resolution in low-abundance transcripts, and can specifically detect m 6A modification quantification.

[0019] The kit further includes a reverse transcription reagent part and a fluorescence quantitative detection reagent part for conventional RT-qPCR technology; an annealing and extension reagent part, a single-base extension and ligation reagent part, and a fluorescence quantitative detection reagent part for single-base extension and ligation qPCR technology;

[0020] Preferably, the reverse transcription reagent part for conventional RT-qPCR technology consists of RNase-Free Water and 5×HiFiScript All-in-one qRT Master Mix;

[0021] Preferably, the fluorescence quantitative detection reagent part for conventional RT-qPCR technology consists of RNase-Free Water, 2×TaqPro Universal SYBR qPCR Master Mix, Gene specific primer F, and Gene specific primer F;

[0022] Preferably, the annealing and extension reagent part for single-base extension and ligation qPCR technology consists of UP Probe, DownProbe, dNTP, and 10×Reaction Buffer;

[0023] Preferably, the single-base extension and ligation reagent part for single-base extension and ligation qPCR technology consists of SELECTTM DNA polymerase and SELECTTM ligase;

[0024] Preferably, the fluorescence quantitative detection reagent part for single-base extension and ligation qPCR technology consists of RNase-Free Water, 2×Taq Pro Universal SYBR qPCR Master Mix, Select F, and Select R.

[0025] The present invention does not make special limitations on other reagents in the kit except for the primers of gene loci and the probes of single-base extension of gene loci related to m6A modification of peripheral blood RNA associated with ASCVD, and those skilled in the art can select according to actual needs.

[0026] Fourthly, the present invention provides a system for auxiliary screening and risk assessment of populations at risk of atherosclerotic cardiovascular disease, and the system includes:

[0027] Sample amplification module: The sample amplification module extracts peripheral blood RNA, performs conventional RT-qPCR on target gene loci using the kit according to any one of claims 4-8, and according to 2 △CTFine-tune the amount of RNA template; perform qPCR for single-base extension and ligation on the adjusted RNA template to detect fluorescence signals;

[0028] Result analysis module: regard the cycle value (CT value) generated by the qPCR technology of single-base extension and ligation as the m 6 A modification level difference of the specific site of the target gene mRNA.

[0029] Fifthly, the present invention also protects the application of the kit described herein and the system in the preparation of products for the auxiliary screening and risk assessment of ASCVD high-risk populations.

[0030] The atherosclerotic cardiovascular disease high-risk populations of the present invention include various populations of extremely high risk, ultra-high risk, high risk, medium risk, and low risk that meet the standards of the Chinese Guidelines for Lipid Management (2023); among them, the extremely high-risk population is patients diagnosed with ASCVD; among the undiagnosed populations, those who meet any of the conditions of LDL-C ≥ 4.9 mmol / L or TC ≥ 7.2 mmol / L and diabetic patients aged ≥ 40 years with CKD stages 3-4 are listed as high-risk populations; for other populations, according to the assessment of the overall ASCVD incidence risk in the next 10 years, based on LDL-C, the presence or absence of hypertension, and the number of other ASCVD risk factors, the average 10-year incidence risk < 5%, 5% - 9%, and ≥ 10% are defined as low-risk, medium-risk, and high-risk populations respectively.

[0031] The specific site m of peripheral blood RNA involved in the present invention 6 The sequence of the A modification molecular marker, the specific m 6 The A modification site sequence of RCAN1 mRNA is shown in SEQ ID No.1, and the specific m 6 The A modification site sequence of VLDLR mRNA is shown in SEQ ID No.2, and the specific m 6 The A modification site sequence of C5AR1mRNA is shown in SEQ ID No.3, and the specific m 6 The A modification site sequence of THBD mRNA is shown in SEQ IDNo.4.

[0032] The present invention has the following beneficial effects: The present invention performs an association analysis on all differentially methylated m 6 A modification peak genes in m6A-RIP-seq and all differentially expressed mRNA level genes in RNA-seq. The results show that there are 2 m 6 A modification peak hypermethylated - downregulated mRNA genes, 2 m 6 A modification peak hypermethylated - upregulated mRNA genes, 31 m 6 A modification peak hypomethylated - downregulated mRNA genes and 22 m6 Genes with low methylation of the modified peak A and upregulated mRNA expression; among them, target genes characterized by "low methylation of peak A - upregulated mRNA expression" were selected; at the peripheral blood levels of single - sample ASCVD - risk populations and healthy controls, the template amount of the target genes was adjusted using conventional RT - qPCR technology, and then, using the qPCR technology method of single - base extension and ligation, the specific site m 6 A modification levels of the target genes were re - screened and verified at the peripheral blood levels of ASCVD - risk populations and healthy controls; it was found that 5 kinds of specific site m 6 A modification molecular markers of peripheral blood RNA in ASCVD - risk populations can be clinically applied in auxiliary screening and risk assessment reagents or kits for ASCVD - risk populations. 6 Continue to obtain through analysis and research that the specific site m

[0033] A modification - differential genes of peripheral blood RNA in 5 kinds of ASCVD - risk populations can be used as molecular markers, showing significant and stable differences in ASCVD - risk populations, with the characteristics of high sensitivity and high specificity. 6 A modification - differential genes of peripheral blood RNA in 5 kinds of ASCVD - risk populations can be used as molecular markers, showing significant and stable differences in ASCVD - risk populations, with the characteristics of high sensitivity and high specificity.

[0034] The present invention can complete the detection of m 6 A at single - base resolution in low - abundance transcripts (≥1 μg), and can specifically perform m 6 A modification quantification on specific sites without antibody enrichment, with the advantages of simple operation, high throughput and low cost.

[0035] The specimen of the present invention is the peripheral blood of the subject, which is easy to obtain, has strong clinical operability and is non - invasive to the subject.

[0036] The specific site m 6 A modification - differential genes of peripheral blood RNA and the detection kit provided by the present invention are of great significance for the auxiliary screening and risk assessment of ASCVD - risk populations through verification in ASCVD - risk populations and healthy controls. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the following drawings are provided for illustration:

[0038] Figure 1 Based on the sequencing results, the m 6 A modification level differences of 5 kinds of target genes characterized by "low methylation of peak A - upregulated mRNA expression" selected in the ASCVD group and the control group. 6 A modification level differences.

[0039] Figure 2 For the 4 kinds of m 6Key genes with significant differences in modification levels were identified, and the sample size was expanded for verification.

[0040] Figure 3 ROC analysis was performed for the RCAN1, VLDLR, C5AR1, and THBD genes individually and in four combinations in the ASCVD group and the control group.

[0041] Figure 4 This is the discovery and validation protocol for the molecular markers. Detailed implementation

[0042] The technical solutions of the embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments that can be obtained by those skilled in the art without creative efforts belong to the protection scope of the present invention.

[0043] For specific technologies or conditions not described in the embodiments, please follow the technologies or conditions described in the literature in this field or follow the product specifications. For reagents or instruments whose manufacturers are not listed, unless otherwise specified, commercially available reagents are used.

[0044] Example 1

[0045] Research subjects: 148 confirmed ASCVD patients admitted to the Department of Cardiology, Eastern Theater General Hospital of the Chinese People's Liberation Army from May 2023 to January 2024. During the same period, 131 healthy control subjects and 48 subjects each with high-risk, medium-risk, and low-risk ASCVD risk were selected from healthy patients visiting the hospital or the physical examination population. Detection indexes and relevant clinical information of the samples were systematically collected. All selected research subjects, including ACS and SCAD patients and controls, were excluded from diseases such as severe liver and kidney insufficiency, malignant tumors, autoimmune diseases, recent trauma or surgery. The specimens collected in this invention were approved by the Medical Ethics Committee of Eastern Theater General Hospital of the Chinese People's Liberation Army, and informed consent of the participants was obtained.

[0046] Example 2

[0047] This example provides a method for detecting the m 6 A modification level at a specific site of peripheral blood RNA in atherosclerotic cardiovascular disease risk populations, and the method includes:

[0048] 1. Sample preparation

[0049] Collect the routine blood test specimens of the first admission of ASCVD patients and healthy controls using vacuum blood collection tubes containing ethylenediaminetetraacetic acid (EDTA) anticoagulant. Centrifuge all blood samples at 3,500 rpm for 5 min at room temperature with a centrifugal radius of 13.5 cm. Collect the upper plasma and lower blood cells of the specimens and store them at -80 °C for later use.

[0050] 2. Peripheral blood RNA extraction

[0051] Take 200 μL of blood cells from each sample, add 1 mL of VeZol Reagent (Vazyme), shake and mix well, let it stand at room temperature for 10 min after sufficient lysis. Add 200 μL of TRIzon Pal TM (CWBIO), shake vigorously to form an emulsion, let it stand at room temperature for 10 min, then centrifuge at 12,000 g for 20 min at 4 °C. After centrifugation, take out the Ep tube, carefully aspirate the upper aqueous phase (about 600 μL) into a new Ep tube, add an equal volume of isopropanol, invert and mix well, and place it in the -20 °C refrigerator overnight. Centrifuge at 12,000 g for 20 min at 4 °C and discard the supernatant. Add 1 ml of freshly prepared 75% ethanol (prepared with DEPC water) for washing, centrifuge at 12,000 g for 10 min at 4 °C and discard the supernatant. Repeat the washing once. Invert and dry on a clean workbench for 10 - 20 min. Add 32 μL of DEPC water to dissolve the precipitate and vortex at room temperature to fully dissolve the precipitate. After measuring the concentration and purity of the extracted RNA, store it in the -80 °C refrigerator or proceed to the next experiment. All the above steps are carried out in a fume hood.

[0052] Example 3

[0053] 1. Select 5 key genes to be verified from the m6A - RIP - seq combined with RNA - seq sequencing results, characterized by "low m 6 A peak hypomethylation - up - regulated mRNA expression". Use 40 ASCVD patients and 40 healthy controls as independent samples, and apply the single - base extension and ligation qPCR technology to verify whether it is consistent with the sequencing results. The sequences of 5 specific - site m 6 A modification molecular markers of peripheral blood RNA are shown in Table 1.

[0054] Table 1: Sequences of 5 specific - site m 6 A modification molecular markers

[0055]

[0056] 2. Conventional RT - qPCR technology

[0057] Conventional RT - qPCR technology includes reverse transcription part and fluorescence quantitative detection part.

[0058] Perform reverse transcription on the target gene locus using reverse transcription reaction, and the specific reaction system is shown in Table 2.

[0059] Table 2: Reverse transcription system

[0060] System components 20 μL system 5×HiFiScript All-in-one qRT Master Mix 4 μL RNA 800 ng / sample concentration RNase-Free Water Make up to 20 μL

[0061] The reverse transcription process uses an Axygen MaxyGene Ⅱ gradient PCR amplifier (Corning), and the reaction conditions are 50 °C for 15 min, 85 °C for 5 sec, 4 °C ∞, and the product is subjected to subsequent PCR reaction.

[0062] Perform amplification on the reverse transcription product using real-time fluorescence quantitative PCR reaction, and the specific reaction system is shown in Table 3.

[0063] Table 3: Real-time fluorescence quantitative PCR reaction system

[0064] System components 10 μL system Template (cDNA) 1 μL 2×Taq Pro Universal SYBR qPCR Master Mix 5 μL Gene-specific primer F 0.2 μL Gene-specific primer R 0.2 μL RNase-free Water 3.6 μL

[0065] The instrument used for the amplification reaction is carried out on a Roche cobas z 480 real-time fluorescence quantitative PCR instrument, and the reaction program settings are shown in Table 4, and other settings are the system default values.

[0066] Table 4: PCR instrument amplification program

[0067]

[0068] Three replicate experiments are set for each sample, and the data is the average value of the three replicate experiments. Check the primer specificity through the melting curve, and a single peak represents specific amplification. According to the CT values of ASCVD patients and healthy controls, fine-tune the next sample loading template amount through the 2 △CT calculation method.

[0069] The above reverse transcription reaction reagents are from CWBIO, 2×Taq Pro Universal SYBR qPCR MasterMix is from Vazyme, and the rest of the reagents are from Epibiotek.

[0070] 3. qPCR technology for single-base extension and ligation

[0071] The qPCR technology for single-base extension and ligation includes an annealing extension part, a single-base extension ligation part, and a fluorescence quantitative detection part.

[0072] Perform annealing extension on the target gene locus, and the specific reaction system is shown in Table 5.

[0073] Table 5: Annealing extension system

[0074]

[0075]

[0076] The reaction conditions for annealing and extension are 90 °C for 1 min, 80 °C for 1 min, 70 °C for 1 min, 60 °C for 1 min, 50 °C for 1 min, 40 °C for 6 min, and 4 °C for ∞. The product is subjected to single-base extension and ligation.

[0077] Single-base extension and ligation are performed on the product of the previous step. The specific reaction system is shown in Table 6.

[0078] Table 6: Single-base extension and ligation system

[0079] System components 10 μL system Product of the previous reaction 8.5 μL SELECTTM DNA polymerase 0.15 μL SELECTTM ligase 0.23 μL ATP 1.12 μL

[0080] The reaction conditions for single-base extension and ligation are 40 °C for 20 min, 80 °C for 20 min, and 4 °C for ∞. The product is subjected to subsequent PCR reaction.

[0081] Amplification is performed using the qPCR method of single-base extension and ligation. The specific reaction system is shown in Table 7.

[0082] Table 7: qPCR reaction system for single-base extension and ligation

[0083] System components 10 μL system Template (cDNA) 1 μL 2×Taq Pro Universal SYBR qPCR Master Mix 5 μL Select F 0.2 μL Select R 0.2 μL RNase-free Water 3.6 μL

[0084] The reaction program is set as shown in Table 5 above.

[0085] Except for the reagents mentioned in the above steps, the remaining reagents are all from Epibiotek.

[0086] Three replicate experiments are set for each sample, and the data are the average values of the three replicate experiments. The primer specificity is checked by melting curve, and a single peak represents specific amplification. According to the CT value, the m 6 A modification levels at specific loci of peripheral blood RNA between ASCVD patients and healthy controls are compared.

[0087] 4. Expression level analysis

[0088] Using SPSS software for result analysis, it is found that among the 5 key genes characterized by "m 6 A hypomethylation - mRNA upregulation" in the peripheral blood of ASCVD patients, the m 6 A modification levels at the RCAN1, VLDLR, C5AR1, and THBD loci are all significantly lower than those of healthy controls, and the difference is statistically significant (P < 0.05). The results are shown in Figure 1 .

[0089] Example 4

[0090] In this example, according to the methods described in Examples 1-3, the sample size was expanded for verification. Another 108 ASCVD patients, 91 healthy controls, and 48 cases each of high-risk, medium-risk, and low-risk ASCVD risk populations were detected to further illustrate the usage effect of the kit described in the present invention.

[0091] Compared with the control group, the m 6 A modification levels at the RCAN1, VLDLR, C5AR1, and THBD gene loci in the ASCVD group were significantly decreased, and the difference was statistically significant (P<0.05). The m 6 A modification levels at specific loci of the RCAN1, VLDLR, and C5AR1 genes in the low-risk group and the medium-high-risk group were significantly decreased, further indicating the role of the present invention in the auxiliary screening and risk assessment of atherosclerotic cardiovascular disease risk populations.

[0092] We further analyzed the diagnostic efficacy of RCAN1, VLDLR, C5AR1, and THBD as ASCVD molecular markers through the ROC curve and the area under the curve (AUC). As shown in Table 8, in the small-sample verification, the AUC of the combined diagnosis of ASCVD by the four markers was 0.800 (95% CI: 0.700 to 0.900, P<0.0001). Further, in the large-sample verification, the AUC of the combined diagnosis of ASCVD by the four markers was 0.751 (95% CI: 0.685 to 0.817, P<0.0001). To distinguish the low-risk and medium-high-risk risk populations from the control group, the m 6 A modification levels at specific loci of the RCAN1, VLDLR, and C5AR1 genes were combined, and their AUCs were 0.884 (95% CI: 0.640 to 0.789, P<0.0001) and 0.714 (95% CI: 0.640 to 0.789, P<0.0001), respectively.

[0093] Table 8: AUC values of the four molecular markers

[0094]

[0095]

[0096] The results showed that the combined detection of the m 6 A modification levels at the RCAN1, VLDLR, C5AR1, and THBD gene loci was helpful as a clinical biomarker to improve the discrimination between ASCVD and healthy controls. In addition, the m 6 A modification levels at these specific gene loci could also be used for the auxiliary screening and risk assessment of atherosclerotic cardiovascular disease risk populations, with clinical value and application advantages.

[0097] In summary, the present invention screened and discovered 4 specific site m 6 A modification molecular markers related to atherosclerotic cardiovascular disease. Based on these markers, a product for auxiliary screening and risk assessment of high-risk populations of atherosclerotic cardiovascular disease was prepared, a method for screening atherosclerotic cardiovascular disease based on these molecular markers was developed, and the above method was verified, as Figure 4 shown. The present invention uses the peripheral blood samples of the subjects, and the specimens are easy to obtain, with strong clinical operability and non-invasive to the subjects. The combined detection of 4 specific site m 6 A modification molecular markers has good diagnostic efficacy for atherosclerotic cardiovascular disease, helps in the auxiliary screening and risk assessment of atherosclerotic cardiovascular disease, and has potential clinical value and application advantages.

[0098] The applicant declares that the above embodiments are only for illustrating the technical solutions of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that within the technical scope of the present invention disclosed herein, any modification by those skilled in the art to the technical solutions described in the foregoing embodiments or / and replacement of some technologies all fall within the protection scope of the present invention.

Claims

1. Detection of specific sites of peripheral blood RNA m 6 The application of a system with A modification level in the preparation of an auxiliary screening and risk assessment product for a population at risk of atherosclerotic cardiovascular disease is characterized in that: The specific site m 6 The A modified gene is selected from one or a combination of RCAN1, VLDLR, C5AR1 or THBD.

2. The use according to claim 1, characterized in that: The RCAN1 mRNA specific 6 The sequence of the A modification site is shown in SEQ ID No. 1, and the VLDLR mRNA specific m 6 The sequence of the A modification site is shown in SEQ ID No. 2, and the C5AR1 mRNA specific m 6 The sequence of the A modification site is shown in SEQ ID No. 3, and the THBD mRNA specific m 6 The sequence of the A modification site is shown in SEQ ID No.

4.

3. The use according to claim 1 or 2, characterized in that: The detection of peripheral blood RNA specific site m 6 A modification level system includes detection of specific sites in peripheral blood RNA m 6 A Modification level of reagents and / or instruments.

4. The use according to claim 1 or 2, characterized in that: The products include systems including reagents and / or devices, and the reagents include formulations or kits.

5. A kit for auxiliary screening and risk assessment of people at risk of atherosclerotic cardiovascular disease, characterized in that: Contains specific sites for detecting peripheral blood RNA 6 A modification level reagent, the specific site m 6 The A modified gene is selected from one or a combination of RCAN1, VLDLR, C5AR1 and THBD.

6. The kit according to claim 5, characterized in that The RCAN1 mRNA specific 6 The sequence of the A modification site is shown in SEQ ID No. 1, and the VLDLR mRNA specific m 6 The sequence of the A modification site is shown in SEQ ID No. 2, and the C5AR1 mRNA specific m 6 The sequence of the A modification site is shown in SEQ ID No. 3, and the THBD mRNA specific m 6 The sequence of the A modification site is shown in SEQ ID No.

4.

7. The kit according to claim 5, characterized in that The kit uses single base extension and ligation qPCR amplification technology to detect specific sites of peripheral blood RNA m 6 A modification level; Preferably, the kit contains annealing and extension reagents for qPCR amplification technology for single-base extension and ligation, single-base extension and ligation reagents, and fluorescent quantitative detection reagents; Preferably, the annealing and extension reagents of the qPCR amplification technique for single base extension and ligation consist of UP Probe, Down Probe, dNTP, and 10× Reaction Buffer; Preferably, the single base extension and ligation qPCR amplification technique single base extension ligation reagent is selected from SELECT TM DNA polymerase, SELECT TM Ligase composition; Preferably, the single base extension and ligation qPCR amplification technology fluorescence quantitative detection reagents consist of RNase-Free Water, 2×Taq Pro Universal SYBR qPCR Master Mix, Select F, and Select R.

8. The kit according to claim 5 or 6, characterized in that The kit also includes conventional RT-qPCR technology reverse transcription reagents and fluorescent quantitative detection reagents; Preferably, the conventional RT-qPCR technology reverse transcription reagent consists of RNase-Free Water and 5×HiFiScript All-in-one qRT Master Mix; Preferably, the conventional RT-qPCR technology fluorescence quantitative detection reagent consists of RNase-Free Water, 2×Taq ProUniversal SYBR qPCR Master Mix, Gene specific primer F, and Gene specific primer R.

9. A system for assisting screening and risk assessment of people at risk of atherosclerotic cardiovascular disease, characterized in that: The system comprises: (1) Sample amplification module: extract peripheral blood RNA, use the kit described in any one of claims 4 to 8 to perform conventional RT-qPCR on the target gene site, according to 2 △CT Fine-tune the amount of RNA template; perform single-base extension and ligation qPCR on the adjusted RNA template, and detect the fluorescence signal; (2) Result analysis module: The cycle value (CT value) generated by the single-base extension and ligation qPCR technology is regarded as the mRNA of the specific site of the target gene 6 A. Differences in modification levels.

10. Use of the kit according to any one of claims 5 to 8 and the system according to claim 9 in preparing a product for auxiliary screening and risk assessment of people at risk of atherosclerotic cardiovascular disease; Preferably, the risk group for atherosclerotic cardiovascular disease is selected from the extremely high risk, ultra-high risk, high risk, medium risk, and low risk groups that meet the standards of the Chinese Guidelines for Blood Lipid Management (2023); wherein, The extremely high-risk population is patients with confirmed ASCVD; among the undiagnosed population, diabetic patients with CKD stage 3 to 4 who meet any of the conditions of LDL-C ≥ 4.9mmol / L or TC ≥ 7.2mmol / L and age ≥ 40 years are classified as the high-risk population; other populations are evaluated based on the overall risk of ASCVD in the next 10 years, and the average risk of 10 years is assessed according to LDL-C, the presence or absence of hypertension and the number of other ASCVD risk factors, and is defined as low-risk, medium-risk and high-risk groups with an estimated average risk of <5%, 5% to 9% and ≥10%, respectively.

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